Involvement of Nifetepimine in Motility and Capacitation of Caprine Spermatozoa

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1 Columbia International Publishing American Journal of Experimental Biology (2014) Vol. 1 No. 2 pp Research Article Involvement of Nifetepimine in Motility and Capacitation of Caprine Spermatozoa Pinki Nandi 1, Bhaswati Banerjee 1, Swatilekha Ghosh 1, Arjit Chakrabarty 1, Kuladip Jana 1, and Parimal C. Sen 1 * Received 1 July 2014; Published online 11 October 2014 The author(s) Published with open access at Abstract Activation of sperm motility and capacitation are essential pre-requisite for successful fertilization. Elucidation of the biochemical mechanisms underlying these processes is central to our understanding of reproductive pathology affecting the male gamete and the development of novel approaches to fertility control. A key element of capacitation is the spontaneous increase in protein phosphorylation observed during sperm capacitation. If this protein phosphorylation event is suppressed, then sperm capacitation does not occur and the spermatozoa lose their ability to respond their physiological agonist essential for successful fertilization. The aim of our present study is to elucidate the effect of a dihydropyrimidone derivative, ethyl- 4-(3-nitro)-phenyl- 6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine) in sperm motility and capacitation using caprine model. Results show that nifetepimine significantly reduces sperm motility by modulating camp dependent protein kinase (PKA) activity and the content and/or localization of motility associated ser/thr/tyr phosphorylated proteins. Nifetepimine significantly decreases the tyrosineserine phosphorylation of proteins and changes their distribution pattern under capacitating condition. Also, the co-localization of nifetepimine with AKAP3 and PKA-RII subunit in caprine spermatozoa illuminate the mechanistic pathway by which nifetepimine modulates sperm function.in the present study we have explored how the sperm motility and capacitation are affected by nifetepimine, a synthetic dihydropyrimidone. Keywords: Caprine spermatozoa; Nifetepimine; Sperm motility; PKA; Ser/thr/tyr phosphorylation; AKAP3 1. Introduction Human and all mammalian spermatozoa are immotile when released from the testis. In order to reach the oocyte and to fertilize, they acquire the ability to actively swim during their transit through the epididymis and the female genital tract under the control of different external and intracellular factors. All these factors are important not only for the development but also for the *Corresponding parimalsen.boseinst@gmail.com; parimal@jcbose.ac.in Phone : * Division of Molecular Medicine, Bose Institute, Kolkata , Department of Chemistry, Maulana Azad Collge, Kolkata, India 61

2 maintenance of sperm motility. In mammalian sperm systems, camp has been proposed as a major factor in the initiation of flagellar movement during sperm maturation and ejaculation and in subsequent stimulation of motility during the fertilization process. Brokaw CJ (1987) has shown that the early studies on camp in sperm movement coincided with the discovery of campdependent protein kinase (camp-pka) as the primary signal transducer for camp in eukaryotes. Protein phosphorylation is a post-translational modification of proteins that allows the cell to control various cellular processes. Mature spermatozoa are highly compartmentalized, transcriptionally inactive and unable to synthesize new proteins. Therefore, the reliance of mature spermatozoa on protein phosphorylation as a means of altering their function is greater than in many other cell types. During fertilization, sperm function is regulated by the activation of intracellular signaling systems that control protein phosphorylation at Serine,Threonine and Tyrosine residues. Visconti PE and Kopf GS (1998) reported that camp-dependent protein kinase A (PKA) plays a central role in sperm function and has been studied in detail. Feliciello A et al. (2001) reported that a portion of PKA activity is tightly bound to the axoneme, possibly by binding to AKAPs, such as those associated with the central pair apparatus (AKAP240) or with the radial spokes (AKAP97) in Chlamydomonas reported by Porter ME and Sale WS (2000). Sperm capacitation is a poorly understood maturation process, which occurs in vivo in the female reproductive tract. Yanagimachi (1994) and references therein has reported that capacitation has been shown to be correlated with changes in sperm plasma membrane fluidity, intracellular ion concentrations, metabolism, and motility, but it is not clear how such changes are necessarily tied to the development of this fertilization-competent state. There are several reports (Urner and Sakkas, 2003; Leclerc et al.,1996; Galantino et al., 1997; Kalab et al., 1998; Pukazhenthi et al., 1998; Si and Okuno, 1999; Urner et al., 2001) showing that protein phosphorylation increases in spermatozoa during capacitation in a number of species including mice, humans, cattle, pigs, hamsters and cats. It has been reported (Urner and Sakkas, 2003; Sakkas et al., 2003) that Protein tyrosine phosphorylation appears to be a necessary prerequisite for a spermatozoon to fertilize an egg. Sakkas et al., 2003 revealed by Immunofluorescence study the localization of tyrosine phosphorylated proteins to the flagellum during capacitation, zona pellucida binding, oolemma binding and gamete fusion. The proportion of spermatozoa presenting phosphorylated proteins in the whole flagellum increases with the capacitation. In both mice and men, approximately 50% of spermatozoa remain non-phosphorylated after capacitation, indicating that there are subpopulations of spermatozoa that exhibit a different susceptibility to undergo tyrosine phosphorylation. Hence protein phosphorylation can be considered as the crucial event during capacitation. Ghosh et al. (2012) have reported that Nifetepimine is a dihydropyrimidone (DHPM) group of compound. According to Li and Liu (2003) in the past decades, Biginelli-type dihydropyrimidones had received a considerable amount of attention due to the interesting pharmacological properties associated with this heterocyclic scaffold. According to Morakinyo et al. (2011) Dihydropyrimidone analogues have exhibited important therapeutic and pharmacological properties as the integral 62

