PURIFICATION AND CHARACTERIZATION

Transcription

1 In: Protein Purification ISBN: Editor: Miguel Benitez and Victoria Aguiree 2012 Nova Science Publishers, Inc. The exclusive license for this PDF is limited to personal website use only. No part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services. Chapter 1 PURIFICATION AND CHARACTERIZATION OF NOVEL SPERM MOTILITY-RELATED PROTEINS Gopal C. Majumder 1, Kaushik Das 2, Sudipta Saha 2, Debjani Nath 3, Arunima Maiti 4, Sujoy Das 2, Nabina Dey 5, Saswati Banerjee 2, Madhabi Barua 6, Mahitosh Mandal 7, Bijay Shankar Jaiswal 8, Rupamanjari Biswas 9, Arpita Bhoumik 2, Debarun Roy 1, Souvik Dey 1, Jharna Som 1, Debdas Bhattacharyya 1 and Sandhya Rekha Dungdung 2 1 Centre for Rural and Cryogenic Technologies, Jadavpur University, Kolkata, India. 2 Indian Institute of Chemical Biology (CSIR-IICB), Kolkata, India. 3 Department of Zoology, University of Kalyani, India. 4 Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, UK 5 New Delhi, India 6 NY US 7 Indian Institute of Technology, Kharagpur, India 8 Department of Molecular Biology, MS 413a, Genentech Inc., CA US 9 Maa Durga Diagnostic Center, Kolkata, India. ABSTRACT Spermatozoa are highly specialised cells that possess flagellar motility which is essential for natural fertilization. Sperm motility measurement is associated with a serious technical problem due to sticking of these cells to the glass surface of haemocytometer required for motility analysis. Our research work has led to the discovery of novel antisticking factors (ASF-I and II) in caprine epididymal plasma (EP) that showed high affinity for inhibiting adhesion of spermatozoa to glass. These factors have been purified to Phone: (Office); (Res); Mobile: , majumdergc42@yahoo.co.in

2 2 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. apparent homogeneity by using multiple biochemical fractionation procedures. ASF-I and II are 47 and 36 kda, heat-stable glycoproteins. The purified ASFs are suitable for eliminating the cell-sticking" artefact in motility assays, as they have no role in sperm motility. Using ASFs in routine sperm motility assays, we have investigated biochemical basis of caprine sperm forward progression. A motility-promoting protein (FMSF) has been purified from buffalo serum using CM-cellulose chromatography, HPLC and native polyacrylamide gel electrophoresis. FMSF is a 66 kda specific monomeric protein. Recently MIP: a motility initiating protein has been purified from EP. It is a 125 kda dimeric protein made up of two subunits: 70 and 54 kda. FMSF is also present in EP. Both the proteins serve as the physiological activators of sperm motility. Studies from our laboratory have identified for the first time a coupled ecto-enzyme system consisting of a protein kinase: CIK, a phosphoprotein phosphatase: PPase and their endogenous specific protein substrate: MPS, that controls the phosphorylation/dephosphorylation state of the endogenous cell-surface proteins, using spermatozoon as the cell model. All the protein components have been purified to apparent homogeneity from sperm membrane. Ecto-CIK is a dimer possessing two subunits: 63 and 55kDa. The CIK is a strongly basic protein. It is a unique membrane protein-specific serine/and threonine kinase. Ecto-MPS is a monomeric protein of 100 kda. Both CIK as well as MPS are required for sperm forward motility and acrosomal reaction. Ecto-PPase is a monomeric 38 kda protein.the coupled-enzyme plays pivotal role in regulating sperm vigorous (vertical) movement. Sperm outer-surface also possesses a novel D-galactose-specific lectin and its receptor that have been purified by using Sepharose and Sepharose-lectin affinity chromatography, respectively. The lectin and its receptor have been implicated to play vital role in the regulation of sperm epididymal maturation and forward movement. A motility inhibiting protein has been purified and characterized from sperm membrane. It is a 100 kda monomeric protein. More recently another motility inhibitor (MIF) has been purified from EP using hydroxylapatite and DEAE-cellulose chromatograph and chromatofocusing. MIF is a heat-labile 160 kda dimeric protein. A protein phosphatase (170 kda) and a low molecular protein (14 kda) have been identified in sperm cytosol and they are motility inhibitors. Sperm cytosol as well possesses a novel bicarbonate-activated soluble adenylyl cyclase that activates flagellar movement by elevating intra-sperm cyclic AMP level. Some of these motility promoting proteins may be extremely useful for improving cattle breeding and breeding of endangered species of animals thereby helping in enhanced production of animal products as well as in the conservation of animals that are almost extinct. The motility-related proteins have also the potential for use as contraceptives to control population growth and for solving some of the problems of human infertility: a global problem of immense dimension. INTRODUCTION Spermatozoon is the male gamete and it is haploid in nature. This unique microscopic motile cell performs an important function in biology: fertilization of ova. Sperm cells are formed in the testes by a process called spermatogenesis which is controlled by the hormones: LH and FSH. Sperm production requires a temperature which is several degrees below (95-97 F) body temperature. The scrotum has a built-in thermostat, which keeps the sperm at the correct temperature while they are being stored. It consists of a head, body and tail. The head is covered by the acrosomal cap and contains a nucleus of dense genetic material from the 23 chromosomes. It is attached from the neck to the body containing mitochondria that supply

3 Purification and Characterization 3 the energy for the sperm's activity. The inner core of the sperm flagella contains microtubules that serve as the basic infrastructure for the ATP-dependent bending of the sperm tail. The flagellar beat kinematics, sperm morphology and surface properties are responsible for the rate of forward progression [Katz et al., 1989]. Upon ejaculation into the female reproductive tract, the male gametes undergo capacitation and acrosomal reaction that are essential for their fertility potential. Cell surface of sperm like the other mammalian cells is believed to play a vital role in the regulation of sperm capacitation, acrosomal reaction and fertilization [Yanagimachi et al., 1994; Ghosh and Datta 2003; Hemchand and Saha 2003; Yoshida et al., 2008; de Lamirande and O, Flaherty 2008]. Mammalian testicular spermatozoa are immotile and infertile and these cells undergo a maturation process in epididymis before they acquire the capacity for forward progression and fertility. The three major parts of mammalian epididymis are: caput, corpus and cauda (Figure 1). The immature testicular spermatozoa acquire testosterone dependent maturity as they pass through different parts of epididymis and finally the mature sperm cells are stored in the terminal part (cauda) of the organ [Glander 1984 et al.; Hoskins et al., 1978; Orgebin- Crist and Tichenor 1972; Prosad et al., 1970]. During the epididymal transit, spermatozoa undergo a variety of biochemical alterations [for reviews see Cooper 1986.; Olson et al., 2002; Jones et al., 2002; Majumder et al., 1990; 2011]. There is a marked increase of intrasperm level of cyclic adenosine monophosphate (camp) and ph during the epididymal sperm maturation suggesting thereby that elevated intrasperm levels of camp and ph have an important role for the in vivo initiation of sperm forward progression [Hoskins et al., 1978; Brokaw et al., 1987; Lee et al., 1983]. Fluidity of the goat sperm plasma membrane (PM) decreases significantly during the epididymal transit of sperm [Poulos and Brown-Woodman, 1975; Rana and Majumder, 1995]. The sperm PM also undergoes major changes in protein composition during epididymal maturation [Olson and Danjo 1981, Austin et al., 1985, Rana and Majumder GC, 1990]. During epididymal transit, the spermatozoa also undergo a change in size, shape, and internal structure of the acrosome [Olson et al., 2002]. In addition, the sperm cell membrane is under constant remodelling with attachment and shedding of molecules in a sequential manner. [Jones et al., 2002; Cuasnicu et al., 2002]. However, the molecular basis of the initiation of flagellar motility in epididymis is still largely unknown. During the last three decades we have established caprine (Capra indicus) sperm as model for investigating the biochemical basis of sperm flagellar motility initiation, regulation and cryopreservation. Methodologies have been developed for the isolation of viable maturing sperm [Halder et al., 1990] and highly purified maturing sperm plasma membranes [Rana and Majumder, 1987; 1989 estimation of sperm motility spectrophotometrically [Majumder and Chakrabarti, 1984] and measuring intactness of isolated sperm using ethidium bromide as the fluorescent probe [Dey and Majumder, 1988]. Some of our recent studies on this cell model is concerned with the structural and functional characteristics of the sperm PM with special reference to lipid analysis [Rana et al., 1990], lipid phase fluidity [Rana and Majumder, 1990], protein phosphorylation/dephosphorylation mechanisms [Dey and Majumder, 1990 a,b; Barua et al., 1990; Barua and Majumder, 1990; Majumder et al., 1990] and cell surface antigens [Chatterjee and Majumder, 1989]. Methodologies have also been developed to analyse sperm adhesion to haemocytometer counting chamber and eliminating the problem of cell-sticking artifacts [Roy et al., 1985; Roy and Majumder 1989; 1990]. Multiple proteins have been identified that play important role in the regulation of mammalian sperm flagellar motility. This article reviews

4 4 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. purification methods and biochemical and functional characteristics of some major motilityrelated proteins with special reference to the caprine sperm model. Figure 1. A photograph of caprine epididymis: The epididymis is closely apposed tubule to the testicles that fuses with the vasa efferentia at their testicular end and the vas deferens at its distal end. It can be roughly divided into three parts; the enlarged proximal end known as the head or caput, the slender middle region or the corpus and the pointed tail end or cauda (Reproduced frommajumder et al 2011) SPERM MOTILITY ANALYSIS It is necessary to have a glimpse of various motility assay methods as the studies that will be discussed here are based on sperm motility. Motility is an important parameter for flagellated or ciliated cells for their survival and propagation. This motility has been found to be necessary for their virulence and motility of different groups can be differentiated by their respective velocities. Velocity level is also directly related to their infectivity [Butler and Camilli, 2004; Lux and Shi, 2004; Appiah et al., 2005]. Sperm velocity is considered as one of the primary determinant factor to predict on the quality and fertilizing ability in vivo [Froman et al., 1999; Bonde et al., 1998]. Moreover, in any in vitro fertilization (IVF) program it is a routine practice to select the best sperms by the swim-up technique [Akerlof et al., 1991; Mortimer et al., 1994]. Microscopic Method Sperm motility was estimated by evaluating forward motility (FM) of spermatozoa using haemocytometer as the counting chamber [Mandal et al., 1989; Mandal et al., 2006]. To eliminate the possibility of artifact due to sperm adhesion to glass, motility assays were

5 Purification and Characterization 5 carried out in presence of Epididymal plasma (EP) (0.6 mg protein/ml) that contained adequate anti-sticking activity to cause nearly 100% inhibition of sperm adhesion to glass [Roy et al., 1985]. Spermatozoa (0.5 X 10 6 cells) were incubated with EP (1.2 mg protein/ml) in the absence or presence of specified amounts of test samples at room temperature (32 C ± 1) for 1 min in a total volume of 0.5 ml of RPS medium (for composition see Majumder et al, 1990). A portion of the cell suspension was then placed in the haemocytometer and the sperm cells showing all sorts of motility (total motility) and only forward motility (FM) were counted under a phase contrast microscope at 250 X magnification. The percentage of motile sperm was then calculated. A unit of activity was defined as the amount of the test sample that induced FM in 10% of the cells under the standard assay conditions. The percentages of motile cells are given as the mean ± SEM of at least three experiments. This method measures essentially sperm horizontal movement. Spectrophotometric Assay of Sperm Motility We have developed a simplified spectrophotometric method for measuring sperm forward motility [Majumder and Chakraborty, 1984] by modifying the method of Sokoloski et al., The microscopic method of motility assay described above take into consideration the number of cells with forward progression but not their velocity, whereas the spectrophotometric method is based not only on the motile cell number but also on their velocity. The weakly motile cells are not detected by this method. The method consists of layering 50 l of freshly extracted cauda epididymal spermatozoa (200x10 6 /ml) mixed with 10% Ficoll in a total volume of 0.5 ml RPS medium with a Hamilton syringe at the bottom of a standard cuvette containing 1.3 ml of RPS medium which was sufficient to cover the entire width of the light beam. Vigorously motile spermatozoa that moved upward in the light beam at any particular time were registered continuously as an increase of absorbance at 545 nm in a spectrophotometer equipped with a recorder (Figure 2). After reaching the maximum absorbance (A eq ) the content of the cuvette was mixed and the absorbance for all the cells was noted (A t ). The percentage of the cells that showed vigorous forward motility was calculated as A eq /A t x100. The change of velocity after treatment with test sample was measured according to change of forward motility activity. One unit of forward motility activity of the most vigorous group of spermatozoa (responsible for the first slope) was defined as an initial linear increase of absorbance of 0.01/min under standard assay condition. Specific activity of a sperm forward stimulating motility is expressed as units of forward progression per 10 7 spermatozoa. [Majumder and Chakraborty, 1984]. Measurement of Sperm Vertical Velocity Subsequently several more objective sperm motility assay methods were developed that are based on light scattering, laser spectroscopy, multiple exposure photography and computer aided semen analyzer (CASA) [Kamidono et al., 1983; Freund and Oliveira 1987; Devi and Shivaji, 1994; Perez-Sanchez et al., 1996; Iguer-Ouada and Verstegen, 2001; Zhang et al., 2002].

6 6 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Figure 2. A representative spectrophotometric tracing, showing the time course of the assay of sperm forward motility. The assays were carried out under standard assay condition without (control) or with 6.8x 10 6 goat spermatozoa from the cauda epididymis. The slope of the initial curve can be calculated from the broken line. A eq = Absorbance at equilibrium. (Reproduced from Majumder and Chakrabarti, 1984). Figure 3. A. Schematic block diagram showing the spectrophotometric system with the positions of the cuvette on vertical movement. B: Schematic diagram of the cuvette describing the buffer solution level, position of the sperm layer in the cuvette and the heights at which the cuvette is exposed to the spectrophotometric light beam. (Reproduced from Saha et. al. 2007).

7 Purification and Characterization 7 Of all the methods CASA is the most widely used one. The presently available motility assay techniques including the CASA, measure only the "horizontal velocity" of spermatozoa. Recently we have developed for the first time; a unique computer-based spectrophotometric system (termed as SPERMA) to determine "vertical velocity" of the spermatozoa (Saha et. al. 2007). It has been developed using the turbidimetric method of sperm motility analysis [Sokoloski et al., 1977; Majumder and Chakrabarti, 1984]. The development comprised a modified spectrophotometer with mechanical up-down movement devise for the cuvette and necessary softwares for cuvette movement, data acquisition and data processing (Figure 3). For fertilizing the ova, spermatozoa have to travel through the hostile environment of nearly the entire female reproductive tract and sometimes in the vertical direction also [Chantler et al., 1989; Rutlant et al., 2005; Gruberova et al., 2006]. Undertaking upward movement is much tougher as compared to horizontal progression because the former involves motion against gravity. Vertical velocity, in comparison with horizontal velocity, is thus expected to be a better identifying parameter for gradation of semen samples according to quality. The novel instrumental system developed by us has thus the potential for immense application in infertility clinics, animal breeding centres, centres for conservation of endangered species, research laboratories, etc. This study is expected to open a new avenue of research regarding molecular basis of cell movement with special reference to dynamics of motile cells in the vertical plane. Measuring vertical velocity or vertical vector of a cell is a novel idea that may be extended to a variety of other motile cells (e.g. protozoa, bacteria, etc.) and particles. It is expected that, once this instrumental system is marketed and gets exposure, various other fields of its usage will be revealed. Considering all its potentialities, national and international patent applications have already been filed [Paul et al., 2004; Paul et al., 2005]. ANTI-STICKING FACTORS Haemocytometer counting chambers are widely used for subjective as well as objective assessments of flagellar motility [Amelar et al., 1980]. While working on the caprine sperm model, we have noted that sperm largely stick to the glass surface of haemocytometer counting chamber which is commonly used for subjective and objective assessments of sperm motility and this phenomenon of "cell-sticking" is posing as a serious artefact in motility assays [Roy and Majumder 1989; 1990]. Both the mature and immature epididymal spermatozoa show high affinity to stick to the glass surface of haemocytometer counting chambers. Similar observation has earlier been reported by Stephens et al. [1981] while working with bovine immature caput spermatozoa. However, the contribution of the process of sperm sticking to the glass surface of the haemocytometer counting chamber has largely been overlooked by the earlier investigators (for reviews see Majumder et al., 1992; 2001; 2011). The presence of variable amounts of ASFs derived from reproductive fluids, blood serum and their fractions in the motility assays may be responsible for varying degrees of sperm adhesion to the surface of the counting chamber thereby causing artifacts in motility determinations. The cell-sticking artifact is likely to be greater in situations where washed spermatozoa or sperm largely devoid of reproductive fluids are used since these fluids possess ASF activity. Consequently the previous data on the biochemical basis of flagellar motility initiation and regulation may be complicated because of the possibility of cellsticking artifacts in motility estimations. It is thus important to identify potent factor(s) that will

8 8 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. inhibit with specificity and affinity for sperm sticking to glass. Such anti-sticking factors (ASFs) when available should be extremely useful to improve the existing motility assays by eliminating the possibility of sperm-sticking artifacts. Assay of Anti-Sticking Activity Goat cauda-ep has been shown to possess anti-sticking activity that prevents nearly completely sperm adhesion to glass [Roy et al., 1985]. Anti-sticking activity of EP and its fractions was measured by the procedure reported earlier [Roy et al., 1985] with minor modifications. An aliquot of the sperm preparation (25 μl containing approx. 0.5 x 10 6 cells) was incubated at room temperature (31 ± 1 C) with or without a specified amount of anti-sticking activity in a total volume. of 0.5 ml of RPS medium. A portion of the cell suspension was then injected into the haemocytometer and the initial cell number was counted (C T ) under a phase-contrast microscope at X250 magnification. Figure 4. Effect of flushing the haemocytometer counting chamber with varying volume of RPS medium on the number of spermatozoa present in the chamber under the standard assay conditions. The concentration of EP when present in the assays was 1.5 mg protein/ ml. The data shown are means of 4 determination ± S.D. (Reproduced from Roy et al 1985). After 5 min incubation, the counting chamber was flushed with an excess of RPS medium (3 X 100 μl) to remove the cells that were in suspension, i.e., those cells that did not stick the glass (Figure 4). The flushing was done by injection of the buffer with a 100 μl auto pipette into the counting chamber from the front side of the haemocytometer, taking care not to disturb the cover-slip. The cells that were left after flushing in the chamber were then counted and these values (C s ) represent the cells that adhered firmly to the glass surface of the haemocytometer. The percentage of cells that did not adhere to the glass surface of the haemocytometer chamber was calculated as [(C T - C S )/C T ] X 100. A unit of activity of ASF was defined as the amount of the factor that prevents sticking of 10% of the cells under the