3 backbone of several calcium channel blockers (CCBs). With the advances in the investigation of human sperm calcium channel, according to the reports (Li and Liu, 2003; Morakinyo et al., 2011) CCBs have been proved to affect not only the shape, activation and acrosome reaction, but also the function of human sperm. This interesting fact led us to study dihydropyrimidones in detail with respect to its effect on spermatozoa function emphasizing particularly on protein phosphorylation events. The exact molecular mechanism how nifetipimine regulates sperm function is still unknown. In this study we have explored for the first time, the role/effect of nifetepimine, a synthetic dihydropyrimidone, on sperm motility and capacitation. 2. Main Research 2.1. Materials and Methods Materials Chemicals were obtained from the following sources: bovine serum albumins, dibutyryl-camp (Bt 2cAMP), 3-isobutyl-1-methylxanthine (IBMX), DCF-DA, H-89, Triton-X-100, EDTA, EGTA, betamercaptoethanol, sodium lactate, SDS, protease inhibitor cocktail, Na 3VO 4, glycerol, leupeptin, aprotinin, PMSF, PVDF membrane, kanamycin, penicillamine, TPCK, TLCK, thioglycerol, TEMED, anti-phosphorylated serine (088K4796), anti-phosphorylated threonine (047K4770) monoclonal antibody, anti-phosphorylated tyrosine (050M4827) polyclonal antibody, Anti-AKAP3 antibody (SAB ), horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, FITCconjugated goat anti-rabbit- IgG, camp assay kit (CA200) from Sigma Chemicals, USA. Rabbit monoclonal antiphosphorylated PKA substrate antibody was purchased from Cell Signaling (Danvers, MA, USA); antibodies against PKA catalytic (Cat. No. PKA-Cα, sc-903) and regulatory (Cat. No. PKA-RIIα, sc-909) subunits were obtained from Santa Cruz (USA). Sodium chloride, sodium carbonate, sodium bicarbonate, calcium chloride, heparin, sodium pyruvate, glucose, magnesium chloride, ATP, DMSO, HEPES, magnesium sulphate, potassium chloride, potassium phosphate, Tris, sucrose were purchased from Sisco Research laboratory (India). Paraformaldehyde, Tween-20, methanol, dimethylformamide, acetic acid, trichloroacetic acid were from Merck, India. Micro BCA protein assay reagent kit was purchased from Genei, Bangalore, India. PKA nonradioactive activity assay kit (V5340) was purchased from Promega Corp., Madison, WI (USA). All other chemicals used were of analytical grade. Deionized milli-q water was used throughout the experiments Collection of caprine epididymis Fresh epididymides of adult goats were obtained from the local slaughterhouse (Haji slaughter house, CK Block, Sector 1, Salt Lake, Kolkata , India) immediately after sacrifice of the animals and brought to the laboratory on ice. Sacrifice of the animals was done following local municipality s guidelines. Spermatozoa were extracted from the tissue within 2 hrs of slaughtering of the animals. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All 63