9 Purification and Characterization 9 standard assay conditions. Systems lacking exogenous anti-sticking activity served as the blank in all assays and the controls showed a very low amount of anti-sticking activity (0-0.3 U). Purification of ASFs The EP-mediated inhibition of sperm adhesion to glass is dependent of the concentration of EP. Sperm antisticking activity of EP is nearly maximal at EP concentration as low as 0.6 mg protein/ml. The activity of the ASF of EP is non-dialyzable. Caprine epididymal plasma (EP) possesses two ASFs (I and II), ASF I being the major one that was purified by the procedure reported earlier (Roy and Majumder 1989). The salient features of this purification procedure have been described below. Step I: EP was concentrated to a great extent with polyethylene glycol and then dialysed against 20 mm Tris-HCl (ph 7.2)/l mm CaCl 2 /l mm MnCl 2 /0.6 M NaCl (buffer 1) before being subjected to affinity chromatography on a Con A-agarose column (1.8 X 15 cm) previously equilibrated with buffer 1. The column was washed with 150 ml of the equilibrating buffer. The anti-sticking activity was eluted first with 150 ml of buffer 1 containing 30 mg/ml of methyl -D-mannoside and then with 90 ml of 50 mm glycine-hcl (ph 3.0)/ 1 M NaCl/ 15 mg/ml methyl -D-mannoside. This Con-Aeluate was neutralized, concentrated by Amicon ultrafiltration technique using PM-30 membrane and dialysed against 10 mm Tris-HCl (ph 9.0). Step-II: The dialysed ASF preparation was applied to a DEAE-cellulose column (1.0 X 15 cm) equilibrated with 10 mm Tris-HCl (ph 9.0). After passage of the sample, the column was washed with 30 ml of the equilibrating buffer. The major peak of the activity (ASF-I) was eluted with 30 ml of 0.1 M Tris-HCl (ph 9.0). The column was then eluted successively with 25 ml each of Tris-HCl (ph 9.0) having concentrations of 0.2, 0.3, 0.5 and 0.7 M and the eluates were discarded. ASF-II was finally eluted from the column with 25 ml of 1.0 M Tris-HCl (ph 9.0)/1.0 M NaCl. The fractions containing ASF-I and -II activities were concentrted by ultrafiltration through Amicon PM-30 membrane and then equilibrated with 5 mm potassium phosphate (ph 7.0). Step III: ASF-I was purified further by using a novel electrophoresis system consisting of polyacrylamide and Sephadex G-200 gels as the matrix (Figure 5). The resolving (7% acrylamide) and stacking (3% acrylamide) polyacrylamide gels were prepared according to the method of Laemmli [1970], except that SDS was omitted. A layer of preswollen Sephadex G-200 (4 X 0.6 cm) was incorporated in the gel tube with resolving gel at the top (4 X 0.6 cm) and the bottom (2 X 0.6 cm) (as shown in Figure 5A). The bottom polyacrylamide gel gave support to the Sephadex layer. Sephadex was dispersed in M Tris-HCl (ph 8.0)/10% sucrose. ASF-I was lyophilized and dissolved in µl 0.06 M Tris-HCl (ph 6.7)/10% glycerol/1% β- mercaptoethanol (approx. 8 mg protein/ml). The gels were pre-electrophoresed for 1 h at 6 C with a current of 5 ma/gel before loading each gel tube with approx μg protein of ASF-I preparation. Electrophoresis towards the anode was carried out for h with a current of 5 ma/gel using Bromophenol blue as the tracking dye. The bottom polyacrylamide gel was then removed by pushing the gel from the top with a plunger. The Sephadex layer containing

10 10 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. the ASF-I band was then pushed out and collected in a small beaker. The Sephadex G-200 materials were pooled from four or five gel tubes and transferred to a small Pharmacia column (inner diameter 9 mm). The anti-sticking activity was eluted with 5 ml of 5 mm potassium phosphate (ph 7.0)/1 mm PMSF. Further purification of ASF-II has been achieved by the fractionation procedures described earlier [Roy and Majumder 1990]. Figure 5. (A) Relative positions of the polyacrylamide and sephadex G-200 in a novel acrylamide- Sephadex gel electrophoresis system. (B) Diagrammatic representation of the polyacrylamide gel electrophoretic pattern of ASF-1 obtained after the DEAE-cellulose chromatography step of the purification under the non-denaturing conditions. After electrophoresis, one gel was stained with Coomassie blue for the detection of protein bands and another gel was sectioned with a Gilson automatic gel slicer, the thickness of each gel slice being 1 mm. For the elution of ASF activity each gel slice was dispersed in 0.5 ml RPS medium overnight at 6 0 C. An aliquot of the supernatant fluid 100 µl from each gel section was used to estimate ASF activity under the standard assay condition (Reproduced from Roy and Majumder, 1989). Some Characteristics of Sperm-Glass Interaction Nearly 100% of the sperm dispersed in a chemically defined RPS medium, stick firmly to the glass surface and these cells cannot be removed by flushing the chamber with excess of RPS medium. Sperm adhesion to glass is a rapid process and it is nearly complete after 5 min of incubation inside the haemocytometer counting chamber. Spermatozoa that stick to glass cannot be dissociated by flushing with excess of EP (2 mg protein/ml) demonstrating thereby that sperm-glass interaction is an irreversible phenomenon. Sperm bound to the glass surface cannot be removed by flushing with 1.0 M NaCl or RPS medium containing 0.3 M NaCl. The results indicate that sperm binds firmly to the glass surface [Roy et al., 1985]. Siliconization of glass with dimethyldichlorosilane has no effect on glass sperm interaction [Roy et al., 1985; Stephens et al., 1981]. Spermatozoa, when treated with 0.1% Triton X-100 do not adhere to the glass surface of the haemocytometer. It is well known that Triton dissolves the cell membrane. The data implicate that demembranated cells do not stick to glass. It thus appears that the cell-adhesive molecules are located on the sperm plasma membrane. Treatment of intact spermatozoa (100

11 Purification and Characterization 11 X 10 6 cells/ml) with trypsin or chymotrypsin (100 μg/ml, each) at 37 C for 60 min, causes marked inhibition (60-70%) of cell-sticking to glass, indicating that sperm external surface possesses protein(s) that mediate adhesion of spermatozoa to glass. p- chloromercuriphenylsulfonic acid (PCMPS) a thiol reagent, is an ideal cell surface probe as it does not penetrate the sperm PM [Roy and Majumder, 1986]. PCMPS has been used to investigate the role of sperm ecto-sh groups in sperm-glass interaction. PCMPS exerts a dose-dependent inhibitory action on sperm attachment to glass. The thiol reagent at 50 µm level inhibits nearly 95% sperm sticking to glass. The data show that the ecto-protein(s) of the sperm surface which are responsible for sperm adhesion to glass, contain free-sh groups that may be essential for their adhesive characteristics. Biochemcal Characteristics of ASFs Purity of ASFs: The summary of the purification of the anti-sticking factor has been shown in Table 1. ASF binds with high affinity to the Con A-agarose column and the activity is eluted slowly first with methyl -mannoside (30 mg/ml) and then with the mannoside containing the acidic glycine-hcl buffer (ph 3.0). By the Con A-affinity chromatographic step, the recovery of ASF activity was nearly 150%, suggesting that there had been removal of some interfering substance(s) from EP by this step of purification. Table 1. Purification of ASF-I from epididymal plasma Fraction Total activity (U.10-3 ) Total protein (mg) Specific activity (U/mg protein.10-3 ) Purification (-fold) Epididymal plasma Con A-agarose eluate DEAE-cellulose chromatography ASF-I ASF-II Acrylamide-Sephadex gel electrophoresis ASF-I (Reproduced from Roy et al 1985). The concanavalin A-eluate of the ASF activity was resolved by DEAE-cellulose chromatography into two distinct peaks, I and II. ASF-I was the major peak, which represents approx. 75% of the epididymal plasma ASF activity. After this step of purifi-

12 12 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. cation, ASF-I was purified approx fold, as opposed to ASF-II, which was purified only 46-fold. This study reports the purification and characterization of the major antisticking factor, ASF-I. While evaluating the purity of ASF-I obtained after DEAE-celulose chromatography under non-denaturing conditions by 7% polyacrylamide disc gel electrophoresis; it was observed that ASF consists of a protein band having an R f value of 0.66 and some contaminating proteins close to the origin (Figure 5). No detectable protein bands were noted in the vicinity of the protein band that migrated well through the gel. Analysis of the gel fractions for ASF activity showed that there is only one peak of activity that co-migrated with the protein band of R f value Since it is very difficult to elute the activity from the polyacrylamide gel, it was decided to develop a novel electrophoresis system containing both polyacrylamide and Sephadex G-200 as the matrix (Figure 5) for the purification of ASF-I. The electrophoretic conditions were manipulated to permit migration of the ASF into the Sephadex layer, so that the protein could be eluted nearly quantitatively from this phase. ASF-I was purified to approx fold (Table 1) and the purified factor, preserved in 5 mm potassium phosphate (ph 7.0)/40% glycerol/1 mm PMSF, was found to be stable for at least 1 month. The new technique of the gel electrophoresis system is simple and it is likely to be particularly suitable for the purification of proteins to homogeneity if the required protein band in the polyacrylamide gel is well separated from the contaminating proteins. The position of the Sephadex G-200 in the gel tube can be altered so that the required protein band is electrophoresed into the Sephadex layer. High resolving capacity of polyacrylamide gels for proteins, DNA, RNA, etc., potential for quantitative elution of the macromolecules from the Sephadex layer and operational simplicity may make this method extremely useful for the purification of a variety of macromolecules. Properties of ASFs: The molecular masses of ASF-I and II as estimated by Sephadex G-100 molecular sieving are 47 and 36 KDa, respectively [Roy and Majumder 1989, 1990]. Similar molecular masses have also been noted by SDS-polyacrylamide gel electrophoresis. Sucrosegradient ultracentrifugation studies show that ASF-I and II have sedimentation constants of 4.25 S and 2.4 S, respectively (Figure 6). Stokes radius of ASF-I is 24.l A and ASF-II 22.0 A. Both the factors possess frictional ratios of 1.0. Anti-sticking activities of the purified ASF-I and II increase proportionately upto approximately 3 units of each factor. At saturating concentration (approximately 40 μg /ml or 1 nm), each factor inhibits sticking of 50-60% of spermatozoa. No further increase in the inhibition of sperm adhesion to glass is observed when both the factors are added together at saturating concentration. Both ASF-I and II are stable to heat treatment at 80 C for 5 min. The anti-sticking factors are sensitive to the actions of trypsin and glycosidases such as L-αfucosidase and α -mannosidase [Roy and Majumder, 1989; 1990] indicating that both the protein and sugar parts of these factors are essential for their anti-sticking activity. In another experiment, protein specificity of ASF-I and II has been investigated [Roy and Majumder, 1989]. Casein, ovalbumin, mucin and phosvitin have little anti-sticking activity. Bovine serum albumin, -lactoglobulin, fetuin and myoglobin show significant amount of anti-sticking activity at relatively high concentrations ( μm) and these proteins have little ASF activity at concentrations below 1 μm. The purified ASFs being maximally active at 1 nm level have protein specificity at least 10,000 times greater than the other proteins examined. ASFs are thus specific proteins and the above-mentioned proteins having anti-sticking activity at markedly higher levels, may be termed as non-specific low affinity ASFs.

13 Purification and Characterization 13 Spermatozoa derived from certain parts of epididymis (e.g., corpus) have been shown to undergo agglutination when incubated under a defined condition at 37 C [Dacheux et al., 1983; Flaherty et al. 1993]. Figure 6. Sedimentation co-efficient of ASF-I and II. Sucrose-density gradient ultracentritugation of [ 125 I] ASFs was carried out as reported earlier (Roy and Majumder, 1989). Ovalbumin (3.6 S) was used as the marker protein (Reproduced from Majumder et al. 1992). Figure 7. Inhibition of sperm-sperm agglutination by ASF-I. The anti-agglutinating action of ASF-I on spermatozoa was studied following the method of Dacheux et. al. (1983) with some modifications. Spermatozoa from the distal corpus region (i.e. the region prior to cauda) of the epididymis were extracted in RPS medium, centrifuged at 450Xg for 5 min. at room temperature to remove the epididymal plasma and then resuspended in RPS medium. Cells were then incubated at 37 0 C for 60 min with or without specified amount of ASF-I in a total volume of 0.5 ml RPS medium. After the incubation, cells are observed under phase contrast microscope and the microphotographs show spermatozoa with a magnification of 270X. (A) Control without (ASF-I). (B) With ASF-I (4ng/ml). (C) With ASF-I (20ng/ml). (D)With ASF-I (50ng/ml) (Reproduced from Roy and Majumder, 1989). Evidence has been presented for the presence of undefined anti-agglutinin protein(s) in EP, seminal plasma and prostatic secretion that inhibit the agglutination of spermatozoa derived from several mammalian species. We have also observed that goat EP has a high

14 14 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. capacity to inhibit agglutination of spermatozoa obtained from the distal end of goat corpus epididymis. As shown in (Figure 7), goat corpus-epididymal spermatozoa when incubated at 37 C for 1 h in RPS medium showed head-to-head agglutination giving rise to large clusters of cells. ASF-I showed a high tendency to inhibit the sperm agglutination. ASF at a concentration as low as 4 ng/ml significantly inhibited this sperm-sperm adhesion as observed by the reduction in size of the cell clusters. ASF at a concentration of 50 ng/ml showed maximal inhibitory effect when most of the cells remained in free suspension. ASF-I (50 ng/ml), when added to the preformed sperm clusters, also showed high efficacy to dissociate the cells from the cell agglutinates thereby showing that it can also reverse the process of sperm-sperm adhesion. The anti-sticking activity of the factors is not due to their coating the glass surface of haemocylometer. Using radioiodinated ASFs several lines of evidence have been obtained to show the presence of specific ASF receptors on the sperm outer surface. The amount of ASF bound to spermatozoa, increases with the concentration of the factor and the binding is saturable at approximately 1.0 nm ASF-I. The Kd of the binding sites is approximately 5.5 x M and nearly 9 fmol of ASF-I bind to 1 x 10 6 cells. The unlabelled ASF-I and II compete with high affinity with the 125 I-labelled ASF-I and II, respectively for sperm binding sites and thus cause displacement of the labelled ASFs bound to sperm cells. The biochemical properties of ASF receptors are largely unknown. Importance of ASFs Postulated role in cell biology: Cell-cell adhesion is believed to play a vital role in cellular regulation [Hughes et al., 1980; Ogita and Takai, 2008]. Whole cells also interact with a variety of surfaces (substratum) of biological or non-biological origin, such as connective tissue elements, glass, plastic, etc. [Hughes et al., 1980]. It is of interest to note that the mechanism of cell-substratum interaction closely resembles that of the cell-cell adhesion [Garrod et al., 1986]. Several cell-surface adhesive proteins (e.g., fibronectin, cadherins, etc.) that may mediate these adhesions have been identified [Schmidmaier and Baumann, 2008]. However, little is known about any specific protein that may regulate these interactions. Occurrence of ASF activity in a variety of tissues [Roy and Majumder, 1989; Banerjee et al., 1990] suggests that ASF may have a biological role not restricted only to spermatozoa. At present, little is known about the physiological significance of ASF. The sperm-glass adhesion phenomenon is an example of cell-substratum adhesion and the above-mentioned finding that ASF is also a potent anti-agglutinin for sperm-sperm adhesion suggests that ASF may have an important role in the regulation of mammalian cell-cell and cell-substratum adhesion by serving as a specific and potent anti-agglutinin. Recently we have demonstrate the occurrence of a Ca 2+ -dependent D-galactose specific lectin as well its receptor on the liver parenchymal cells and the observed autoagglutination event is caused by the interaction of the cell-surface lectin with its receptor of the neighbouring homologous cells [Banerjee and Majumder 2010]. More recently we have purified an anti-sticking protein from buffalo blood serum and the serum ASF has shown high efficacy to inhibit parenchymal cell-cell adhesion in addition to sperm-sperm interaction discussed above. [Banerjee and Majumder: unpublished results]. These data strengthen further the above notion that ASFs may serve a pivotal role in biology by modulating cell-cell adhesion/recognition/communication mechanisms.

15 Purification and Characterization 15 Usefulness for blocking cell-sticking artifacts: The ASFs may inhibit sperm adhesion to a neighboring sperm or to the inner surfaces of the male and female reproductive tracts and thereby may permit the male gametes to remain in a free state (Figure 8), which is essential for manifesting their motility and fertility potential. ASFs may thus play a very important physiological role in sperm biology. The effect of ASFs on sperm forward motility has been estimated spectrophotometrically - an objective method of assessing sperm motility that largely eliminates the possibility of cell-adhesion artifacts in motility assays [Majumder and Chakrabarti, 1984]. Figure 8. Schematic diagram showing the postulated role of anti-sticking factors (ASFs) in reproduction. Figure 9. Effects of anti-sticking factors (2.5 nm each) on sperm forward motility estimated spectrophotometrically. The spectrophotometric motility assay method (Majumder and Chakrabarti, 1984) consists of layering 50 µl of goat cauda-epididymal sperm preparation containing 1.0% Ficoll-400, at the bottom of an optical cuvette containing RPS medium. Vigorously FM-cells that moved upwards against the gravity into the light beam were registered as an increase of absorbance at 545 nm in a spectrophotometer connected to a recorder (Reproduced from Majumder et al, 1992).

16 16 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. As shown in (Figure 9), the sperm preparation has approximately 30% vigorously forward motile cells with and without ASF-I or II (2.5 nm, each). The initial slope of the curve that represents an index of the velocity of the fastest moving sperm population is not affected by the ASFs. The data demonstrate that ASFs have no effect on the sperm velocity and that they are incapable of inducing forward motility in non-motile/weakly motile sperm. ASFs are thus ideally suitable for improving the existing motility assays by ruling out the possibility of cell-sticking artifacts in motility estimation. This contention is strengthened by our observation that goat and ram EP possess ASF activity towards homologous and heterologous spermatozoa. EXTRACELLULAR SPERM MOTILITY REGULATING PROTEINS Forward Motility-Promoting Protein (FMSF) Motile spermatozoa dispersed in seminal or epididymal plasma, lose flagellar motility upon dilution with an isotonic synthetic medium and this loss of sperm movement can be prevented to a large extent with the addition of seminal/epididymal plasma and blood serum. These studies provided evidence for the occurrence of undefined extracellular motility regulating factors in these fluids. As reviewed earlier [Mandal et al., 1989; Majumder and Mandal, 1992; Majumder et al., 2001], there are several reports on the occurrence of various types of sperm motility regulating protein factors in male reproductive fluids and blood serum. A motility maintenance factor has been demonstrated in rat seminal vesicle [Morita and Chang, 1971] and a sperm survival factor in hamster epididymal plasma (EP) [Morton and Chang, 1973]. Motility promoting factors have also been reported in human serum [Yanagimachi et al., 1970; Morton and Chang, 1973; Bavister et al., 1975; Morton et al., 1979], human seminal plasma [Gaur and Talwar, 1975], and human epididymal homogenate [Sheth et al., 1981]. However, these motility proteins have not been adequately purified and characterized. It is noteworthy that the earlier investigators have not evaluated the effect of celladhesion to the counting chamber on sperm motility assays. As elaborated above, earlier studies from our laboratory [Roy and Majumder 1989, 1990] have identified novel antisticking factors from goat EP which is a rich source of these factors. The presence in the motility assays of variable amounts of ASF derived from biological fluids [Roy and Majumder, 1989; 1990] may cause variable amounts of sperm adhesion to the glass surface of the hemocytometer chamber and thus may lead to artifacts in motility assays. We have, therefore, taken interest to investigate the presence of genuine motility-regulating factors in biological fluids by using an experimental condition that will largely eliminate the possibility of cell-adhesion artifacts and the following section reviews some of our findings in this direction [Roy and Majumder 1989; Roy et al., 1985; Sheth et al., 1981; Hoskins et al., 1981]. We have conducted investigation in search of motility promoters in serum using goat caudaepididymal sperm model [Mandal et al., 1989; 2006]. Motility assays were performed in presence of goat epididymal plasma (1.2 mg protein/ml) that has enough ASF activity to

17 Purification and Characterization 17 eliminate the possibility of sperm-sticking artifacts in motility estimation. The motility promoting protein designated as FMSF has been purified and its biochemical and functional properties have been delineated [Mandal et al., 2006]. Some aspects of this investigation have been outlined in the following section: Assay of FMSF Activity The activity of FMSF-I of intact spermatozoa was measured by the procedure described earlier [Mandal et al., 1989]. Spermatozoa (0.5 x 10 6 cells) were incubated with EP (0.6 mg protein) in absence or presence of specified amount of plasma/serum or its fractions at room temperature (32 0 C 1) for 1 min in a total volume of 0.5 ml of Medium B free of Ca 2+. A portion of the cell suspension was then injected into the haemocytometer. Immediately spermatozoa that showed well defined forward motility (excluding cells that moved in small or large circles) (FM cells) and total cell numbers were counted under a phase contrast microscope at x 250 magnification. The percentage of FM cells was then calculated. System lacking FMSF served as control in all the assays. A unit of activity of the FMSF activity was defined as the amount of the factor that induced FM in 10% of the cells under the standard assay conditions Purification of FMSF As buffalo serum is the richest source of FMSF, it has been purified from buffalo blood serum by the procedure described earlier [Mandal et al., 1989]. Step-1: The buffalo blood plasma proteins were fractionated by using 0-30%, 30-60% and 60-80% saturations of ammonium sulphate. In each step, the mixture of protein and salt was centrifuged for 15 min at 18,000 x g and the sedimented protein pellet was dissolved in RPS medium while the suspernates were subjected to further saturation by addition of solid salt. The fractions were dialysed extensively against Medium B prior to assay for FMSFactivity. FMSF-activity was largely precipitated (about 90%), by using 60-80% saturation of (NH 4 ) 2 SO 4. The active fraction was then concentrated with polyethylene glycol compound and dialysed against 10 mm Na-acetate buffer, ph 5.6. Step-2: The resulting dialysed FMSF-fraction was subjected to ion-exchange chromatography in a column (1 x 30 cm) of CM-cellulose previously equilibrated with 10 mm Na-acetate ph 5.6. After passage of the sample, the column was washed with 30 ml of 10 mm Na-acetate buffer, ph 5.6. The column was then eluted successively with 30 ml each of 10 mm Na-acetate buffer, ph 5.6 with NaCl having concentrations of 0.2 M, 0.4 M, 0.6 M and 1M. The major amount of FMSF-activity was eluted with 10 mm Na-acetate containing 0.2 M NaCl buffer, ph 5.6. Before activity measurement, each fraction was dialysed against Medium B. The active fraction was concentrated with polyethylene glycol and dialysed extensively against 0.1M phosphate buffer ph 6.9. Step-3: The concentrated FMSF preparation was chromatographed by HPCL on a gel filtration column: LKB Ultrofac, TSK 3000 SWG (21.5 x 600 mm) with constant monitoring of absorbance at 280 nm in a spectrophotometer (481 LC, Waters) equipped with a 745 B data module (Waters). Mobile phase used was 0.1 M phosphate buffer ph 6.9 at a flow rate of 2.2 ml/min. In this step, FMSF-activity resolved into two peaks. Retention times of FMSF-I and II were 43.5 min. and 30 min, respectively. Both of them were concentrated by filtration through Amicon PM-10 membrane.

18 18 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Step-4: FMSF-I was purified further by using 7.5% native polyacrylamide tube gel electrophoresis according to Laemmli [Laemmli, U.K., 1970]. Before sample application, the gel was pre-run for 15 min. After electrophoresis, one gel was stained with Coomassie Blue for the detection of protein bands and another gel was sectioned to slices, the thickness of each gel slice being 0.5 cm. For the elution of FMSF-activity, each gel slice was dispersed in 0.5 ml of Medium B overnight at 4 0 C. After staining the gel showed two major protein bands of R f values 0.3 and 0.6 respectively and from activity measurement it was observed that protein of R f value 0.6 showed major FMSF-activity. For preparative work, several tube gels were run and FMSF of R f value 0.6 was eluted. The pooled fractions were concentrated by filtration through Amicon PM-10 membrane and then stored at C. Characteristics of Forward Motility-Stimulating Factor Purity: Table 2 summarises the purification of FMSF. When ammonium sulphate fractionation was performed, recovery of FMSF-activity was around 95% in the 60-80% saturated (NH 4 ) 2 SO 4 fraction [Mandal et al., 2006]. By this step, FMSF was purified to 13- folds. The active fraction from this fractionation was purified further by CM-cellulose ionexchange chromatography. FMSF binds to the resin and the major amount of FMSF-activity was eluted with 10 mm Na-acetate M NaCl buffer, ph 5.6 [Mandal et al., 2006]. By preparative HPLC, FMSF resolved into two distinct peaks: FMSF-I and II (Figure 10). FMSF-I was the major peak which represent approx. 60% of the plasma FMSF-activity. Analysis of FMSF-I derived from HPLC step of purification by native PAGE, showed the presence of two motility-promoting protein bands. Nearly 75% of the FMSF-activity was associated with the protein band having R f value of 0.6. This major FMSF activity (FMSF-I) eluted from the gel was purified to approx. 600-fold. The purifed FMSF showed apparent homogeneity as tested by native gel electrophoresis, HPLC and isoelectricfocussing (Figure 11). Step Fraction Table 2. Purification of FMSF-I from buffalo blood plasma Total activity (Units) Total protein (mg) Specific activity (Units/mg protein) Recovery (%) Blood plasma 39,200 20, Fold purification I Ammonium sulphate 37,100 1, precipitation (60%-80% saturation) II CM-cellulose 25, chromatography (0.2 M NaCl eluate) III HPLC : FMSF-I 11, (Retention time 43.5 min.) IV Native PAGE: FMSF-I (Protein Band: Rf = 0.6) 6, , Motility-promoting factor was isolated from 200 ml of buffalo blood plasma. (Reproduced from Mandal et al, 2006).

19 Purification and Characterization 19 Properties: FMSF is a 66 KDa monomeric protein. The Stoke s radius and the frictional ratio of FMSF-I were found to be A and 1, respectively indicating that the protein is spherical. Figure 10. Separation of two forward motility stimulating factors (FMSF-I and FMSF-II) by HPLC: FMSF activity obtained after CM cellulose chromatography was subjected to HPLC (Reproduced from Mandal et al, 2006). Figure 11. To determine the isoelectric point of FMSF-I analytical isoelectric focussing was carried out according to the instruction manual, Pharmacia Fine Chemicals. The broad pi calibration kit was used on a 5% acrylamide gel containing pharmalyte ph Markers used were: 1. Trypsinogen (pi 9.30), 2. Lentil lectin basic band (pi 8.65), 3. Lentil lectin middle band (pi 8.45), 4. Lentil lectin acidic band (pi 8.15), 5. Horse myoglobin basic band (pi 7.35), 6. Human carbonic anhydrase B (pi 6.55), 7. Bovine carbonic anhydrase B (pi 5.85), 8. β-lactoglobulin-a (pi 5.20), 9. Soyabean trypsin inhibitor (pi 4.55), 10. Amylo-glucosidase (pi 3.50) (Reproduced from Mandal et al, 2006).

20 20 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. This protein contained a high proportion of aspartic acid, glutamic acid and leucine (Table 3). Relatively higher level of acidic amino acids in FMSF-I is consistent with the finding that it has pi in the acidic region (Figure 11). Nearly 20-30% spermatozoa of the freshly extracted goat cauda sperm preparations showed forward progression when analyzed in absence of exogenous FMSF-I by the microscopic method of motility assay. Addition of FMSF-I, enhanced markedly sperm forward motility (Figure 12). Figure 12. Effect of FMSF-I, theophylline and bicarbonate at different concentrations on sperm motility under the standard assay conditions. (- -): FMSF-I, (- -): Theophylline + Bicarbonate, (- -): Theophylline, (- -): Bicarbonate (Reproduced from Mandal et al, 2006). The numbers of forward motile cells increased markedly with the increase in the concentration of FMSF-I. A proportional increase in FMSF-I activity was observed up to approx. 3 units (approx M or 10 g/ml) of FMSF. The factor showed maximal activity at a concentration as low as 0.5 M (33 g/ml) when it induced forward motility in nearly 50% of the cells. The motility-promoting action of the factor is extremely rapid and its action is nearly complete in approx. 60 sec. Mg 2+ caused dose-dependent marked increase of the motility-promoting activity of FMSF (Figure 13). Mg 2+ at 600 M level caused maximal activation (approx. 3-fold) of FMSF-activity. FMSF is a specific protein having markedly higher protein specificity as compared to other proteins tested. FMSF-I is a glycoprotein as demonstrated by PAS-staining and its ability to bind to Con A-agarose. The derived N- terminal amino acid sequence of FMSF-I is DTHKSEIAHRFKDL The derived FMSF sequence showed maximum homology with amino acid residues located at positions (from N-terminal end) of BSA [Primary Accession Number: P02769]. However, the sequence of the motility protein did not show any homology at all with the amino acid residues located at the N-terminal of BSA.

21 Purification and Characterization 21 Table 3. Amino acid composition of FMSF Amino acid Mole percent Aspartic acid + Asparagine Glutamic acid + Glutamine 9.13 Serine 8.21 Glycine 8.51 Histidine 4.23 Arginine 5.04 Threonine 4.88 Alanine 6.59 Proline 6.05 Tyrosine 3.61 Valine 4.64 Methionine 1.33 Cystine 1.09 Isoleucine 3.01 Leucine Phenylalanine 5.18 Lysine 6.89 Amino acid analysis of FMSF-I was carried out by the Department of Physiology, Tufts Medical School, USA, using waters PICO-TAG method (Millipore). (Reproduced from Mandal et al, 2006). Table 4. Action of glycosidases on FMSF activity Enzyme treatment FMSF-I-activity (unit) Nil (Control) 1.9 -Galactosidase (200 unit/ml) 1.4 -L-Fucosidase (0.25 unit/ml) 1.8 -Mannosidase (16 unit/ml) 0 -Glucosidase (12 unit/ml 1.0 -N-Acetylglucosaminidase (1.6 unit/ml) 1.9 Neuraminidase (20 unit/ml) 1.8 FMSF-I (18 g protein each) was pretreated with specified concentrations of glycosidases in a total volume of 80 l of RPS-medium at 37 0 C for 2 hrs. After incubation the mixture was heated at 80 0 C for 5 min. to destroy the glycosidase activities and the samples were assayed for FMSF-I activity under standard assay conditions. The glycosidases had no forward motility stimulating activity. (Reproduced from Mandal et al, 2006). The factor lost activity completely when treated with -mannosidase showing that the sugar part of the protein is essential for its biological activity (Table 4). To estimate the binding sites of FMSF-I on the sperm surface, FMSF-I was radiolabelled with [ 125 I] using iodobead. Figure 14 shows the dose course of the binding of [ 125 I]-FMSF to intact spermatozoa. The amount of FMSF-I bound to spermatozoa increased with the concentrations of the factor and the binding was nearly saturable at approx. 200 nm FMSF-I. Scatchard analysis of the data show that the Kd of the binding sites was approx. 4.6 x 10-8 M and nearly 215 fmoles of FMSF-I bind to 1 x 10 6 cells. The unlabelled FMSF-I competed with the radiolabelled factors for sperm binding sites and thus caused displacement of the labelled

22 22 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. FMSF-I bound to the cell. The results demonstrate that binding of FMSF-I to spermatozoa is specific. FMSF has no species specificity for its motility-activating potential. It is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. It is well known that theophylline and bicarbonate activate sperm motility by elevating intra-sperm cyclic AMP level. As shown in Figure 12, motility promoting efficacy of FMSF-I is markedly higher than theophylline or bicarbonate or their combination. At saturating level FMSF stimulation of sperm FM is as high as 80% in contrast to those of bicarbonate (42%), theophylline (45%) and their combinations (47%). In addition, FMSF activates sperm motility much more rapidly than theophylline and bicarbonate. Figure 13. Effect of various concentrations of MgCl 2 on the activity of FMSF-I. Standard assay conditions were used except for the specified amounts of MgCl 2. Amount of FMSF-I used was 9 µg/ml. (- -): FMSF-I, (-Δ-): Control, (- -): - FMSF-I activity (Reproduced from Mandal et al, 2006). Figure 14. Effect of various concentrations of 125 I-labelled FMSF-I on their binding to intact spermatozoa (2 X 10 7 cells / assay) under the standard assay conditions. The insert shows the Scatchard analysis of the data (Reproduced from Mandal et al, 2006).

23 Purification and Characterization 23 Physiological Significance of FMSF Molecular weight of BSA and FMSF-I are similar but they differ markedly in several physical and biochemical properties. pi of BSA is 5 [Putnam, Academic press, 1975] whereas pi of FMSF-I is around 3.7 (Figure 11). Amino-acid composition of FMSF-I is also different from that of BSA [Putnam, Academic press, 1975]. From immunological studies it is also evident that they are different proteins. Specific activity of BSA is markedly lower compared to that of FMSF-I. Hoskins and his associates have partially purified a 37 KDa glycoprotein (FMP) from bovine seminal plasma that induces motility in the immature caput-epididymal sperm [Acott and Hoskins, 1978]. The serum FMSF is clearly different from FMP because the molecular mass of the former is markedly higher than the latter. Sperm- bound proteins such as 34 KDa hyaluronic acid binding protein [Ghosh et al., 2002], 36 KDa ectophosphoprotein phosphatase [Barua et al., 2001] and 100 KDa ecto-phosphoproteins,, the substrate of sperm outer surface cyclic AMP-dependent protein kinase [Maiti et al., 2004] has been implicated to activate sperm flagellar motility. A 52 KDa glycoprotein from porcine follicular fluid and 58 kda porcine blood serum antithrombin III also enhance sperm motility [Lee et al., 1992; Lee et al., 1994]. However, little is known about the efficacy and other characteristics of these proteins from the point of motility regulation. The observation that FMSF-I is present in testis and epididymal plasma suggests that the motility factor has a regulatory role on sperm physiology. FMSF through its localization on the sperm surface may provide continuous stimulation to sustain sperm motility during the long journey through the female reproductive tract. In vitro initiation of forward motility in the immature caput-epididymal sperm requires four exogenous parameters: theophylline, an epididymal plasma protein, bicarbonate and alkaline ph [Hoskins et al., 1978; Jaiswal and Majumder, 1996; Jaiswal and Majumder, 1998; Kann and Serres, 1980; Cornwall et al., 1986]. Bicarbonate works by elevating the intrasperm level of cyclic AMP [Jaiswal and Majumder, 1998; Okamura et al., 1985; Tajima et al., 1987; Rojas et al., 1992] whereas theophylline enhances sperm level of cyclic AMP but by inhibiting cyclic phosphodiesterase [Hoskins et al., 1978; Jaiswal and Majumder, 1998]. Of these four components, theophylline is the most important one because in its absence forward motility cannot be induced by the other three factors either individually or in combination [Hoskins et al., 1978; Jaiswal and Majumder, 1998]. But in the in vivo mammalian system there is no theophylline as it is a plant product. The finding that at saturating concentration, FMSF is a much powerful activator of sperm motility than the combined action of both bicarbonate and theophylline (Figure 12), suggests that FMSF acts by a mechanism which is largely different from those of theophylline / bicarbonate. This view is supported by the kinetic data showing that FMSF acts much more rapidly than bicarbonate and theophylline. During the epididymal maturation process in vivo, cyclic phosphodiesterase level of goat sperm decreases sharply [Jaiswal and Majumder, 1996] showing thereby that the sperm motility induction is associated with downregulation of cyclic phosphodiesterase activity resulting in decrease of the breakdown of cyclic-amp which is essential for sperm forward motility [Jaiswal and Majumder, 1998; Okamura et al., 1985; Tajima et al., 1987; Rojas et al., 1992; Garbers and Kopf, 1980; Yeung, 1984; Vijayraghavan et al., 1985]. It is still not clear as to how the FMSF-receptor interaction triggers the flagellar motility. Like the EP- motility protein [Jaiswal and Majumder, 1996], FMSF may as well act by elevating sperm level of cyclic AMP. It is thus compatible with the view that FMSF - receptor

24 24 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. interaction releases an undefined 2 nd messenger which in turn generates a natural substitute of theophylline for downregulation of cyclic phosphodiesterase: the key enzyme for maintaining the threshold level of cyclic AMP [Jaiswal and Majumder, 1996] needed for flagellar motility induction. Applied Potential of FMSF The data from this study raised the possibility that the serum FMSF may be extremely useful as a diluent for the preservation of not only buffalo semen but also semen from other species. The wide spread occurrence of FMSF in blood sera of different species and lack of species specificity for FMSF-action (Table 5) and the fact that serum FMSF does not require systemic administration for its effectiveness as a motility-promoter, are some of the favourable parameters justifying the uses of FMSF as an enhancer of male fertility in animal breeding farms and human infertility clinics. Animal products have an important role in the global economy and any improvement in animal breeding will lead to production of more and better quality animal products such as milk, butter, meat, wool, leather etc. FMSF has also the potential for improving the breeding of the wild animals and the species that are almost extinct thereby helping in the conservation of endangered species of animals. Among humans, nearly 15% couples are believed to be infertile. Human infertility brings about personal miseries to millions of people and it is a social stigma in all human races. As mentioned above FMSF will also be useful in human infertility clinics to solve some of the problems of human infertility. The males are responsible for nearly 40% of the infertility problems and more than 50% of the male infertility is due to low order of sperm motility. Although several sophisticated Assisted Reproductive Technologies (e.g. IVF: In vitro Fertilization, ICSI: Intracytoplasmic Sperm Injection) are available to solve the problems, but these technologies are highly expensive and the success rate is very low. Purified FMSF is expected to have potentially important applications since it will improve the quality of human semen by activating sperm motility essential for fertility of the male gametes in vivo. Because of the immense applied potential of FMSF-I, it has been patented in India and USA [Majumder et al., 2001; Majumder et al., 2003]. Table 5. Effect of FMSF on forward motility of spermatozoa of different species Source of sperm Forward motility (%) (Mean SEM) Percentage of FMSF-I mediated motility Control + FMSF-I stimulation Goat Rat Hamster Human FMSF-activity was measured under the standard assay conditions. The data were representative of Mean SEM of three separate experiments. FMSF-I concentration used 9 g/ml. (Reproduced from Mandal et al, 2006).