4 experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, Registration Number: 95/99/CPCSEA) Synthesis and characterization of nifetepimine Nifetepimine was synthesized from 3-nitrobenzaldehyde, ethylacetoacetate, and urea in the presence of HCl as catalyst as previously described (Ghosh et al. 2012). The crude product was crystallized from methyl alcohol to obtain the pure ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4 tetrahy-2 dropyrimidine-5 carboxylate (nifetepimine) (1.6 g, 40%; melting point C) Microscopic and CASA analysis of sperm motility In the present study, highly motile sperm populations were taken to determine the motility inhibiting effect of nifetepimine. Sperm cells were incubated with different doses (from µM) of nifetepimine. Cells were assessed for viability by Trypan blue staining and for motility inhibition with haemocytometer under microscope. Analysis was also performed using the CASA system (Version: 12, HTM-CEROS CASA System, Hamilton Thorne Research, Inc., Beverly, MA, USA) for assessing the sperm motility following incubation with appropriate concentration of nifetepimine. Briefly, a 5 ml aliquot of the prepared sperm sample was placed on a Mackler chamber. At least 200 spermatozoa were counted with CASA to evaluate the sperm motility variables including Average Path Velocity (VAP), Straight Line Velocity (VSL) and Curvilinear Velocity (VCL). The CASA settings were followed according to the manufacturer s instruction Reactive Oxygen Species (ROS) measurement in sperm cell 2',7'-dichlorodihydrofluorescein diacetate (H 2DCFDA) was used to determine the reactive oxygen species in sperm cell preincubated with nifetepimine for 45 min. Working solution of 20 mm H 2DCFDA in DMSO was aliquoted and stored in the dark at C. On the day of the experiment, sperm were mixed with H 2DCFDA (final concentrations: 1-3x 10 6 sperm/ml; 200 µm H 2DCFDA). Sperm were incubated for 60 min at 25 0 C followed by fluorescence assay in a FACS Calibur flow cytometer (BD Bioscience, Mississauga, ON) with a 488-nm excitation source. A total of 10,000 individual sperm-sized events were selected based on forward and side scatter; H 2DCFDA was detected with 530/30-nm filter. Using the statistical package of the FACS, evident sperm populations were gated and the mean fluorescence intensity and number of sperm in each population were determined Protein kinase A (PKA) activity assay To assess the effect of nifetepimine on sperm PKA activity, it was measured under normal as well as capacitating condition using a nonradioactive PKA assay kit following manufacturer s instructions. Following incubation with nifetepimine, sperm were washed twice with cold PBS and homogenized on ice in 0.5 ml PKA extraction buffer. After centrifugation at 14,000g for 5 min at 40 0 C, the supernatant was removed. The reaction system contained 5 µl PepTag PKA reaction buffer, 5 µl 64