25 Motility Initiating Protein (MIP) Purification and Characterization 25 As alreadymentiedabovin vitro initiation of forward motility in the immature caputsperm requires four exogenous parameters: theophylline, epididymal plasma, bicarbonate and alkaline ph Evidence has been provided to support the view that epididymal plasma possesses an unknown protein factor that works in concert with theophylline for the induction of forward motility in the immotile sperm [Acott et al. 1978; Hoskins et al., 1978; Pinto et al., 1984; Jaiswal and Majumder, 1998]. Hoskins and his associates [Acott et al., 1978; Hoskins et al., 1978] have partially purified the active principle from bovine seminal plasma and epididymal plasma and designated it as forward motility protein (FMP). It is thus clear that although many reports have appeared on the occurrence of undefined motility regulating proteins in male reproductive fluids, the motility proteins have not been adequately purified and characterized. Recently we have reported for the first time purification to apparent homogeneity of a novel motility initiating protein (MIP) from a male reproductive fluid (epididymal plasma) using the caprine model [Jaiswal et al 2010]. Some of the salient features of this protein have been indicated below: Assay Method of MIP Activity The activity of MIP that causes induction of forward motility in immature caput sperm was measured by the procedure described earlier [Jaiswal and Majumder, 1998] with some modification. Caput spermatozoa (25 x 10 4 cells) were incubated with 30 mm theophylline, ASF (250 g/ml) and in absence or presence of specified amount of purified MIP at room temperature (32 C 1) for 10 min in 250 l of RPS medium. A portion of the cell suspension (5 l) was then injected into the haemocytometer. Immediately spermatozoa that showed well defined forward motility (excluding cells that moved in small or large circles) and total cell numbers were counted under a phase contrast microscope at 400X magnification. The percentage of forward motile cells was then calculated. System lacking MIP served as the control in all assays. A unit of MIP activity was defined as the amount of the factor that induced forward motility in 10% of the immature cells under the standard assay conditions. Purification of MIP MIP has been purified to apparent homogeneity according to [Jaiswal et al., 2010]. Details of this purification procedure have been shown below: The epididymal plasma proteins were fractionated using 0-30%, 30-50% and 50-70% saturation of ammonium sulphate. In each step, the mixture of proteins suspension and salt was centrifuged for 15 min at 18,000 x g and the sedimented protein pellet was dissolved in 10 mm potassium phosphate, ph 8.0 while the supernatant fluids were subjected to further saturation by addition of the solid salt. MIP-activity was largely precipitated (about 90%) by 30-70% saturation of (NH 4 ) SO 4. The active fraction was dialyzed against 10 mm potassium phosphate buffer, ph 8.0. The resulting ammonium sulfate fraction of MIP was applied at a constant flow rate of 1 ml/min to DEAE-anion exchange column (7.5 x 0.75 cm) on Waters HPLC system that was pre-equilibrated with 10 mm potassium phosphate, ph 8.0. The column was then eluted at a flow rate of 1 ml/min using the following regime: (1) a linear gradient of 10 to 200 mm potassium phosphate (ph 8.0) over a volume of 15 ml, (2) 200 mm potassium phosphate, ph 8.0 for a volume of 10 ml, (3) another reverse linear gradient of

26 26 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al mm potassium phosphate ph 8.0 for a volume of 2 ml, (4) 10 mm potassium phosphate ph 8.0 for a volume of 20ml. Protein elution was monitored at 280 nm and peaks were recorded manually and assayed for MIP activity after dialysis against RPS medium. The active MIP fractions were then pooled and concentrated by Amicon ultrafiltration with PM-30 membrane. Concanavalin A immobilized on Sepharose, is known to have high affinity for binding D-mannose and D-glucose residues of the glycoproteins. ConA-Sepharose column (1x12 cm: bed volume 10 ml) was equilibrated with buffer I (20 mm Tris-HCl, ph 7.2 containing 1.0 mm CaCl 2, 1.0 mm MgCl 2 and 1.0 mm MnCl 2 ). The above-mentioned MIP fraction was dialysed extensively against buffer I, prior to its loading on the ConA-Sepharose affinity column. As the sample passed through the column was washed with 15 ml of buffer I. The elute from the column represents the unretained fraction (fraction A). The column was washed further with 50 ml of the equilibrating buffer to remove the residual amount of unbound material. Finally, MIP bound to the affinity column, was eluted with 60 ml buffer I containing 0.5 M -methyl-d-mannopyranoside (fraction B). All the fractions were dialysed extensively against RPS medium prior to assay of MIP activity. MIP was purified further by chromatofocusing on PBE 94 [Sluyterman and Wijdenes, 1978]. A column of ion exchange resin, PBE 94 (0.7x10 cm), was equilibrated with 25 mm imidazole buffer ph 7.4 and the sample was applied to it. After the sample passed through, the column was washed with the eluting buffer. The MIP activity was eluted with polybuffer 74 (Pharmacia Fine Chemicals), ph 4.0 (1:8 dilution) in 1ml fraction. The elution was monitored at 280 nm absorbance and ph of each fraction was measured. Active fractions were pooled, concentrated and finally dialysed against 10 mm potassium phosphate ph 6.9 before activity measurement. Pooled and concentrated MIP sample after chromatofocusing was subjected to C -Alumina gel adsorption. 10 ml alumina gel suspension (2.5 g solid) was centrifuged at 500 x g for 10 min at 4 C. The pellet was then washed twice successively with 10 mm potassium phosphate, ph 6.0. The MIP preparation was mixed with the above gel and left in ice with constant stirring for 45 min. The mixture was then centrifuged at 500 x g for 10 min and the resulting supernatant fraction was discarded. Bound proteins were then eluted successively with 20 ml each of 0.1 M, 0.25 M, 0.5 M and finally with 1 M potassium phosphate, ph 6.9 containing 1 M NaCl. Before activity measurement, all the fractions were dialyzed against RPS medium and concentrated. Major portion of MIP-activity was eluted with 0.5 M potassium phosphate, ph 7.0. The MIP preparation was then loaded onto a gel filtration column (0.9 x 50 cm) previously equilibrated with RPS medium. The fractions (1.0 ml/tube) were collected in a LKB fraction collector. The active fractions were pooled, concentrated and stored at C. Biochemical Characteristics of MIP Purity of isolated MIP: Summary of the purification of MIP has been shown in (Table 6). By this step MIP was purified to 8-fold. MIP binds to DEAE-HPLC resin and was eluted with the linear gradient of potassium phosphate ( mm) buffer, ph 8.0. MIP activity binds to the Concanavalin A-Sepharose affinity matrix and the bound MIP was eluted with methyl - D-mannopyranoside (Figure 15). When the active MIP from ConA-Sepharose was subjected to chromatofocusing, one major and two small protein peaks were obtained. Assay of MIP activity showed that MIP activity was present in the major protein peak. The isoelectric point of MIP was found to be Nearly 90% of the MIP activity was adsorbed by alumina gel and the activity could be recovered from the gel by elution with 0.5 M potassium phosphate

27 Purification and Characterization 27 buffer, ph 6.9. The MIP preparation was further purified by Sephacryl S-200 gel filtration. The isolated MIP was approximately 500-fold purified. MIP showed a single protein band under all electrophoretic conditions. The molecular weight of MIP estimated by Sephacryl S- 200 gel filtration was 125 kda. A single sharp peak of activity was obtained when MIP was subjected to Sephacryl S-200 gel filtration and HPLC (Figure 16). By both the techniques a single symmetric activity peak was obtained in the MIP preparation. These data establish apparent homogeneity of the purified MIP. Figure 15. Affinity chromatography of MIP on concanavalin A-Sepharose. An extensively prewashed 10 ml column (1.0x12 cm) of concanavalin A-Sepharose was equilibrated with buffer I. Active MIP fraction eluted from DEAE-HPLC column and subsequently dialyzed against buffer I, was subjected to affinity column.(reproduced from Jaiswal et al, 2010). Figure 16. HPLC gel filtration profile of purified MIP. The purified MIP was chromatographed on TSK-G3000 SW column (7.5 mm x 3 cm) using the waters HPLC system. The column was previously equilibrated with the RPS medium at a flow rate of 0.8 ml/min. Protein elution was monitored at 280 nm. The MIP activity was eluted as a sharp peak and its molecular weight was determined from the plot shown in the inset. Molecular weight calibration of the column was done by using the standard proteins. The standards used were: 1. apoferritin (443kDa); 2. -amylase (200 kda); 3. alcohol dehydrogenase (150 kda); 4. bovine serum albumin (66 kda); 5. carbonic anhydrase (29 kda) and 6. cytochrome C (14.2 kda). The log molecular weight (in kda) was plotted against Ve/Vo (inset), where Ve is the elution volume of each protein and Vo is the void volume of the column.(reproduced from Jaiswal et al, 2010).

28 28 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Table 6. Purification of motility initiating protein from goat epididymal plasma Purification steps Total Protein (mg) Total activity (Unit) Specific activity (Units/mg) Recovery (%) Caudal EP %(NH4) 2SO DEAE-HPLC ConA-Sepharose Chromatofocusing Alumina gel Sephacryl S-200 gel filtration Fold purification Motility initiating protein was isolated from 250ml of diluted goat caudal epididymal plasma (approx. 10 mg protein/ml). (Reproduced from Jaiswal et al, 2010). Figure 17. SDS-PAGE of purified MIP using 10% polyacrylamide gel. Lane a: marker proteins - - galactosidase (116 kda), phosphorylase b (97 kda), BSA (66 kda), ovalbumin (45 kda), carbonic anhydrase (29 kda), trypsin inhibitor (20 kda). Lane b: Purified MIP (Reproduced from Jaiswal et al, 2010). Biochemical properties of MIP: MIP when subjected to denaturing SDS polyacrylamide electrophoresis resolves into two protein bands: 70 kda and 54 kda that were detected by the silver staining method of [Wray et. al., 1981] (Figure 17). MIP is thus a heterodimer of 70 kda and 54 kda subunits. MIP is an acidic protein with isoelectric point of Freshly extracted goat caput-sperm (immature) preparations do not show forward motility when analyzed in absence of exogenous MIP. Addition of MIP induces forward motility to a significant population of goat caput-sperm (immature).the numbers of forward motile cells increased markedly with the increase in the concentration of MIP. The factor showed maximal activity at concentration as low as 30 g/ml when it induces forward motility in nearly 22% of the immature spermatozoa. The motility promoting activity of the factor is nearly complete within 2 min. indicating extreme rapidity of MIP action. MIP (80 µg/ml) enhanced forward motility of mature (cauda) goat sperm to the extent of nearly 140% with

29 Purification and Characterization 29 respect to the control. The factor does not lose its activity even when heated at 100 C for 5 min [Jaiswal et al., 2010]. MIP has high degree of protein specificity thereby showing that MIP is a specific protein. The effect of ph on the activity of MIP has been shown in (Figure 18). Sperm forward motility promoting potency of MIP was nearly maximal at ph 8.0. MIP has little efficacy to initiate forward motility at neutral ph (ph 7.0). This finding provides biochemical basis of the earlier observation that external alkaline ph favors forward motility initiation in the immature caput-sperm in vitro in presence of crude epididymal plasma [Jaiswal and Majumder, 1998]. Figure 18. Effect of ph on the activity of MIP. Standard assay conditions were used except for the variation in ph of the RPS medium. Amount of MIP used was 30 g/ml. The data shown are mean SEM of three experiments. (Reproduced from Jaiswal et al, 2010). MIP binds to Con A-Sepharose indicating thereby that MIP is a glycoprotein having at least one D-mannose and/or D-glucose residue at its sugar side chain. Figure 19 elicits lectin specificity of purified MIP as analysed by Ouchterlony double diffusion system using different lectins such as Con A, WGA, RCA 2, and kidney bean lectin. A distinct precipitation line appeared only with Con A. The results demonstrate that MIP is a glycoprotein that interacts with Con A with high affinity. Sugar residues of MIP were analyzed by gas liquid chromatography. MIP contains mannose, galactose and N-acetylglucosamine in the ratio of 6:1:6. MIP activity is lost completely upon treatment with the glycosidase: -mannosidase and -N-acetylglucosaminidase. The data demonstrate that mannose and -Nacetylglucosamine of MIP sugar sode chains are essential are essential for the biological activity of the motility promoter [Jaiswal et al., 2010]. The specific activity of MIP in a variety of caprine tissue extracts have been investigated by ELISA (Figure 20). MIP level was rather low in the tissues. The concentration of MIP in epididymal fluid was remarkably high. The level of MIP in epididymal plasma was approx. 15-fold higher than that in bone marrow. Although both MIP and FMSF are present in the epididymal plasma [Mandal et al., 2006], the former is the major motility promoting protein in epididymal plasma. The data provide further evidence to support the view that MIP is the major physiological activator of sperm motility. Treatment of the caput sperm with isolated MIP significantly elevate the intrasperm level of cyclic AMP (Table 7). Mechanism of action of MIP is still not clear. It may elevate sperm cyclic AMP level by causing decrease of the cytosolic cyclic phosphodiesterase activity and/or by activating membrane-bound/cytosolic adenylate cyclase.

30 30 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Figure 19. Ouchterlony double diffusion test using different lectins: central well, purified MIP (20mg). Amount of lectin used per well is 50mg: A, Con A; B, WGA; C, RCA2; D, kidney bean lectin.(reproduced from Jaiswal et al, 2010). Figure 20. Immunodetection of MIP in goat organ tissue extracts and epididymal plasma by ELISA. Reproduced from Jaiswal et al, 2010). Table 7. Effect of purified MIP on cyclic AMP content of goat caput sperm Treatment Cyclic AMP concentration (pmol/10 9 cells) Mean SEM Control MIP (6 nm) * The control assay system contained caput sperm incubated in RPS medium containing 30mM theophylline. Cyclic AMP content of sperm incubated in presence or absence of MIP has been measured. The data shown are mean SEM of 5 experiments. * P <0.05 when compared with control. (Reproduced from Jaiswal et al, 2010).

31 Purification and Characterization 31 Importance of MIP Population explosion is a major problem in all developing countries. As MIP is highly immunogenic, has high immunological specificity for epididymal plasma and its antibody strongly inhibits forward motility of mature sperm. It has the potential to serve as a contraceptive vaccine. Another global social problem of immense dimension is human infertility [Hull et al. 1985]. One of the reasons of human infertility is due to low order of sperm motility. As MIP has high efficacy for inducing motility in the immature spermatozoa and stimulating motility of the mature male gametes, it has great potentiality for rectifying some of the problems of human infertility utilizing various Assisted Reproductive Technologies [Boone et al., 2007]. MIP as well has the potential for improving cattle breeding and preservation of endangered species. Because of the immense applied potential of this novel protein, it has recently been patented in India [Majumder and Jaiswal, 2004], USA [Majumder and Jaiswal, 2001a; 2001b] and Japan [Majumder and Jaiswal, 2003]. Motility Inhibiting Factor (MIF) Presence of undefined motility inhibitors has been reported in seminal plasma (SP) derived from several mammals [De Lamirande et al., 1984]. Rat seminal vesicle secretion possesses both motility promoting as well as inhibitory protein factors as resolved by gel filtration on Bio-gel P-150 [Peitz 1988]. Usselman and Cone [1983] has demonstrated a high molecular glycoprotein, called Immobilin that immobilizes rat sperm mechanically by increasing the viscoelastic drag of rat cauda epididymal (CE) fluid. In our previous report [Dungdung and Majumder, 1995], we have described preliminary studies on the occurrence of a motility inhibiting factor from caprine EP. Jeng et al., [1993] have purified two sperm motility inhibitors (SMI-1 and SMI-2) from porcine seminal plasma. A sperm motility inhibitor from boar seminal plasma was also purified [Iwamoto et al., 1992]. Bass et al., 1983] found some non-dialyzable factors in bovine seminal plasma that affect the viability and motility of spermatozoa. Human seminal plasma also contains a sperm motility inhibitor (SPMI) that originates from seminal vesicles as a 52kDa precursor form and is degraded into smaller peptides by prostatic proteases shortly after ejaculation [Iwamoto and Gagnon, 1988; Robert and Gagnon, 1996]. But there are no reports on the purification and characterization of these factors from epididymal plasma. Investigation has therefore been undertaken to purify to apparent homogeneity a potent sperm motility inhibiting protein factor (designated as MIF- II) from caprine epididymal plasma [Das et al., 2010]. Purification procedure and some of its biochemical and functional properties have been depicted below: Though microscopic method is the most widely used subjective method for sperm motility analysis [Mandal et al., 2006], here, other than this method, spectrophotometric methods were also used to estimate motility in terms of change in absorbance or optical density [Majumder and Chakrabarti, 1984]. Another sophisticated instrument Computer aided semen analyzer (CASA) was utlilised that is based on microscopic video photographic method and used for estimating sperm horizontal velocity [Devi and Shivaji, 1994]. To determine "vertical" velocity of spermatozoa a unique computer-based spectrophotometric system was developed in our laboratory recently [Saha et al., 2007] and used for the purpose. This instrument has been named as SPERMA. Undertaking upward movement against gravity is much tougher as compared to horizontal movement; average vertical velocity is

32 32 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. expected to be a much better identifying parameter for assessing quality of spermatozoa. All these assay procedures have been used to study the accurate level of the MIF-II activity on sperm forward motility as well as its velocity. Assay of MIF MIF activity of EP was estimated by evaluating forward motility (FM) of spermatozoa using the microscopic method of [Mandal et al., 2006] that has already been outlined above. Spermatozoa (1 x 106 cells) were incubated with boiled EP (0.6 mg protein/ml) in the absence or presence of specified amounts of test samples (goat EP and crude plasma membrane of cauda sperm) at room temperature (32 C ± 1) for 5 min in a total volume of 0.5 ml of RPS medium. A portion of the cell suspension was then placed in the haemocytometer and the forward motile (FM) sperm and total number of sperm were counted under phase contrast microscope at 400X magnification. The percentage of FM sperm was then calculated. A unit of activity of the MIF was defined as the amount of the factor, which inhibited FM in 10% of the cells under the standard assay conditions. The calculated percentages of FM cells are given as the mean ± SEM of at least three experiments. As mentioed above, spectrophotometric sperm motility assay [Majumder and Chakrabarti, 1984] is a more objective method as compared with the microscopic method. This method has also been used for assaying the activity of MIF. As elaborated above,, to determine "vertical" velocity of spermatozoa a unique computer-based spectrophotometric system has recently been developed in our laboratory [Saha et al., 2007]. This sophisticated instrument has been named as SPERMA, has also been untilled for assessing the potency of MIF. Purification Strategy for MIF Dialysed epididymal plasma was subjected to hydroxylapatite gel adsorption chromatographic column (2.5 X 1.5 cm) pre-equilibrated with 10mM K-phosphate buffer, ph 7.0. After passing the sample, column was washed with 10 mm K-phosphate buffer, ph 7.0, and eluted successively with 0.1 M, 0.25 M, 0.5 M and finally with 1 M K-phosphate, ph 7.0. Active fraction was concentrated with polyethylene glycol and then dialyzed extensively against 10mM K-phosphate buffer, ph 7.5, for the next step. The resulting dialyzed MIF-II fraction was subjected to ion exchange chromatography column of DEAE-cellulose previously equilibrated with 10 mm potassium phosphate buffer, ph 7.5 (Figure 21). After passage of the sample, the column was extensively washed with 10mM K- phosphate buffer, ph 7.5 and eluted with 0.1 M, 0.2 M, 0.5 M and finally with 1 M K- phosphate, ph 7.5. The active fraction was concentrated and dialyzed against start buffer 0.025M imidazol, ph 7.4 and subjected to chromatofocusing coloumn (0.7 X 10 cm or 3 ml) using PBE-94, previously equilibrated with M imidazol, ph 7.4. Activity was eluted by polybuffer 74-HCl ph 4. The elution was monitored by measuring ph of each fraction as well as activity of MIF-II. Active fractions were pooled and concentrated and dialyzed against RPS medium and kept at -20 C with protease inhibitors. To check the homogeneity, the fractions obtained from the each steps was analyzed by PAGE under non-denaturing conditions.

33 Purification and Characterization 33 Figure 21. Purification of MIF-II by using different chromatographic methods: A) Epididymal plasma MIF-II activity was subjected to hydroxylapatite gel adsorption column The MIF-II activity was eluted with 0.5 M K-phosphate buffer (ph 7.0). B) Active MIF-II fraction eluted from first step was subjected to DEAE-cellulose ion exchange chromatography. MIF-II activity was eluted with 0.2 M K-phosphate buffer at the ph 7.5. C) Chromatofocusing of MIF-II on PBE-94 (0.7 X 10 cm) chromatography coloumn. (Reproduced from Das et al 2010). Physical and Biochemical Properties of MIF-II Table 8 shows summary of the purification of MIF. The factor has been purified to more than 700-fold by using multiple biochemical fractionation procedures as specified above. The native molecular mass of the purified MIF-II is approx. 160kDa as estimated by Sephacryl S- 300 gel filtration (Figure 22A). MIF-II when subjected to denaturing SDS polyacrylamide gel electrophoresis, resolves a single protein band of 80kDa (Figure 22B), thereby showing that the native MIF-II is a homodimer possessing two subunits each having a molecular mass of 80kDa. The isoelectric point of MIF-II is around 5.1 as obtained in chromatofocusing. Figure 22. Determination of molecular weight of MIF-II: A) The molecular weight of MIF-II was estimated using a column of Sephacryl S-300 (1 X 60 cm). The Mol. Wt. Markers used as standard were thyroglobulin (669 kda), apoferritin (443 kda), -amylase (200 kda), alcohol dehydrogenase (150 kda), BSA (66 kda), carbonic anhydrase (29 kda) and cytochrome C (12.4 kda). B) SDS-PAGE of MIF-II using 10% polyacrylamide gel. Markers used as standard were -galactosidase (116 kda), phosphorylase b (97 kda), bovine serum albumin (66 kda), ovalbumin (45 kda) and carbonic anhydrase (29 kda). (Reproduced from Das et al 2010).

34 34 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Figure 23. Dose course of action of purified MIF-II under the standard assay conditions. (Reproduced from Das et al 2010). Figure 24. Time course of the action of MIF-II under the standard assay conditions. Amount of MIF-II used was 2µg/ml. The data shown are mean ± SEM of three experiments. (Reproduced from Das et al 2010). MIF-II is a heat labile protein and lost its activity when heated at 100 o C for 2 min. The motility inhibiting activity of purified MIF-II increased linearly up to 6.5 units at the concentration of 1μg/ml (6.25 nm). The inhibitory effects of the factor showed maximal activity (approx. 92%) at 2μg/ml (12.5 nm) concentration (Figure 23). Figure 24 shows the time course of the MIF-II activity. The inhibitory action of the factor is very rapid and nearly 100% inhibition has been noted in appropx. 5 min. Interestingly, MIF-II not only inhibits goat cauda sperm forward motility, but also vertical velocity which is far better index of sperm quality. MIF-II also inhibits human sperm forward motility as well as vertical velocity almost completely at a concentration of 2µg/ml. It implicates that though its source is caprine but its activity is not species specific.

35 Purification and Characterization 35 Table 8. Purification chart of MIF-II Fractions Total Total Protein Specific Fold Recovery (%) Activity (mg) Activity Purification EP* Hydroxylapatite gel adsorption DEAE Ion-exchange Chromatography Chromatofocusing *Motility inhibiting factor was isolated from 110 ml (2.54 mg protein/ml) of epididymal plasma (Reproduced from Das el al 2010). Applied Potential of MIF-II Population explosion is a major problem in all developing countries. As MIF-II strongly inhibits forward motility of mature sperm and its action is very specific, it has the potential to be served as a contraceptive. MIF-II may be served as a candidate for developing contraceptive vaccine. SPERM EXTERNAL SURFACE (ECTO) MOTILITY REGULATING PROTEINS It is well documented that mammalian external cell surface proteins, play vital role in the regulation of cell functions by modulating cell-cell interactions, effector-receptor interactions, transmembrane signalling, etc [Yoshida et al., 2008; de Lamirande and O,Flaherty 2008; Marshall et al 2003; Pokutta and Weis, 2007; Schmidmaier and Baumann, 2008; Majumder et al 2011]. It is essential to follow several guidelines to establish localization of a protein on outer cell surface i. e. to determine the ecto nature of a protein. Some of the important guidelines have been outlined in Table 9. The concerned mammalian cell should be viable and the cell membrane should be intact. There should be little leakage/secretion of the biomolecule in the surrounding extracellular fluid. The occurrence of the protein on the external cell surface should be tested using well documented surface probes such as p- chloromercuriphenylsulfonic acid (PCMPS) and diazonium salt of sulphanilic acid (DSS). Finally, it is necessary to confirm the ecto-nature of the protein using the antibody of the purified cell surface protein. While working with the sperm cells there is an added advantage: vigorously motile spermatozoa (that are nearly100%viable) can be separated from the less motile /non-motile cells, by the swim-up technique (Majumder et al 1988; Dey andmajumder, 1990b). The existing scientific literature in the area of ecto-proteins in sperm and other cells is rather confusing as in many studies the above guidelines have not been followed. Studies from our laboratory have established the presence of multiple proteins on caprine sperm surface and their ecto-nature has been documented by strictly following the above guidelines. Some of these studies have been summarised here:

36 36 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Table 9. Evidences for the ecto nature of sperm-surface enzymes/proteins A Coupled Ecto Enzyme System: Regulator of Phosphorylation and Dephosphorylation of Sperm Outer Surface Proteins Sperm External Surface Cyclic AMP-Independent Protein Kinase (Ecto-CIK) The first two reports on the localization of a protein kinase (ecto-kinase) on the external surface of mammalian cells were published in the year 1976 [Schaefer and Kohler, 1976; Mastro and Rosengurt, 1976] demonstrated the presence of a camp-dependent ecto-protein kinase on the rat C-6 glioma cells whereas [Mastro and Rosengurt, 1976] showed that the outer surface of the cultured 3T3 cells possess a protein kinase that causes phosphorylation of the membrane-bound proteins. Since then many investigators have provided evidences for the occurrence of several types of protein kinase in a variety of cell types. Presence of campdependent ecto-protein kinases have been demonstrated on the external surface of spermatozoa of several species [Majumder, 1978; 1981+; Schoff 1982; Pariset et al.,, 1983; Atherton et al., 1985; Dey and Majumder, 1990b], 3T3 fibroblast cells [Boman et al., 1984; Chiang. et al., 1979], HeLa cells [Kubler et al., 1989; Mastro and Rosengurt., 1976], rat adiposite cells [Kang. et. al., 1979]. Cyclic AMP- independent ecto-protein kinases (ecto- CIK) have been documented in many cell systems like goat spermatozoa [Halder and Majumder, 1986; Halder, et al., 1986; 1990], PC12 neuronal cells [Pawlowska et al., 1993], HeLa cells [Jordon et al., 1994; Kubler et al., 2001; Walter et al., 1994], rat myoblast cells [Chen and Lo, 1991], neutrophils [Skubitz et al., 1991], human leukemic cells [Pass and Fishelson, 1995], 3T3 fibroblast cells [Imada et al., 1988] and RBL 2H3 cells [Teshima et al., 1999]. The ecto-cik groups of enzymes are the most extensively studied enzymes from the stand point of the phosphorylation of the endogenous membrane proteins. These intact cellbound enzymes showed high efficacy for the phosphorylation of the serine/threonine residues of phosphoproteins. Preliminary studies of several investigators using the cell-bound uncharacterized ecto-kinase models, have implicated that these enzymes may participate in the regulation of a variety of cell functions such as: cytokine functions [Al-Nedawi et al., 1999], neural differentiation [Pawlowska et al., 1993], myogenic differentiation [Chen and

37 Purification and Characterization 37 Lo, 1991a], myogenesis [Chen and Lo, 1991b], etc. However, precise biochemical identity of the ecto-kinases is largely unknown as no study has yet been reported on the purification of these enzymes to apparent homogeneity. Recently we have reported for the first time the purification of an ecto-cik to apparent homogeneity using caprine sperm as the model [Nath et al., 2008]. Some salient features of this investigation with special reference to purification procedure, physical and biochemical properties and physiological significance have been discussed here. Assay of Purified Ecto-CIK The standard assay system contained 200 nmoles of ATP containing 20-50x10 4 cpm, 2 µmole of magnesium chloride, 1 mg of casein and 200 ng of isolated enzyme in a total volume of 0.2 ml of 50 mm tris-hcl buffer ph 9.0. The incubation was carried out at 37 C for 5 min. When casein was used as substrate, the reaction was stopped by adding 0.1 ml 0.5% casein as carrier protein containing 250 mm K-phosphate, 10 mm ATP and 2 ml 10% TCA. The radiolabelled protein was recovered by filtration through Whatman no. 1 filter paper washed with 5% TCA dissolved in scintillation fluid and counted for radioactivity as describd earlier [Halder and Majumder, 1986]. One unit of CIK activity has been defined as the amount of the enzyme that catalyzes transfer of 10 pmoles of 32 P from [γ- 32 P] ATP to casein. Purification of Caprine Sperm Membrane-Bound Ecto-ClK Highly purified plasma membrane was isolated from the mature cauda and maturing corpus and caput spermatozoa by an aqueous two-phase-polymer method [Rana and Majumder, 1987; Rana and Majumder, 1989]. Membrane purity was judged by estimating marker enzymes: alkaline phosphatase, 5 / nucleotidase, acrosin, cytochrome -oxidase, glucose-6-phosphatase and by electron microscopic study. The specific activities of the plasma membrane (PM) bound 5 / nucleotidase and alkaline phosphatase were folds higher in the isolated PM than in the cell debris, indicating marked membrane enrichment. There was no detectable amount of acrosin and glucose-6-phosphatase in the isolated PM and specific activity of cytochrome oxidase was nearly 7-fold lower in membrane than in the cell debris. The data show that there is little contamination of PM with acrosome, mitochondria and endoplasmic reticulum. Electron microscopic studies also showed high degree of purity of the isolated sperm PM. The membrane preparation was finally dispersed in 25 mm potassium phosphate buffer, ph 7.0, containing 1 mm PMSF, 2 mm -mercapto ethanol, 1 mm EDTA 30% (v/v) glycerol and were stored at -20 C. The protein content of the plasma membrane was estimated using BSA standard [Bensadown and Weinstein, 1976]. The initial three steps (I, II, III) were modified from [Mitra et al., 1994]. Upto that step, the enzyme was partially purified. Further two steps were adopted for apparent homogenous recovery of the isolated kinase. According to the first three steps, the isolated plasma membrane (concentrated approximately 10 mg/ml) were solubilized using 1% Triton X-100 and kept in ice for 1 hr with intermittent stirring. Then the sample was centrifuged at 27000x g for 90 min at 4 C. The resulting supernatant was loaded on a DEAE cellulose column (1x10 cm) equilibrated with 5 mm K-PO 4 ph 7.0 containing 1 mm PMSF, 2 mm - mercaptoethanol, 20% glycerol and 0.1% triton X-100 (buffer A). The activity peak was eluted in unretained fraction. The unretained fraction was then chromatographed over a

38 38 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. casein-sepharose 4B column (0.9x5 cm), primarily equilibrated with the same buffer used in DEAE column or buffer A. The column was eluted with discontinuous gradient of 0.1, 0.2, 0.3, 0.5 and 1M NaCl. The activity peak was eluted with 0.2 M NaCl-buffer A. Pooled fraction of isolated kinase eluted at 0.2 M NaCl was concentrated by Diaflo Ultrafiltration using PM 30 membranes (Amicon). The concentrated elute was dialysed against pharmalyte- HCl ph 8.0 (1:45) or eluent buffer and loaded on an ion exchange resin PBE 118 (0.7x 10 cm or 3 ml), equilibrated with M triethylamine-hcl ph 11.0 [Sluyterman and Elgeroma, 1978]. After passing the sample, the column was washed with eluent buffer in 1 ml fraction. The elution was monitored by measuring ph of each fraction using a ph meter as well as activity of enzyme. The CIK activity fractions were pooled and concentrated using PM 30 Diaflo ultrafiltration membrane. The concentrated fraction, containing the CIK activity of step IV was subjected to Casein-Sepharose 4B column again to remove the ampholyte as well as for further enrichment of CIK activity. The affinity column was primarily equilibrated with buffer A. After passing the sample, the column was washed thoroughly with buffer A and activity fraction was eluted using 0.2 M NaCl-buffer A like step III. The fractions containing CIK activity were concentrated using PM-30 diaflo ultrafiltration membrane (Amicon) and dialysed against buffer A and supplemented with 50% glycerol and kept at -20 C until used. All the purification steps were carried out at 0-4 C. Physical and Biochemical Characteristics of Ecto-CIK Purity: The summary of the purification of the CIK has been shown in (Table 10). The activity peak of casein-sepharose 4B column (Step III) elution showed heterogeneity as shown in [Mitra et al., 1994] in PAGE (native electrophoresis). This protein peak, when subjected to chromatofocussing, showed single peak at the pi value of approximately depending on the enzyme activity. Purification in this step was nearly 140 fold higher for the kinase. The activity peak represents about 50% recovery of the plasma membrane CIK activity. Non-denaturing condition of electrophoresis showed a fine contaminating protein band of this activity peak. So, this active protein was further subjected to casein-sepharose 4B affinity matrix (re-affinity) for further purification as well as removal of ampholine. In the final step the isolated kinase showed approximately 150 fold purification and 48% recovery of enzyme activity. A considerable loss of the total activity occurred during the purification procedure and this hindered a reliable determination of the specific activity increase of CIK. CIK as membrane kinase, is very difficult to handle and to maintain the membrane microenvironment, we have added different preservatives including high concentration of glycerol to overcome the possibility of loss of activity in isolated condition. The purified CIK (after step VI) showed a single protein band in all the three lanes indicating apparent homogeneity and co-migration of activity peak with the band also confirms the homogeneity of the isolated enzyme. The purity has also been confirmed by the results of SDS gel electrophoresis, gel filtration chromatography, sucrose density gradient ultracentrifugation. Properties of CIK: The molecular weight of the purified CIK as estimated by Sephacryl S-200 gel filtration was approx. 120 ± 15 kda. SDS gel electrophoresis of purified CIK (5 µg, 10 µg, 25 µg) showed that the enzyme was made up of two subunit of about 63 kda and 55 kda (Figure 25). Apparent stoichiometric analysis indicated that the subunits were in monomeric forms and the values were also consistent with the molecular weight obtained by

39 Purification and Characterization 39 the gel filtration chromatographic techniques. One activity peak was obtained when isolated CIK from step VI was subjected to sucrose density gradient according to [Mitra et al., 1994]. The fraction having ph 10.1 showed the activity (CIK) peak. Figure 25. (a) SDS-PAGE of CIK using 10% polyacrylamide gel. Markers were β-galactosidase (116 KDa), Phosphorylase b (97 KDa), bovine serum albumin (66KDa), Ovalbumin (45 KDa) Carbonic anhydrase (29 KDa), Trypsin inhibitor (20 KDa). Purified CIK 5 µg (lane a), 10 µg (lane b), 25 µg (lane c), were loaded in three successive lanes. (b). Determination of mol.wt. of CIK by SDS PAGE (Reproduced from Nath et al 2008). Step Table 10. Purification of CIK from isolated plasma membrane Total activity Units x10-3 Total protein (mg) Specific activity Unit/mgx10-3 Recovery(%) Fold purificatio n Plasma membrane Triton extract DEAE unretained Casein Sepharose 4B affinity Chromatography Chromatofocussing Re-affinity chromatography (Reproduced from Nath et al 2008).

40 40 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. The enzyme showed maximal activity at ph However further increase in ph upto 11.0 caused greater decreases in the activity of CIK. The apparent Km value for ATP of CIK was approx 33.3 µm. CIK was activated maximally by Mg 2+. Co 2+, Mn 2+ could activate it but could not replace Mg 2+. Ca 2+ had no effect on enzymatic activity. Zn 2+ (10 mm) inhibited about 45% of enzymatic activity in presence of Mg 2+. The apparent Km value for Mg 2+ was approx. 5 mm. EDTA (10 mm), NaF (10 mm), VO 4 (50 µm, 100 µm) had no effect (Table 11). The kinase was neither activated by camp (5 µm) or cgmp (5 µm), calmodulin (5 µg) and Ca 2+ (100 µm). The kinase was also not activated in the presence or absence of CaCl 2 (100 µm) and / or phosphatidylserine (25 µg/ml), phosphatidylcholine (25 µg/ml) phosphatidylethanolamine (25 µg/ml) phosphatidylinositol (25 µg/ml) or in presence of phosphatidylserine plus 1-3 Diolein (5 g/ml). Table 11. General properties of the CIK Assay system Enzyme activity units Enzyme activity % Complete Mg Mg 2+ +EDTA (5mM) Mg 2+ +Co (5mM) Mg 2+ +Co (20mM) Mg 2+ +Mn (2mM) Mg 2+ +Mn (20mM) Zn 2+ (5mM) Zn 2+ (10mM) NaF (10mM) Na3VO4 (100μM) EGTA (200μM) Ca 2+ (100μM) Ca 2+ (500μM) Ca 2+ (1mM) Ca 2+ (100 μm)+calmodulin (5 μg) camp (5 μm) camp (10 μm) cgmp (5 μm) cgmp (10 μm) Standard assay conditions were used except for the alterations indicated. The data are representative of 5 such experiments. (Reproduced from Nath et al 2008). The purified protein kinase showed markedly higher specificity for the phosphorylation of the acidic proteins: casein and phosvitin as compared to the basic proteins like histones and protamine. The isolated ecto-cik also showed high efficacy for the phosphorylation of proteins bound to the sperm plasma membrane. MPS, the purified sperm ectophosphoprotein, was tested for its efficacy as a protein substrate of the kinase (Figure 26). The apparent Km value of the enzyme for MPS is approximately 100 µg/ml (1 µm). In

41 Purification and Characterization 41 contrast the apparent Km for casein was approximately 0.8 mg/ml (approx. 35 µm). The data demonstrate that the ecto-kinase has a remarkably high affinity and specificity for phosphorylating MPS: its natural substrate localized in the micro-environment of the kinase. Figure 26. Effect of different concentrations of MPS on the activity of CIK. Standard assay conditions were followed using (A) isolated: MPS or (B) casein: as the substrate. The insert shows the Lineweaver-Burk plot of these data. Ecto-CIK causes phosphorylation of the serine and threonine residues of outer cellsurface phosphoproteins (Figure 27). CIK is a strongly basic protein with an isoelectric point Indirect immunofluorescence studies demonstrate that during the epididymal maturation, ecto-cik undergoes a remarkable lateral movement on the outer-cell surface culminating in capping on the acrosomal tip of the sperm. (Figure 28). CIK is localized in the outer cell surface micro-environment of the kinase. The antibody of CIK markedly inhibits sperm surface protein phosphorylation as well as flagellar motility. The results demonstrate that the ecto-cik is an unique membrane protein-specific serine / threonine kinase and this appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane bound ectophosphoproteins.

42 42 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Role of Ecto-CIK in Sperm Functions Effect of CIK on forward motility of spermatozoa: Microscopic motility assay method described above, did not show did not show any significant effect on the percentage of forward motility (% FM) in cauda mature sperm cells when treated with CIK antibody with 1:500 dilution at different time scale (15 min, 30 min, 45 min, 1 hr and 1.5 hr) as compared to the control serum treated sperm (Nath et al 2008). Figure 27. Identification of the phosphorylated amino acids. Plasma membraneprotein (MPS) (lane a) and casein (lane b) were phosphorylated under standard assay condition and 32 P-labeled products were analysed by cellulose thin layer chromatography. The position of authentic internal standards are indicated as p-ser, phosphoserine, p-thr, phosphothreonine, p-tyr, phosphotyrosine. (Reproduced from Nath et al 2008). Figure 28. Immunofluorescence of goat epididymal maturing spermatozoa. Sperm were isolated from (a) caput, (b) corpus (c) cauda part of epididymis. Cells were labelled with a rabbit polyclonal antibody of goat sperm CIK followed by FITC-labelled goat anti-rabbit IgG. Spermatozoa were examined by fluorescence microscope at 1000x magnification. Preimmune rabbit sera treated cells were used as the control cells. (Reproduced from Nath et al 2008).