5 PepTag A1 peptide, 5 µl camp, 1 ml peptide protection solution, and 4 ml sample homogenate, and added water to a final volume of 25 µl. The reaction was carried out for 30 min at room temperature, and stopped by boiling for 5 min. The phosphorylated peptides were separated by electrophoresis on a 0.8% agarose gel at 100 V for 15 min and visualized under UV light. The bands that appeared in the gel gave a qualitative estimate of the relative amount of the PKA activity in the samples Determination of sperm intracellular camp concentration Cyclic AMP level of spermatozoa was determined using camp Enzyme Immunoassay Kit, (EIA) Direct. The EIA Direct cyclic AMP kit is a competitive immunoassay for the quantitative determination of cyclic AMP in samples treated with 0.1 M HCl. Samples (i.e. spermatozoa, 50x10 6 cells/ml) after treatment with HCl were acetylated as acetylation of the samples increases the sensitivity of the assay. The kit uses a polyclonal antibody to bind camp in a competitive manner. Samples, alkaline phosphatase conjugate, and antibody were simultaneously incubated at room temperature in a secondary antibody coated multiwell plate. The excess reagents were then washed away and substrate p- nitrophenyl phosphate was added. After a short incubation the enzyme reaction was stopped and absorbance was recorded at 405 nm. Das et al. (2010) have reported that the intensity of the yellow colour was inversely proportional to the concentration of camp in either the standards or the samples. The measured optical density was used to calculate the concentration of camp Induction of capacitation Highly motile swim-up spermatozoa were extracted at room temperature from the cauda epididymis in Tyrodes medium (100 mm NaCl, 3.1 mm KCl, 1.5 mm MgCl 2, 0.29 mm potassium dihydrogen phosphate, 21.6 mm sodium lactate, 1 mm sodium pyruvate, 20 mm HEPES, 50 µg/ml kanamycin, ph 7.4). Control samples (non capacitated, NC) were taken immediately following swim-up procedure for processing by immunoblotting and immunofluorescence. In vitro capacitation was performed according to the reports (Therien et al.,1997; Miller et al.,1990; Parrish, 1988) by incubating the swim-up samples (2x10 8 cells/ml) in modified Tyrodes medium (above Tyrodes medium plus 2mM CaCl 2, 7mg/ml BSA and 25 mm NaHCO 3, 10μg/ml heparin ph 7.4) for 3 hr at 37 0 C in a humidified incubator with 5% CO 2 in air (capacitated, C). To test the effect of nifetepimine on capacitation, sperm were incubated in presence or absence of the compound for different time periods depending on the experimental design SDS-PAGE and immunoblotting Western blots were conducted as described by Krapf et al. (2010). Briefly, after treatment, sperm were collected by centrifugation at 7500g for 5 min, washed twice in 1 ml phosphate-buffered saline, resuspended in sperm lysis buffer, vortexed and immediately placed on a 95 0 C heating bath for 5 min. Samples were then centrifuged at 7500g for 15 min, and supernatant collected. After protein estimation following BCA protein assay method, 5% β-mercaptoethanol and 1% glycerol 65

6 were added to the supernatants, and the mixture was boiled again for 5 min. The sperm extracts, containing approximately µg of protein, were diluted in Laemmli s reducing sample buffer (62.5 mm Tris ph 6.8, 10% glycerol, 2% SDS, and 200 mm dithiothreitol), incubated at 95 C for 5 min and loaded onto a 10% polyacrylamide-bisacrylamide gels followed by electro-transferred to PVDF membranes (Millipore ) at 150 ma for 2 hr at 4 C. Membranes were treated with 5% fat-free skimmed milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (T-TBS) for anti-pkacatalytic, anti-pka-regulatory immune-detections and with 5% bovine serum albumin (Sigma) in T-TBS for anti-y-p, anti-s-p and anti-t-p immunodetections. Antibodies were diluted (1:1000) in 0.5% BSA in T-TBS unless otherwise stated. After incubation with HRP-conjugated secondary antibodies (1/10,000 in T-TBS), enhanced chemiluminescence detection was performed according to the manufacturer s instructions. For reprobing with different antibodies, PVDF membranes were stripped at 65 C for 15 min in 2% SDS, 0.74% β-mercapto-ethanol, 62.5 mm Tris, ph 6.5, and washed six times for 5 min each in T-TBS and re-probed with specific primary antibodies according to Escoffier et al., (2011). γ-actin, a protein known to be constitutively expressed in all cells, served as a loading control. For all experiments, molecular masses are expressed in kda Indirect immunofluorescence Spermatozoa, incubated with or without nifetepimine, under normal or capacitated condition was collected by centrifugation and washed thrice in PBS medium. After washing, cells were fixed for 30 min in 3.7% paraformaldehyde, washed, permeabilized with 0.25% Triton-X-100 for 15 min, washed thrice with PBS. Non-specific binding sites were blocked with 3% BSA in PBS for 1 hr at 37 0 C in a humid chamber. After blocking, cells were washed three times with PBS and incubated with specific primary antibodies for PKA-catalytic, PKA-regulatory, AKAP, tyrosine, serine and threonine phosphorylated proteins (all in 1:20 dilution) overnight at 4 0 C. These samples were treated according to the Nandi et al., (2012), briefly, washed thrice with PBS, and then incubated with FITC-conjugated secondary antibody (1:100) for 1 hr at 37 0 C followed by three 5-min washes with PBS, smear prepared on a glass slide with a drop of cell suspension and allowed to dry. 10 µl antifade mounting media (invitrogen) was added on the dried smear, covered with a coverslip and sealed with nail polish. Spermatozoa were examined by confocal microscopy (Zeiss laser scanning confocal microscope, LSM-510). To localize the binding site of nifetepimine on sperm cell, the fixed-permeabilized cells were incubated with RITC (Rhodamine isothiocyanate) conjugated nifetepimine for 1hr, washed extensively with PBS and used for smear preparation and subsequent analysis as stated above. To co-localize RITC - nifetepimine and AKAP/PKA, fixed-permeabilized cells were first incubated with the tagged compound for 1hr, washed thrice with PBS, subsequently treated with primary and secondary antibodies followed by analysis by confocal microscopy Statistical analysis Values are shown as standard error of mean (SEM) except otherwise indicated. Data were analyzed and, when appropriate, significance of the differences between mean values was determined by Student s t test. Results were considered significant at *p<

7 2.2. Results Synthesis and characterization of nifetepimine Synthesis and characterization of nifetepimine was done as reported recently from our laboratory by Ghosh et al., (2012) Motility and viability of caprine spermatozoa: The effect of nifetepimine Microscopic analysis In the present study, sperm cells were incubated with different doses (from 30µM -100µM) of nifetepimine. Cell motility was assessed with haemocytometer. The results showed that this compound at a concentration of 90µM/10 7 cells led to the maximum inhibition of ~ 67% on sperm progressive motility without producing any toxic effect (Fig.1A). Highly motile sperm populations were taken for microscopic analysis to determine the motility inhibiting effect of nifetepimine Analysis by Computer Assisted Sperm Analyser (CASA) Addition of nifetepimine to the sperm preparation caused significant decrease in sperm forward motility. Two different doses, 60 and 90 µm nifetepimine /10 7 cells were used to examine the effect on motility variables by CASA. There was approx. 29% decrease in VAP, 30% in VSL and 18% in VCL at higher concentration of nifetepimine (Fig.1B) Reactive Oxygen Species (ROS) generation in sperm cell: effect of nifetepimine 2',7'-dichlorodihydrofluorescein diacetate (H 2DCFDA) was used to determine the reactive oxygen species in sperm cell. It has been observed from the data (Fig.1C) that upon treatment with 90µM nifetepimine/10 7 cells, there was an increase (20%) in ROS generation in sperm population compared to control cells Identification of PKA regulatory and catalytic subunits in caprine spermatozoa Current knowledge indicates that the phosphorylation in sperm cells is critically controlled through modulation of PKA. So, the presence of PKA subunits and its localization in sperm cell are important in understanding their role in sperm cell activity. Immunoblot analysis of sperm proteins with affinity-purified isoform-specific antibodies detected the presence of PKA RIIα regulatory subunit and Cα catalytic subunit in caprine sperm. No significant change was observed in case of PKA-RIIα subunit expression upon treatment with nifetepimine. There was a slight decrease in protein level of PKA-Cα subunit when they were treated with 90µM nifetepimine/10 7 cells (Fig. 2A). 67

8 Fig. 1. Effect of nifetepimine on the forward motility, viability, and ROS generation of caprine spermatozoa: (A) Microscopic analysis. Spermatozoa (1x 10 7 ) were incubated with nifetepimine at two different doses (60µM and 90µM) for 60 min at room temperature (32 0 C ± 1) and forward motility of sperm was monitored. Data shown represent the mean of four experiments ± SEM. Units: nm = non-motile sperm, vm = vibrating sperm, pm = progressively motile sperm. (B) Data Shows the effect of nifetepimine on different CASA parameters. Data represents mean of three experiment ± SEM. Units: VAP (Average Path Velocity) = µm/sec, VSL (Straight Line Velocity) = µm/sec, VCL (Curvilinear Velocity) = µm/sec. (C) Effect of nifetepimine on ROS generation in sperm cells. Flow-cytometric analysis of fluorescence intensity of caprine sperm stained with H 2DCFDA following 45 min incubation with 90µM nifetepimine. Exposure to the compound increased sperm H 2O 2 concentration upto 20%. Values are means of DCF intensity ± SEM (n =4), * p < Localization of PKA catalytic subunit in caprine spermatozoa and effect of nifetepimine The intracellular distribution of PKA catalytic subunit was studied with intact caprine spermatozoa fixed with formalin, permeabilized with Triton X-100. When immunostained with anti PKAα- 68