43 Purification and Characterization 43 However the spectrophotometric assay method showed a drastic retardation of forward motility with time when treated with CIK antibody as compared to control serum treated sperm. At 1:500 dilution of antibody forward motility percentage decreased about 30%, 50%, and 75% after 30min, 1 hr, and 2 hr of incubation respectively (Figure 29). Figure 29. (a) A representative spectrophotometric tracing showing the effect of CIK antibody on sperm forward motility percentage assayed spectrophotometrically. The experiment was carried out under standard assay conditions using 10 7 cells/assay. The slope of the initial curve can be calculated from the broken line. Absorbance of all the cell suspension after mixing the content of the cuvette (At) was A, B, C. are control serum treated cells for 30min, 1hr, 2hr respectively and A B C are CIKantibody treated cells respectively.( b) Effect of antibody on percentage of forward motility of mature cells as measured by spectrophotometric method. Sperm added with antibody was studied at different time scale ( ) against preimmuned sera added cells (o). These data are representative of seven such experiments. (Reproduced from Nath et al 2008). No recovery of forward motility was observed when purified enzyme was added to CIK antibody-treated (1hr) sperm cells. Forward motility activity as measured by the initial slope of the curve, also decreased appreciably upon treatment with CIK antibody. The difference between these two methods of motility assessment is due to the fact that microscopic method takes into consideration only the number of cells with forward progression (not the velocity); whereas the spectrophotometric method shows not only the motile cell numbers but also their velocity and thus the latter method gives more objective assessment of sperm forward motility index. CIK antibody drastically inhibited the purified enzyme activity when casein used as the exogenous substrate for phosphorylation (Figure 30). About 80% activity was

44 44 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. inhibited using 1:500 dilution of antibody in assay medium. The control rabbit serum from non-immunized rabbit did not have any significant effect on the phosphorylation reaction. So the observed effect of the CIK antibody on sperm motility is due to its inhibitory action on the activity of the protein kinase. Figure 30. Comparative analysis of inhibitory effect of CIK antibody on the mature cell surface protein phosphorylation as well as on specific activity of forward motility or velocity by spectrophotometric assay method depending on time. Protein phosophorylation in presence of CIK-antibody ( ); phosphorylation in presence of preimmune rabbit sera (o); specific activity of forward motility in presence of antibody ( ) and specific activity of forward motility in control sera treated cells ( ). (Reproduced from Nath et al 2008). Figure 31. Effect of ecto-cik antibody on acrosome reaction of goat cauda spermatozoa as monitored by the Rose Bengal staining method. Acrosome reaction was carried out under the standard assay conditions and the cells after staining with Rose Bengal were observed under microscope at 1000x magnification. (A) Sperm cells treated with preimmune sera. (B) Cells treated with ecto-cik antibody. ( ) represents acrosome reacted (acrosome not intact) sperm and ( ) represent acrosome un-reacted (acrosome intact). The acrosome unreacted cell has a tiny colored spot on the tip of the sperm head whereas the acrosome reacted cell has no such colored spot. The insets showing sperm cells at higher magnification give clearer vision of the acrosome reacted and un-reacted cells. (Reproduced from Maiti et al 2009).

45 Purification and Characterization 45 Effect of ecto-cik on acrosome reaction: The effect of CIK antibody was assessed on the sperm acrosome reaction [Maiti et al., 2009]. Prior to the addition of calcium ionophore approx. 12% of the sperm cells underwent acrosome reaction whereas following treatment with the ionophore, nearly 40% of the untreated spermatozoa showed acrosome reaction (Figure 31). Treatment of sperm cells with CIK antibody (dil. 1:500) caused a significant decrease (approx 50%) in percentage of acrosome reacted sperm compared to the PBS-BSA treated or control sera-treated sperm. The control rabbit serum did not show any significant effect on the percentage of acrosome reacted sperm as compared to the PBS-BSA control. Another well-defined biochemical index for assessing acrosome reaction is the release of acrosin from the acrosomal sac of spermatozoa. (Figure 32) shows the time course of the acrosin release from the sperm acrosome during the acrosome reaction. In absence of Ca ++ ionophore, rate of release of acrosin in the medium was very low. Onset of the acrosome reaction i.e. after the addition of Ca ++ ionophore in the preincubated cells caused a remarkable increase in the rate of released acrosin in the medium from the normal sperm, the major amount of this release being nearly complete during the first 15 min of incubation. However, CIK antibody treatments (1:100, 1:500 and 1:1000 dilutions) caused a significant decrease in the release of this enzyme: 1:100 dilutions being most effective in this respect. The control rabbit IgG from normal rabbit serum did not show any significant effect on the acrosome release of sperm as compared to the PBS-BSA control Approx. 50% acrosin release was inhibited of by antibody at a dilution of 1:500. Figure 32. Effect of CIK antibody on the release of acrosin during acrosome reaction. Highly motile spermatozoa were pre-incubated in the BWW medium for 180 min prior to the addition of calcium ionophore for the induction of acrosome reaction. Acrosin released from the sperm samples was assayed. Acrosin was measured as change of OD at 253 nm. (ʘ) change of OD before addition of ionophore A (10 M); ( ) change of OD after addition of ionophore in preimmune sera treated sperm ;( ) 1:1000 dil of antibody ;( ) in 1: 500 dil of antibody; ( ) with 1: 100; ( ) Change of OD in absence of ionophore. The data shown are Mean SEM. (Reproduced from Maiti et al 2009). MPS: Major Protein Substrate of Ecto-CIK Several studies have been reported on the occurrence and functional significance of protein kinases and their endogenous substrate phosphoproteins in a variety of cell types and

46 46 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. they are known to play key role in downstream signalling and cellular functions through phosphorylation-dephosphorylation of substrate phosphoproteins. For example, CSQ phosphorylation in heart is associated with intracellular transport [Ram et al 2004], mitochondrial phosphoproteins are involved in critical mitochondrial functions [Reinders and Sichmann, 2007], tight junction permeability is regulated through occluding phosphrylation in pancreatic epithelial cells [Rajasekhran et al., 2007], Ezh2 is phosphorylated by cyclindependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner [Kaneko et al., 2010], phosphorylation of Ser312 is required for p53 to function fully as a tumor suppressor in vivo [Slee et al., 2010]. Phosphoproteins derived from plasma membrane of a variety of mammalian cells, have been implicated to play a vital role in regulation of membrane functions [Majumder and Turkington, 1972; Uno et al., 1977; Boman et al., 1984; Kang and Chiang, 1986; Naik et al., 1991; Lytle and Forbush, 1992; Sarrouilhi et al., 1992]. Phosphorylation and dephosphorylation of specific protein, have been found to play vital role for entry of cells into S phase of cell cycle [Ramanadham et al., 1984], platelet function [Kang and Chiang, 1986], neuronal adhesion molecules (N-CAMs), etc through their substrates [Ehrlich et al 1986]. Presence of phosphoproteins have been demonstrated in the sperm plasma membrane of humans [Huacuja et al., 1997], cattle [Noland et al., 1984; Chaudhury and Casillas, 1989], hamsters [Devi et al., 1997], and goats [Halder and Majumder, 1986; Mitra and Majumder, 1991]. Cyclic AMP-dependent protein kinase has been demonstrated on the outer surface of spermatozoa derived initially fron rat [Majumder 1978; Atherton et al., 1985] and successively from human [Schoff 1982, Pariset et al., 1983] cow [Noland et al., 1984] and goat [Dey and Majumder 1990a, 1990b]. However, the camp dependent ecto- protein kinases are incapable of phosphorylating the endogenous plasma membrane proteins [Halder and Majumder, 1986], [Dey and Majumder 1990b]. A cyclic AMP independent protein kinase (ecto-cik) has also been demonstrated on the outer surface of sperm plasma membrane of cattle [Noland et al., 1984], goat [Chaudhury and Casillas, 1989], [Mitra and Majumder, 1991] that causes phosphorylation of multiple outer-membrane bound phosphoproteins. The ecto-protein kinase of the intact cell as well as the membrane bound enzyme phosphorylates the serine and threonine residues of endogenous membrane associated phosphoproteins. Our laboratory has investigated the role of cyclic AMP independent protein kinase (ecto- CIK) on goat sperm model. Ecto-CIK of goat sperm plasma membrane phosphorylates with high affinity the serine and threonine residues of several endogenous phosphoproteins and the rate of phosphorylation of these proteins undergo marked modulation during epididymal sperm maturation [Nath and Majumder, 1999]. As already elaborated above, the ecto-cik has been purified to apparent homogeneity and characterized [Nath et al., 2008]. The major physiological substrate (MPS) of ecto-cik has also been identified [Maiti et al., 2004; 2008; 2009] and outlines of these studies have been shown below. Assay of MPS Anti-serum against the purified MPS was raised in rabbit as described earlier [Maiti et al., 2009]. The immunoglobulin of the immune serum was precipitated twice with 50% ammonium sulfate. The final precipitate was dissolved in PBS (ph 8.0) and dialyzed overnight against the same buffer. Standard ELISA technique was used for analyzing MPS levels in various sperm fractions. For determining the antibody titre value, 50 l of MPS solution (containing 100 ng protein/triton X-100 solubilized plasma membrane) in was added

47 Purification and Characterization 47 in the wells of microtitre plates and incubated overnight at 4 0 C. After washing with PBS, the wells were blocked with PBS containing 3% BSA and incubated at 37 0 C for 1hr. Then the 1 st antibody (MPS antibody/cik antibody) in PBS containing 1% BSA was added at different dilutions. Incubation and washing were done as before followed by the addition of HRPconjugated goat anti rabbit IgG (2 nd antibody at a dilution of 1:1000 in PBS containing 1% BSA). The plate was then incubated at 37 0 C for 1hr. Finally colour development was done by using 3 mm orthophhenyldiamine (OPD) in 24 mm citric acid-50 mm sodium hydrogen phosphate containing 0.04% H 2 O 2 (ph ) in PBS (Wisdom 1976). Development of colour was stopped after 30 min with 4(N) H 2 SO 4 and absorbance was measured at 492 nm by ELISA reader. Purification of MPS Highly purified sperm plasma membrane was found to possess ecto-cik activity that caused phosphorylation of endogenous phosphoproteins. MPS was purified from caprine sperm PM by the procedure developed earlier in our laboratory (Maiti et al 2004).To purify these surface external substrate proteins,the plasma membrane-bound proteins were radiolabelled with endogenous kinases using [ - 32 P]-ATP. (Step 1).The solubilised labelled plasma membrane was applied to Sephacryl S-300 column (1.4 x 75 cm) equilibrated previously with 5 mm Tris-Cl, ph 8 containing 20% (v/v) glycerol, 1 mm PMSF, 2 mm - mercapto ethanol, 1 mm EDTA and 0.05% (v/v) Triton X-100 (Buffer A). The fractions (1 ml) were collected after void volume with monitoring 32 P radioactivity of each fraction (Step 2). Three radioactive protein peaks were obtained (Figure 33). Figure 33. Molecular sieve chromatography of solubilised [ 32 P] labeled membrane. The fraction size was 1 ml and an aliquot of each fraction was used to measure radioactivity ( ). (Reproduced from Maiti et al 2004). The first two peaks comprised of only 10% of the total radioactivity. The third one contained 90% of the total radioactivity and this peak contains the major substrate protein (MPS) of ecto-cik. The fractions containing the highest amount of radioactive protein peak (from the earlier step), were then subjected to DEAE cellulose ion exchange column (1 x 5 cm) previously equilibrated with buffer A. The radioactive fraction was eluted by linear

48 48 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. gradient of NaCl in buffer A. The major radioactive peak was eluted at 60 mm salt concentration (Step 3) (Figure 34). Chromatofocusing: Fractions eluted at 60 mm NaCl in buffer A was concentrated to a great extent and dialysed against PBE-74-HCl (ph 4) or eluent buffer and subjected to chromatofocusing using PBE-94 (0.7 x 10 cm) equilibrated with M imidazole-hcl, ph 7 (Sluyterman and Elgersma, 1978) The elution was monitored by measuring ph as well as radioactivity of each fraction (Step 4). Three radiolabelled MIP fractions were separated (Figure 35) those were concentrated using PM-30 Diaflo ultrafiltration. All procedures were performed at 4 C and the concentrated purified MPS was preserved in buffer A at 20 o C. Figure 34. Ion-exchange chromatography of the major radioactive peak obtained by gel filtration described earlier. The elution was monitored by measuring the radioactivity ( ) of each fraction and also by concentration of NaCl in buffer-a measured by the conductivity ( ) of each fraction. The data is representative of 4 such experiments. (Reproduced from Maiti et al 2004). Figure 35. Chromatofocusing of the radioactive fraction eluted from ion-exchange column using PBE- 94. The elution was monitored by measuring ph ( ) as well as radioactivity ( ) of each fraction (Reproduced from Maiti et al 2004). Purity of MPS As shown in Table 12 MPS has been purified to approx. 190-fold with 40% recovery. The MPS of ecto-cik has been purified to apparent homogeneity as revealed by multiple analytical fractionation methods. A considerable loss of substrate occurs during the

49 Purification and Characterization 49 purification procedure. MPS is a membrane protein and very difficult to handle. To maintain the microenvironment and to overcome the difficulty we have added preservatives (Tritron- X-100 and glycerol) and protease inhibitors in the purification buffer. Autoradiography at different steps of purification procedure demonstrated the presence of one major 32 P- labelled protein band of 100 kda and a few minor bands in the soluble fraction of phosphorylated membrane. The 100-kDa band became more and more prominent with the advancement of the purification and in the last step of purification all the minor protein bands were undetectable (Figure 36). Step Table 12. Summary of purification of MPS Total Protein ( g) Total Counts/ min (cpm) cpm/ g of Protein Fold Purification % Recovery Plasma membrane x x Triton Extract (Step I) x x Sephacryl S-300 molecular sieve chromatography x x Peak C (Step II) DEAE-ion exchange chromatography x x (Step III) Chromatofocusin g-peak-3 (Step IV ) x (Reproduced from Maiti et al 2004). Figure 36. Autoradiography of MPS at different steps of purification. Lane 1, at Triton extract (step1), lane 2, at Sephacryl S-300 molecular sieve chromatography (step2), lane 3, at DEAE-cellulose ionexchange chromatography (step3), lane 4, at chromatofocusing (pi 5.14) (step4), About 250 g of protein was loaded in each lane. (Reproduced from Maiti et al 2004).

50 50 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Properties of MPS The molecular weight of purified MPS as estimated by Sephacryl S-200 gel filtration was approximately 100 kda. The same molecular mass was also obtained when MPS was subjected to SDS gel electrophoresis thereby showing that it is a monomeric protein (Maiti et al 2004). Upon chromatofocusing MPS resolves into three peaks and these isoforms of MPS have pi of 6.37, 6.05, and All these isoforms served as the specific substrate of ecto- CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate (Figure 26). Each of the three chromatofocussed fractions showed molecular weight of 100 kda when analyzed by SDS gel electrophoresis [Maiti et al., 2004]. The data suggest that all these peaks represent the same protein having different levels of phosphorylation. This is due to the fact that phosphate groups carry negative charges, so variance in phosphorylation leads to the difference of in the number of negative charges and hence differences in pi were observed. The preimmune rabbit serum (control) did not show any agglutination effect on the mature sperm. After incubation of the mature, motile sperm cells with MPS antibody at 37 o C for 1 hour at antibody dilution of 1:100, head-to-head agglutination was observed under phase contrast microscope (Figure 37). No head-to-tail or tail-to-tail agglutination was observed. Distribution of MPS on the sperm surface as well as its localisation was analysed by the indirect immunofluorescence technique. Binding of the MPS antibody on sperm surface was visualised by the binding of FITC-conjugated IgG with MPS antibody (Figure 38). MPS antibody was found to bind intensely with acrosomal area of sperm head in cauda sperm cells. But the other parts of the spermatozoa are completely devoid of MPS. Negative control using the same amount preimmune rabbit serum instead of MPS antibody lead to no detectable florescence on the head of cauda sperm cells. The observation gave support to the view that MPS was localised on the acrosomal region of the mature goat spermatozoa. Figure 37. An Effect of MPS antibody on intact spermatozoa. Washed cells derived from cauda epididymis were treated with MPS antibody at (1:100) dilution for 60 min. and then visualized under phase contrast microscope at 1000x magnification. Cells with preimmune sera under same condition served as control (Reproduced from Maiti et al 2004).

51 Purification and Characterization 51 Figure 38. Immunofluorescence of goat epididymal spermatozoa. Cells were treated with (a) preimmune sera (b) polyclonal MPS antibody followed by FITC-labeled goat-anti-rabbit IgG according to the procedure described in Materials and Methods. Spermatozoa obtained thus were examined by fluorescence microscope at 1000X magnification. (Reproduced from Maiti et al 2009). Role of MPS in Sperm Functions Effect of MPS on sperm motility: MPS is immunogenic to rabbit. To prove the immunological specificity of the MPS antibody in vitro, the membrane phosphorylation experiment using endogenous ecto-cik and [ - 32 P]-ATP was performed in presence of different dilutions of antibody raised against the major physiological substrate. The same experiment performed in presence of pre-immune sera served as the control. The extent of inhibition was 100%, 89%, 84%, 72% and 42% at 0, 1:10, 1:100, 1:500, 1:1000 dilutions, respectively. MPS antibody markedly decreased sperm membrane proteinphosphorylation. Inhibition of ecto-cik activity in presence of the MPS antibody was also observed. A drastic fall in motility was observed when mature motile sperm suspension in RPS medium was incubated with monovalent MPS antibody (Fv/Fab fragments: prepared by digesting it with papain). There was also inhibition of forward motility in presence of low concentration of the polyvalent MPS antibody. The data show that MPS plays an important role for activating sperm motility. Presence and concentration of MPS was estimated in forward motile and motile + non motile cells (separated by the procedure described above) by ELISA using MPS antibody and HRP-conjugated anti-rabbit-igg. [Maiti et al., 2004). It was found that concentration of MPS was approximately half in less motile and nonmotile cell population compared to the vigorously forward motile cell population (Figure 39). To investigate further the functional role of MPS in regulation of sperm flagellar movement, we took advantage of cell electroporation method [Maiti et al., 2008]. We increased the intra-sperm level of MPS by using this technique. Cell electroporation is a novel biochemical tool that causes temporary disruption of membrane permeability on application of electric pulses [Tsong et al., 1991]. This is a simple method in which transient micropores are generated in the plasma membarne of cells on application of electric field. Exchange of molecules from either side takes place. The pores reseals on the removal of electric field. But the cells retain the molecules [Weaver et al., 1995]. The method of electroporation was standardised in goat sperm system. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2X10 8 /ml) to external electric field of intensity 1.5 kv/cm and capacitation of 25 mf at 4 0 C and post-pulse incubation at 37 0 C for 1 hr. MPS insertion caused a remarkable increase of approximately 34% total motility (P<0.01) and 32% forward motility (P<0.01) with respect to the motility before electroporation. At

52 52 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. maximum (75%) MPS incorporation, total motility and forward motility increments were also maximal (Figure 40). Incorporation of MPS was also a time-dependent procedure. The incorporation increased linearly with post-electroporation incubation time. Incorporation, motility and forward motility increments were maximum when the post-pulse incubation time was 60 min. Thus MPS incorporation caused an increase of approximately 34% motility (P < 0.01) and 32% forward motility (P < 0.01) as compared to the motility before electroporation [Maiti et al., 2008]. Figure 39. Immunodetection of MPS in forward motile and other (motile+ nonmotile) cells. Cells were separated by swim up technique and plasma membranes were isolated, solubilised in Triton X-100 and centrifuged. The supernatant was used in ELISA. Presence and amount of MPS was detected with MPS antibody (Reproduced from Nath et al 2008). Figure 40. Effect of MPS incorporation on motility (dose course). Spermatozoa (2 x 10 8 cells/ml) were electroporated under standard assay conditions in presence of variable amounts of [ 32 P] MPS (0 300 pmol). Amount of incorporation of MPS was assayed by precipitating the cells with 10% TCA and estimating the radioactivity in the cells in a liquid scintillation counter. Motility and forward motility were determined by the microscopic method. The results showed the mean of five separate experiments. (Reproduced from Maiti et al 2008).

53 Purification and Characterization 53 Table 13. Effect of MPS antibody on acrosome reaction and acrosin release of goat sperm Treatment Acrosome reacted sperm (%) Mean ±SD MPS antibody (1:500 dilution) 20± ±4.11 MPS antibody (1:100 dilution) 10± ±2.9 Control (preimmune sera) 42±1.1 86±1.15 PBS-BSA control 40± ±1.8 Acrosin activity in supernatant Mean ± SD Assays were performed on various days using sperm collected from at least 6 different tissues. Acrosin activity was expressed as U of acrosin/10 7 sperm cells. (Reproduced from Maiti et al 2009). Role of MPS in sperm acrosome reaction: To assess the role of MPS on acrosomal reaction, we have used two methods: a direct method which is based on the release of acrosin: the proteolytic enzyme from the acrosomal sac and an indirect method commonly known as Rose Bengal method which is based on the staining of the residual amount of acrosomal content left inside the sac following membrane fusion [Maiti et al., 2009]. The effect of MPS antibody was also assessed on the sperm acrosome reaction, which is essential for fertilization. After incubation with MPS antibody (1:100 dilutions) for 1 hr. and subsequent treatment with capacitation buffer and calcium ionophore A23187, the sperm samples demonstrated a significant decrease in percentage of acrosome reacted sperm compared to the PBS-BSA treated control or control rabbit IgG treated sperm (Table 13). The control rabbit serum did not show any significant effect on the percentage of acrosome reacted sperm as compared to the PBS-BSA control. Treatment with MPS antibody demonstrated a lower concentration of acrosin release in the supernatant than did PBS-BSA or preimmune sera treated (control) sperm. It was found that acrosin activity was 51% and 76% less at 1:500 and 1:100 dilutions respectively compared to the preimmune sera control. 52% and 76% acrosome un-reacted spermatozoa at 1:500 and 1:100 dilutions, respectively were observed under microscope at 1000X magnification compared to the pre-immune sera control (Table 13). The control rabbit serum did not show any significant effect on above two cases as compared to the PBS-BSA control. The concentration of MPS was estimated in sperm cells before and during acrosome reaction at different time intervals following initiation of acrosome reaction with calcium ionophore. It was found that concentration of MPS increases significantly with time, up to 20 min of incubation. During this period the membrane-bound MPS as estimated by ELISA, increase by nearly 100% (Figure 41). The results demonstrate that MPS is required for the sperm acrosome reaction and the acrosin release from spermatozoa. The biochemical basis of this altered level of MPS during acrosomal reaction is not clear. In one of our earlier publication [Maiti et al., 2009], we have reported that MPS present in the cytosol finally gets localized to the external sperm surface. It is thus possible that during the acrosomal reaction more of cytosolic MPS gets translocated to the outer cell surface. Alternatively during the acrosomal reaction phase there may be a major restructuring of the cell membrane leading to greater availability of exposed MPS located on the external cell surface. This enrichment of MPS following acrosome reaction strengthens the above view.

54 54 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Figure 41. Relationship of MPS concentration with acrosome reaction of spermatozoa. MPS concentration (by ELISA of solubilized membrane) were determined in isolated plasma membrane of acrosome reacted and unreacted cell population, according to the procedure describe of five such experiments (Reproduced from (Maiti et al 2009). Goat Sperm Ecto-Phosphoprotein Phosphatase Activity (Ecto-PPase) As discussed above, phosphoprotein have been demonstrated on the surface of a variety of mammalian cells including spermatozoa and they have been implicated to have important role in regulating cell function [Uno et al., 1977; Boman et al., 1984; Noland et al., 1984; Kang and Chiang 1986; Halder and Majumder 1986; Chaudhry and Casillas 1989; Mitra and Majumder 1991; Naik et al., 1991; Lytle and Forbush 1992; Sarrouilhi et al.,. 1992; Devi et al., 1997]. Isolated plasma membrane from several mammalian cells also have been shown to possess PPases that cause dephosphorylation of exogenous as well as endogenous membranebound phosphoproteins [Maeno and Greengard 1972; Kabayashi and Ozawa 1981; Church et al., 1988; Gruppuso 1990; Han and Dokas 1991; Liao et al., 1991; Williams 1991; Goldenring et al., 1992; Yang et al., 1992; Klumpp et al., 1994; Kubokawa et al., 1995; Vijayaraghavan et al., 1996; Devi et al., 1999; Smith et al., 1999]. Goat epididymal spermatozoa possess a camp-independent protein kinase on the outer cell surface that causes phosphorylation of multiple endogenous ecto-phosphoproteins [Mitra and Majumder 1991; Mitra et al., 1994; Nath and Majumder 1999]. Evidence has been presented to support the localization of phosphoprotein phosphatase on the goat sperm outer surface (ecto-ppase) that causes dephosphorylation of exogenous proteins such as histone, casein, phosvitin and protamine [Barua et al., 1985; Barua and Majumder. 1987]. The ecto-ppase localized on the outer surface of spermatozoa also dephosphorylates sperm outer-surface phosphoproteins [Barua and Majumder, 1990; Barua et al., 1990]. Multiple ecto- phosphoproteins of the goat cauda-epididymal intact spermatozoa have been shown to undergo dephosphorylation in vitro by endogenous PPase(s) located on the sperm outer surface [Barua and Majumder, 1990]. The cell surface dephosphorylation reaction is not dependent on divalent metal ions. The ecto-ppase has been solubilized from the isolated sperm plasma membrane and partially purified [Barua et al., 1999]. It is a specific phosphatase that dephosphorylates phosphoserine/phosphothreonine residues of a variety of proteins. Subsequently the caprine sperm ecto-ppase has been purified to apparent homogeneity and characterized [Barua et al.,

55 Purification and Characterization ]. The following section describes some salient features of its purification and biochemical and functional characteristics of this sperm external surface enzyme. Assay of Ecto-PPase Activity The ecto-ppase activity that causes dephosphorylation of [ 32 P] histones was measured by the procedure described before [Barua et al., 1985] with some modifications. The standard assay system contained 225 g of [ 32 P] histone and 1 g (10 l) of the isolated PPase in 0.25 ml 50 mm Tris-HCl buffer, ph 8.0. The incubation of isolated PPase was carried out at 37 C for 10 min and the reaction was stopped by chilling and with the addition of 0.1 ml of 1.5% casein and 0.25 ml 50% trichloroacetic acid (TCA). The tubes were then processed for the assay of [ 32 P] radioactivity in the TCA-filtrates as described earlier [Barua and Majumder, 1987). The unit of the PPase activity was defined as the amount of the enzyme, which catalysed liberation of 1 pmole of 32 Pi from [ 32 P]-histones under the standard assay conditions. The concentration of Triton X-100 (derived from the enzyme preparation) in the assay system was 0.004% and the detergent at this level had no effect on the enzymatic activity. By the same assay method, PPase activities of the isolated plasma membrane and its fractions were also estimated. Purification of Sperm Ecto-PPase Previous reports from our laboratory [Barua et al., 1999] have shown that the sperm PMbound PPase can be solubilized under alkaline conditions (ph 11.4) in the absence of any detergent. However, the alkali-solubilized enzyme could only be partially purified. Although the isolated PPase showed only one protein band under non-denaturating polyacrylamide gel electrophoresis (PAGE), it resolved into multiple protein bands under denaturating PAGE (in presence of SDS : sodium dodecyl sulphate) presumably due to aggregation of PPase with other proteins. An improved protocol of PPase purification has been developed by incorporating in the isolation media Triton X-100, a non-ionic detergent that block proteinprotein aggregation phenomenon [Barua et al., 2001]. Although 0.5% (v/v) Triton X-100 strongly inhibits the PPase activity [Barua et al., 1999], this inhibition is reversible. Consequently, the presence of Triton X-100 in the purification media had no adverse effect on the activity of the purified PPase. Isolated plasma membranes were treated with 0.5% (v/v) Triton X-100 with stirring on ice for 1 hr. The suspension was then centrifuged at x g for 90 mins. The resulting supernatant fluid was concentrated using a PM-10 diaflo ultrafiltration membrane. The concentrated fraction was loaded on a Sephadex G-75 (1x120 cm) column equilibrated with buffer A containing 0.1% (v/v) Triton x-100. The activity peak, as monitored by absorbance at 280 nm as well as PPase activity measurement, was collected and concentrated by PM-10 Diaflo ultrafiltration membrane. The resulting PPase preparation was dialyzed against 20 mm Tris-HCl, ph 7.2 containing 1 mm CaCl 2, 1 mm MnCl 2, 0.6 M NaCl, 0.5 mm - mercaptoethanol, 0.5 mm PMSF (Buffer I) and 0.1% Triton X-100 and then subjected to affinity chromatography on a concanavalin A-Sepharose affinity column (1 x 14 cm) previously equilibrated with buffer I. The column was washed thoroughly with the same buffer. The PPase activity bound to the resin was eluted with -methyl mannoside (30 mg/ml). The enzyme fraction was concentrated immediately by PM-10 diaflo ultrafiltration and dialyzed against 50 mm Tris-HCl ph 7.2 containing 10% glycerol, 1 mm PMSF, 2 mm

56 56 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. -mercaptoethanol and 0.1% (v/v) Triton X-100. The resulting PPase was applied to a DEAE-cellulose column (1x10 cm) previously equilibrated with 50 mm Tris-HCl, ph 7.2 containing 10% glycerol, 1 mm PMSF, 2 mm -mercaptoethanol, 0.1% (v/v) Triton X-100. The activity peak was eluted with the same buffer containing 0.12 M NaCl. The active fraction was concentrated by PM-10 diaflo ultracentrifugation and finally stored at -20 o C. All the steps of the enzyme purification were carried out at 6 C. Purity of Ecto-PPase The summary of the purification of the PM-bound PPase has been shown in Table 14. Highly purified sperm PM possessed PPase-activity that caused dephosphorylation of [ 32 P]- histone and the specific activity of the PPase was as high as approximately 530 units/mg protein. To solubilize the ecto-ppase activity, isolated sperm membrane was treated with 0.5% of different ionic and non-ionic detergents like Trion X-100, Tween-20, sodium deoxycholate, Nonidet P-40, lubrol PX (data not shown). Triton X-100 was most effective in solubilizing the PPase activity from the membrane. To optimize the concentration of the detergent for maximal solubilization, different concentrations of Triton X-100 were used. At 0.5% concentration, the detergent gave the best solubilization result. The PPase was purified from the Triton-extract by using molecular sieving chromatography on a Sephadex G-75 column, affinity chromatography on ConA-Sepharose and DEAE-cellulose ion-exchange chromatography. A single peak of PPase activity was detected by each of the chromatographic techniques. The enzyme binds with high affinity to ConA-Sepharose and could be eluted with methyl -mannoside. The isolated PPase showed one protein band when subjected to polyacrylamide gel electrophoresis under non-denaturing conditions (Figure 42). Only one peak of PPase activity was eluted when the gel slices were analysed for the PPase activity. Step Table 14. Purification of PPase from isolated sperm plasma membrane Total activity (Units) Total protein (mg) Specific activity (Units/mg protein) Recovery (%) Plasma membrane Fold purification Triton X-100 solubilization Sephadex G-75 gel filtration chromatography ConA-Sepharose affinity chromatography DEAE-Cellulose ion-exchange chromatography (Reproduced from Barua et al 2001).

57 Purification and Characterization 57 Figure 42. Non-denaturing polyacrylamide gel electrophoresis of purified membrane PPase (Reproduced from Barua et al 2001). Biochemical Properties of Purified Ecto-PPase A single PPase activity peak was observed when the isolated enzyme was subjected to gel filtration on a Sephadex G-75 column (1x120 cm). By this gel exclusion chromatography, molecular mass of the enzyme was estimated to be 36 KDa (Figure 43). No subunits of the enzyme were detected when it was subjected to SDS-gel electrophoresis thereby demonstrating that PPase is a monomeric protein. Chromatofocusing of the isolated PPase has been depicted in Figure 44. As demonstrated earlier [Barua et al., 1999] the sperm ecto-ppase causes dephosphorylation of both phosphoserine as well as phosphothreonine (Table 15). The approximate pi of this isolated PPase was Distribution of PPase on sperm surface as well as change of its localization during epididymal maturation were analysed by the indirect immunofluorescence technique (Figure 45). Figure 43. Determination of molecular weight of PPase by Sephadex G-75 gel filtration chromatography (Reproduced from Barua et al 2001).

58 58 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. Figure 44. Chromatofocusing of PPase in a column of PBE 94. PPase activity obtained from DEAEcellulose chromatography was subjected to chromatofocusing. (Reproduced from Barua et al 2001). Figure 45. Immunofluorescence of goat epididymal maturing spermatozoa. Spermatozoa were isolated from caput, corpus and cauda parts of epididymis and labeled with rabbit polyclonal antibody (against the goat sperm membrane PPase) by FITC-goat anti rabbit IgG. Spermatozoa were examined by fluorescence microscope at 1000xmagnification A. caput-spermatozoa, B. corpus-spermatozoa, C. cauda-spermatozoa. Percentages of caput, corpus and cauda spermatozoa showing the characteristic fluorescence pattern were approx. 75%, 80% and 100%, respectively (Reproduced from Barua et al 2001). Table 15. Dephosphorylation of phosphoserine and phosphothreonine residues of histones by the partially purified ecto-ppase 32 P-labeled bands Distribution of radioactivity (counts per minute) Control +PPase M-I-treated (o min) (15 min) O-Phosphoserine* O-Phosphothreonine* *Values were corrected for loss during the acid hydrolysis according to Takeda et al [1971]. Isolated PPase M-I was incubated for O-min (control) and 15 min under the standard assay conditions and the reaction was arrested by chilling and by the addition of 20%TCA. The precipitated histone was collected by centrifugation at 5000 x g for 10 min in cold. The resulting precipitate was washed extensively with diethyl either to remove TCA from the labeled histone. The [ 32 P] histone was then hydrolyzed in 2N HCl in a boiling water bath for 15 hrs [Takeda et al, 1971] HCl was removed under vacuum, the hydrolysates were applied to whatman No.1 paper strips (4cm x 54cm) and then subjected to papere lectro phoresis with 8% (v/v) formic acid as the electrophoresis buffer [Barua and Majumder,1987]. Unlabeled O-phosphosphoserine, O- phosphothreonine and Pi were used as markers during electrophoresis. These bands were cut out and counted for 32 P in a toluene-based scintillation liquid. (Reproduced from Barua et al 1999).

59 Purification and Characterization 59 It was found to bind intensively with the neck and post acrosomal region of the caput cells and other parts of head showed moderate staining. The tail showed the presence of PPase only on the principal piece region. In the corpus-sperm cells the PPase was concentrated on acrosomal region of the head. In the cauda sperm the fluorescence was present only on the posterior region of head and no staining was observed on the other parts of the head. The principle piece also regained its fluorescence intensity like caput spermatozoa. Negative control using the same amount of preimmune rabbit serum instead of purified PPase-antibody led to no detectable amount of fluorescence on the head or tail region. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the composite spermatozoa from where the former cells were isolated. [Barua and Majumder, 1987]. This PPase is at least one of the ecto-ppases causing dephosphorylation of the sperm outer-surface proteins phosphorylated by an endogenous camp- independent ecto-protein kinase. This remarkable maturation-dependent modification of ecto-ppase activity as well as its distribution on sperm surface suggests that the ectoenzyme may play an important role in sperm function by regulating phosphorylation states of the membrane-associated and reproductive fluid phosphoproteins substrates. Importance of the Novel Coupled Ecto-Enzyme System in Cell Biology It is clear from the above-mentioned discussion that although several investigators have provided evidences for the occurrence of ecto-protein kinases and their protein substrates in a variety of mammalian cells, we have for the first time purified to apparent homogeneity a membrane protein-specific ecto-protein knase (ecto-cik) and its endogenous ecto-protein substrate : MPS and characterized them. We have also for the first time purified and identified an ecto-ppase on sperm surface that causes dephosphorylation of the ecto-mps. These studied thus establish conclusively the localization of a novel coupled ecto-enzyme system (consisting of CIK, PPase and their substrate protein: MPS) on the external surface of a mammalian cell using spermatozoon as the cell model. Summerizing the above-mentioned findings, it may be concluded that ecto-cik and its substrate protein: MPS play vital role in the regulation of sperm forward progression as well as acrosome reaction. The substrates of protein kinases are known as functional proteins in various cellular systems [Ram et al., 2004; Williams and Lisanti, 2005; Reinders and Sichmann, 2007; Rajasekaran et al., 2007]. It thus appears that CIK through its substrate protein: MPS plays an important role in the regulating sperm functions such as flagellar motility and acrosomal reaction. In other words MPS is the direct regulator of sperm functions. It is of interest to note that sperm vigorous forward motility (i.e. vertical velocity) is associated with higher level of both ecto-cik as well as ecto-ppase (Table 16). As depicted in the schematic diagram (Figure 46), the dynamic state of high level of sperm forward movement is associated with rapid phosphorylation and dephosphorylation of specific sperm surface protein : MPS and this process is mediated by enhanced levels of the ecto-cik and ecto-ppase, respectively. CIK phosphorylates the dephospho- form of MPS to generate phospho-mps while the cell surface PPase acts on the phospho-mps to regenerate dephospho- MPS on the outer sperm surface. This diagram lucidly explains the functional concept of the coupled ecto-enzyme system. All the protein components of the system work in a synchronous manner leading to induction and sustaining of a specific sperm function. It is fascinating to note that based on this postulated model, two functionally opposite enzymes (CIK and PPase) are working in concert to maintain adequate level of biologically active form

60 60 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. of MPS ( i. e. phospho-form) which serves as the driving force for sperm functions : motility and acrosomal reaction. Based on this model, sperm velocity is dependent on the speed of operation of the above-mentioned MPS phosphorylation and dephosphorylation cycle. This model is consistent with all the observations discussed above. Figure 46. Schematic representation of the actions of the coupled-enzyme system for the regulation of sperm forward motility. The sperm outersurface coupled-enzyme system is made-up of CIK, ectocyclic AMP-independent protein kinase; PPase, ecto-phosphoprotein phosphatase and their endogenous proteinsubstrate: MPS. The conformation and biological activity of the ecto-proteins are likely to undergo alteration as a consequence of their phosphorylation and dephosphorylation. Symbol (+) represents activation. Table 16. Specific activity of Ecto-Enzymes in intact forward motile spermatozoa Ecto-enzyme No. of experiments Specific Activity of ecto-enzymes (units/10 7 cells) Composite cells (Mean ± SEM) Forward motile cells (Mean ± SEM) Cyclic AMP-dependent protein kinase (Ecto-RC) Cyclic AMP-independent protein kinase (Ecto-CIK) Phosphoprotein phosphatase (Ecto-PPase) ± ± 7.9* ± ± 5.8* ± ± 0.28* Vigourusly forward motile spermatozoa were separated from non-motile and weakly motile cells of composite spermatozoa based on their capacity to move upward against the gravity.the ectoenzymic activities of these cells were estimated as described before [Barua et al 1985 and Halder et al 1986]. *p<0.01;when compared with the composite cells. (Reproduced from Majumder et al, 1988).

61 Purification and Characterization 61 Sperm forward motility is required for transport in female tract and to produce the thrust for penetration of cumulus cells and zona pellucida [Kubler et al., 1983]. Acrosome reaction is followed by egg recognition and zona penetration. It is of interest to note that both ecto- CIK as well as MPS are located on the tip of sperm head overlaying the acrosomal cap. It is thus tempting to postulate that these ecto-proteins with special reference to MPS may play vital role in the sperm-egg recognition mechanism as visualised in diagram shown in Figure 47. Figure 47. Schematic diagram showing the possible role of MPS and ecto-protein kinase in sperm - egg interaction. More recent study from our laboratory demonstrates that ecto-cik is also present in all the mammalian tissues tested and it is predominantly localized in the plasma membrane (Unpublished results). The ecto- membrane protein-specific kinase may thus play important role in cellular regulation by modulating membrane biology through serine/threonine phosphorylation of specific membrane proteins located in the ecto-domains of a variety of mammalian cells. Sperm External Surface Lectin and Its Receptor The goat epididymal spermatozoa during epididymal transit specifically at the distalcorpus stage undergo head-to-head autoagglutination when incubated in vitro in a chemically defined medium (Banerjee et al., 1992; Dacheux et al., 1983). In guinea pig, sperm-sperm adhesion occurs during epididymal maturation that results in the formation of rouleaux in which the sperm heads are stacked one upon the other (Flaherty et al., 1993). Biochemical basis of the autoagglutination event has subsequently been investigated in our laboratory (Banerjee et al 2006;). Results of the subsequent studies suggest that maturing spermatozoa at the distal-corpus stage of maturity possesses an undefined D-galactose-specific lectin and its receptor that may be responsible for the autoagglutination phenomenon. The effect of different sugars on the autoagglutination phenomenon has been analyzed. Of all the sugars

62 62 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. tested only D-galactose (50 mm) served as a potent inhibitor of sperm-sperm adhesion process. The sperm autoagglutination process is dependent on Ca 2+. Asialofetuin with its terminal D-galactose residue can replace galactose for its inhibitory action (Banerjee et al 2006). Maturing spermatozoa derived from different parts of epididymis were evaluated for their efficacy to bind the labelled asialofetuin. The immature caput and mid-corpus spermatozoa had little efficacy to bind the labeled glycoprotein whereas the maturing distal corpus as well as the mature cauda spermatozoa were potentially active for binding the radioiodinated asialofetuin. The data demonstrate that galactose-specific lectin is nearly undetectable in the immature caput and mid-corpus sperm and it specifically expresses on the sperm surface at the distal corpus stage of sperm maturation. There are several reports on the occurrence of lectins on the surface of mature spermatozoa of a few species and some of these lectins have been implicated to play a vital role in fertilization by mediating sperm-ovum fusion (Goluboff et al, 1995; Yuuji et al. 1992; Tulsiani and Abou-Haila, 2001; Libia et al. 1992; Rodriguez-Martinez et al., 1998; Loeser and Tulsiani, 1999; Rodeheffer and Shur, 2004). Sugar moieties on the sperm surface have been shown to undergo a variety of speciesspecific alterations during the epididymal sperm maturation as demonstrated with commercially available exogenous lectins (Edelman and Millette, 1971; Enders et al, 1983; Sarkar et al, 1991; Mburu, 1999). However, little is known about the levels of the endogenous lectin and their receptors on the outer surface of sperm undergoing maturation during epididymal transit. For the first time a novel D-galactose-specific lectin and its receptor have been identified on the maturing caprine sperm surface (Banerjee et al 2006) and the following section delineates some of their biochemical properties and physiological significance; Assays of Lectin Activity Heamagglutination studies of the lectin were carried out using rabbit erythrocytes in a 96- well microtiter plate. The purified protein solution [50 µl (0.2 mg/ml)] was placed in the first well and serially diluted into the successive wells with phosphate-buffered saline, ph 6.9. Then, 50 µl of 4% rabbit erythrocyte suspension was added to all the wells. Hemagglutination was visualized in the plate after 30 min. of incubation at 37 C. Lectin activity also measured by sperm autoagglutination phenomenon described earlier (Banerjee et. al., 1992). Washed intact maturing spermatozoa derived from caput segments of goat epididymis were tested for their efficacy to undergo autoagglutination when dispersed in RPS medium. The standard assay medium contained 10x10 6 sperm cells in a total volume of 0.5ml RPS medium and the cell suspensions were incubated at 37 0 C for 60 minutes with gentle shaking. The cells were also inspected under a phase contrast microscope at 400x magnification for their agglutination characteristics. The cell agglutination data expressed with + signs. The symbol 4 + represents maximal cell agglutination when more than 90% of the spermatozoa were agglutinated whereas the symbol 1+ represents minimal detectable sperm agglutination. Assays of Lectin Receptor Activity Antiagglutinin action of receptors was estimated using goat distal corpus epididymal sperm autoagglutination model (Banerjee et al. 1992). Intact washed spermatozoa ( ) were incubated with and without the test substance in a total volume of 0.5 ml RPS medium at 37 C for 60 min. The degree of sperm aggregation was then assessed as described above. Antiagglutination potency of sample was expressed as its efficacy to inhibit sperm autoagglutination phenomenon. Antiagglutinin-mediated decrease of cell agglutination from

63 Purification and Characterization 63 the initial 4+ values to 2+, 1+ and 0+ values have been assumed to represent inhibition of cell agglutination to the extents of 50%, 75% and 100%, respectively. Purification of Cell Surface D-Galactose-Specific Lectin Activation of Sepharose-4B: Acid treated Sepharose-6B was prepared as described by Ersson et al. (1973) by washing the gel with cold 0.2 M-HCl and adding the acid washed gel (10ml) to 0.2 M HCl (20ml). This suspension was placed in a gently shaking water bath at 50 C for 2 hours. The gel was washed with 0.15 M-phosphate-buffered saline [composition (g/liter): NaCl, 8; Na 2 HPO 4 (anhydrous), 1.15; KCl, 0.2; KH 2 PO 4 (anhydrous), 0.21], ph 7.2, and was ready for use. Sepharose-6B affinity chromatography: For solubilizing the plasma membrane of mature spermatozoa, washed cauda sperm cells were dispersed in RPS medium containing 0.2% w/ v Triton-X-100. The suspension was kept in ice for 30 min with intermittent stirring prior to centrifugation at 12,000 g for 15 min at 4 C. Sepharose-6B possesses D-galactose residues that bind with high affinity to D-galactose-specific lectin. For isolating the D-galactosespecific lectin (Das K., Majumder G.C. and Dungdung S.R., unpublished data), the Triton X- 100 solubilised crude sperm PM was subjected to a Sepharose-6B affinity chromatography column (1.4cm X 6.4cm) previously equilibrated with RPS medium (modified Ringer s solution): 119mM NaCl, 5mM KCl, 1mM CaCl 2, 1.2mM MgSO 4, 10mM glucose, 16.3mM potassium phosphate, ph 6.9 and penicillin-g (50 units/ml) containing 0.05% Triton X-100. The column was then washed extensively with the equilibrating buffer and 0.5 M NaCl. Finally the lectin bound to the affinity column was eluted with RPS medium containing D- galactose (10mg/ml), 0.05% Triton X-100, 1 mm CaCl 2 and 0.5 M NaCl. Eluted fraction was concentrated and dialyzed extensively against potassium phosphate buffer (ph 7.5). Lectin activity of the fractions was estimated based on its agglutination action on immature (caput) spermatozoa. Purification of Cell Surface D-Galactose Specific-Lectin Receptor Activation of Sepharose-4B: Cyanogen bromide activated sepharose-4b was prepared as described by Kohn and Wilchek (1982). Sepharose-4B were washed with 30% acetone and suspended in 60% acetone. The mixture was cooled to 20 0 C and then according to the described degree of activation the required volume of cyanogen bromide solution (1M) was added to the Sepharose-4B suspension. With cooling and stirring, the corresponding volume of tri-ethylamine solution (1.5M in 60% acetone) was added drop wise over a period of 1-3 minutes. Then the reaction mixture was quickly poured into ice cold washing medium (100% Acetone: 0.1N HCl = 1:1) for about 1hour. Sepharose-4B affinity chromatography: The immature washed caput-spermatozoa were dispersed in RPS medium containing 0.2% w/v Triton X-100 for solubilizing the crude sperm plasma membrane (Banerjee et al 2006). D-galactose-specific sperm lectin (Das K., Majumder G.C. and Dungdung S.R., unpublished data) was conjugated to cyanogen bromideactivated Sephrose-4B according to Kohn and Wilchek (1982). Triton X-100 soluble caput sperm PM was subjected to a column of Sepharose 4B-D-galactose-specific lectin affinity matrix (4 cm X 1.5 cm) previously equilibrated with RPS medium. The column was then washed extensively with the equilibrated buffer and 0.5 M NaCl. Finally the lectin receptor that bound to the affinity column was eluted with RPS medium containing D-galactose (10

64 64 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. mg/ml), 0.05% Triton X-100, 1 mm CaCl 2 and 0.5 M NaCl. Prior to the assay of lectin receptor activity, eluted fraction was concentrated and dialyzed extensively against K- phosphate Buffer (ph 7.0) using the antiagglutinin assay method based on the distal corpus sperm autoagglutination model. Properties of Lectin and Its Receptor Lectin: We have purified a D-galactose specific lectin from mature cauda sperm plasma membrane by Sepharose-6B affinity chromatography (Das K., Majumder G.C. and Dungdung S.R., unpublished data).this lectin agglutinated nearly 100% of the maturing (caput) spermatozoa (Figure 48). Lectin mediated agglutinated cells were dissociated most potently by 50 mm D-galactose (Figure 49). As shown in Figure 50, the isolated lectin showed high efficacy for inhibiting the distal-corpus sperm autoagglutination event. The sperm lectin elicited maximal anti-agglutinin activity at approx. 100μg/ml level. As a regulator of cell-cell adhesion/interaction, lectin may play important role for recognizing the egg-surface receptor during fertilization. Figure 48. Effect of partially purified D-galactose-specific sperm lectin on the agglutination of the sperm cells derived from different epididymal segments. Assays were carried out without (- -) and with lectin (100 g/ml) (- -) under the standard assay conditioned. The results showed the mean S.E.M. of three experiments, P<0.01 (Reproduced from Banerjee et al., 2006). Figure 49. Dose course of D-galactose for its inhibitory action on autoagglutination of goat distalcorpus epididymal sperm. The results showed the mean SEM of three experiments, P<0.01. (Reproduced from Banerjee et al., 2006).

65 Purification and Characterization 65 Figure 50. Inhibition of distal-corpus sperm autoagglutination phenomenon on addition of different concentrations of purified sperm lectin (- -) and its receptor (- -), under the standard antiagglutinin assay conditions. The results showed the mean S.E.M. of three experiments, P<0.01. (Reproduced from Banerjee et al., 2006). Lectin receptor: Goat sperm surface receptor of D-galactose-specific lectin has been purified (Das K., Majumder G.C. and Dungdung S.R., unpublished data).the receptor has high affinity to serve as an antiagglutinin for the sperm autoagglutination model (Figure 50). As reported earlier, commercially available proteins have no appreciable antiagglutinin and motility inhibiting efficacies (Banerjee et al., 1992; Mandal et al., 2006). Immotile caput spermatozoa have undetectable level of lectin and towards the terminal maturation phase, it increases dramatically whereas reverse is true in case of the lectin receptor. Failure of preand post-distal corpus sperm to show any appreciable autoagglutination property is due to lack of lectin and its receptors, respectively on the outer surface of sperm head. Role of Lectin and Its Receptor in Sperm and Cell Biology The terminal stage of sperm maturation, i.e., induction of flagellar motility is associated with a sharp disappearance/inactivation of the lectin receptor and appearance of the lectin (Figure 51). This study demonstrates that synchronous modulation of sperm surface D- galactose-specific lectin and its receptor constitutes a novel mechanism for the initiation of sperm motility during the epididymal maturation event. The biochemical basis of this modulation of sperm outer surface lectin and its receptor is not known. It may be due to enzyme catalyzed modification caused by proteolytic breakdown, glycosylation or deglycosylation, specific release of the molecule from the cell surface or uptake from the surrounding epididymal plasma. This postulated cellular regulation (Banerjee et al 2006), is a novel mechanism for the control of the cellular functions. It appears from our results that the lectin-like molecule acquired by the mature sperm may induce sperm motility whereas the receptor may suppress the motility-mediating potential of the lectin. Our recent observation that addition of isolated sperm lectin receptor inhibits with high affinity forward motility of mature goat sperm (Das K., Majumder G.C. and Dungdung S.R., unpublished data), supports this view. As flagellar motility is essential for fertilization in vivo (Majumder et al, 1999), the lectin and receptor has the potential for the management of human/animal fertility. The galactosespecific lectin-like molecule acquired by the maturing spermatozoa are retained by the mature

66 66 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. male gametes, and this lectin may play pivotal role in fertilization as implicated by the earlier investigators mentioned above. Localization of D-galactose-specific lectin on the goat sperm head surface is constituent with this view as sperm head directly interacts with ovum during fertilization. Figure 51. Correlation of sperm levels of lectin (- -) and its receptor (- -) with autoagglutination phenomena (- -) and induction of total motility (- -) and forward motility (- -) in the maturing goat epididymal spermatozoa. The sperm levels of the lectin (- -) in maturing spermatozoa were estimated by incubating the cells with radioiodinated asialofetuin. D-galactose-specific lectin receptor levels (- -) of the maturing sperm have been expressed as percentage of sperm agglutination caused by the isolated sperm surface lectin. Spermatozoa at different stages of epididymal maturation were assessed for autoagglutination (-Ο-). Maturing sperm total motility (- -) and forward motility (- -) were estimated by a microscopic method. The data indicated the mean S.E.M. of three experiments, p<0.01. (Reproduced from Banerjee et al., 2006). OTHER MOTILITY REGULATING PROTEINS Membrane-Bound Motility Inhibitor Protein As mentioned above, plethora of sperm-surface proteins have been implicated to play important role in augmenting sperm motility. Isolated sperm plasma membrane showed high efficacy for inhibiting sperm forward motility as judged by microscopic as well as spectrophotometric motility assays. Studies from our laboratory have led to the identification of a new membrane-bound motility inhibitor protein (MIF) in caprine sperm (Dungdung and Majumder 2003). Method of isolation and some of the characteristics of this purified protein have outlined below: Assay of Motility Inhibitor Protein Motility inhibitor activities of sperm plasma membrane, its fractions and purified motility inhibitor protein were estimated by measuring sperm forward motility using the microscopic method of [Mandal et al., 2006] that has already been elaborated above. Spermatozoa (1 x 10 6 cells) were incubated with boiled EP (0.6 mg protein/ml) in the absence or presence of specified amounts of test samples at room temperature (32 C ± 1) for 5 min in a total volume

67 Purification and Characterization 67 of 0.5 ml of RPS medium. A portion of the cell suspension was then placed in the haemocytometer and the forward motile sperm and total number of sperm were counted under phase contrast microscope at 400X magnification. A unit of activity of the motility inhibitor was defined as the amount of the factor, which inhibited FM in 10% of the cells under the standard assay conditions. Forward motility inhibiting activity of spermatozoa was also estimated quantitatively by the spectrophotometric method discussed above. Purification of Motility Inhibiting Protein from Caprine Sperm Plasma Membrane 0.2% Triton X-100 -solubilised sperm plasma membrane was subjected to Sepharose-6B affinity chromatography column previously equilibrated with RPS medium. Motility inhibitor-activity that bound to the affinity column was eluted with RPS medium containing D-galactose (10 mg/ml). The resulting dialyzed active fraction was subjected to DEAEcellulose ion-exchange chromatography column previously equilibrated with 10 mm K- phosphate buffer ph 7.5 containing 0.05 % Triton X-100. After washing the column, the inhibitor protein was eluted successively with gradient elution of 0.16 M to 0.18 M K- phosphate buffer ph 7.5 containing 0.05 % Triton X-100 (Figure 52). By using these methods membrane inhibitor protein was purified to 227-fold with 40% recovery. The summery of the purification of membrane inhibitor from cauda sperm plasma membrane, has been shown in the Table 17. Fractions Table 17. Purification of membrane inhibitor protein from Goat Sperm Plasma Membrane Total activity (units) Total protein (mg/ml) Specific activity (units/mg) Recovery (%) Crude Plasma Membrane Sepharose-6B Affinity chromatography DEAE-cellulose ionexchange chromatography (Reproduced from Dungdung and Majumder 2003). Fold Purificatio n Characterization of Motility Inhibitor Protein A sperm motility inhibiting glycoprotein was purified to apparent homogeneity from goat sperm plasma membrane by using Sepharose-6B affinity chromatography and DEAEcellulose ion-exchange chromatography (Figure 52). The molecular weight of the isolated factor was estimated 100 kda by analytical HPLC. The purified inhibitor protein has the capability to inhibit completely the sperm forward motility at 140nM (14 g/ml) level (Figure 53). The sperm forward motility was totally inhibited after 2 min in presence of the inhibitor protein Study of varying ph on the activity of the inhibitor protein showed that there is no effect of ph on the activity of the factor. Motility inhibiting factor is heat stable. Bivalent metal ions do not play any role on inhibitor protein activity. Purity of the factor has been checked by analytical HPLC. Native molecular mass of membrane inhibitor is 98 kda (Figure 54). The protein has the capability to inhibit sperm motility of all the species tested (Table

68 68 Gopal C. Majumder, Kaushik Das, Sudipta Saha et al. 18). On SDS-polyacrylamide gel electrophoresis,.motility inhibitor protein gave a single band of 100 kda, indicating that the factor is a monomeric protein (Figure 55). Figure 52. DEAE-cellulose ion-exchange chromatography of them membrane inhibitor protein: MIF. Partially purified MIF obtained after Sepharose-6B affinity chromatography, was subjected to a DEAEcellulose ion-exchange column and the activity was eluted with gradient elution at 0.15 to 0.18 M potassium phosphate. (Reproducd from Dungdung and Majumder 2003). Figure 53. Dose course of action of purified membrane inhibitor protein (MIF) under the standard assay conditions. (Reproducd from Dungdung and Majumder 2003).

69 Purification and Characterization 69 Figure 54. HPLC gel filtration profile of purified membrane inhibitor protein: MIF. The purified membrane inhibitor protein was chromatographed on TSK-G2000 SW column (7.5 x 300 mm) using the Waters HPLC System. The column was previously equilibrated with the 10 mm potassium phosphate, ph 7.0 at a flow rate of 2 ml/min. Protein elution was monitored at 280 nm. The MIF activity was eluted in a single peak as a 98 kda protein as determined by the plot. The markers used as standard were: 1, apoferritin (443 kda); 2, β- amylase (200 kda); 3, alcohol dehydrogenase (150 kda); 4, BSA (66 kda); 5, carbonic anhydrase (29 kda); and 6, cytochrome C 12.4kDa). (Reproducd from Dungdung and Majumder 2003). Figure 55. SDS-PAGE of membrane inhibitor protein: MIF using 10% polyacrylamide gel. Markers used were β-galactosidase (116 kda), phosphorylase b (97 kda), BSA (66 kda), ovalbumin (45 kda) and carbonic anhydrase (29 kda). (Dungdung and Majumder 2003).