9 catalytic subunit antibody, the midpiece exhibited a bright fluorescence. Fluorescence was also observed at principal piece of sperm tail (Fig.2B, topmost panel). Here the staining was restricted to the flagellar segment, and the head was negative. The flagellum exhibited a characteristic gradient of staining intensity, with the midpiece being the most brightly stained and a gradual decline of fluorescence intensity was noted proceeding from the midpiece to the end of the flagellum. Control spermatozoa immunostained with non-immune rabbit IgG were completely negative (Fig.2B, lowermost panel). When nifetepimine treated cells were similarly immunostained with the same antibody, the distribution of fluorescence was same as control cells, but intensity was very poor (Fig.2B, 3 rd panel). Cells treated as solvent control exhibited a similar pattern of fluorescence as the control one (Fig.2B, 2 nd panel). Fig. 2. Expression, localization and activity of PKA in sperm cell: Effect of nifetepimine. (A) Approximately 50 µg protein lysate from control, solvent control and nifetepimine treated 69

10 caudal sperm were separated by SDS-gel electrophoresis and subjected to western blot analysis. The antibody dilutions used were (A) PKA-RIIα (1:1000) and PKA-Cα (1:1000). Actin was used as control for equal loading. ECL followed by incubation of the blots with horseradish peroxidase-conjugated anti-rabbit second antibody. (B,C) Matched phase contrast (gray background) and fluorescence (black background) photomicrographs of formalin-fixed, Triton-permeabilized spermatozoa immunostained with antibodies to the Cα/RII-α subunits of PKA and with non-immune rabbit IgG. Control spermatozoa stained with PKA-Cα exhibit specific staining of the flagellum with intensely stained midpiece, and the lesser staining intensity of the principal piece (pp) segments. Nifetepimine treatment maintained the pattern of fluorescence but intensity decreased significantly in both cases. Sperm immunostained with control IgG exhibited no fluorescence in any case. Bar=10 µm in each photomicrograph Localization of PKA regulatory subunit in caprine spermatozoa and the effect of nifetepimine To identify the intracellular distribution of the regulatory subunit of PKA, intact spermatozoa were fixed with formalin, permeabilized with Triton X-100, and then immunostained with a type-specific R-subunit antibody. Spermatozoa immunostained with anti-riiα exhibited fluorescence at midpiece (Fig.2C, topmost panel). In contrast to PKACα, which exhibited fluorescence at midpiece and principal piece of sperm tail, the staining was restricted to midpiece only. Control spermatozoa immunostained with non-immune rabbit IgG were completely negative (Fig.2C, lowermost panel). When nifetepimine treated cells were similarly immunostained with the same antibody, the distribution of fluorescence was same as control cells, but intensity was relatively low (Fig.2C, 3 rd panel). Cells treated as a solvent control exhibited similar pattern of fluorescence as the control one (Fig.2C, 2 nd panel) Protein serine, threonine and tyrosine phosphorylation: effect of nifetepimine Pharmacological and physiological evidence indicate that protein phosphorylation is involved in the initiation and regulation of flagellar motility; the presence and anchoring of several kinases and phosphatases in the axoneme also suggest a tight control in the regulation of motility. When nifetepimine-treated sperm cells were analysed by Western blot for protein phosphorylation level, a marked decrease in tyrosine phosphorylation of proteins in the range kda was observed compared to the control (Fig.3A). Decrease was also observed in serine phosphorylation of a protein having molecular weight about 48 kda (Fig.3B). No significant change in threonine phosphorylation of any protein was observed when compared with the control (result not shown). 70

11 Fig. 3. Expression and localization of proteins phosphorylated at ser/tyr residues in caprine spermatozoa: effect of nifetepimine. Immunoblot analysis of the effect of nifetepimine on caprine sperm protein phosphorylation in tyrosine (A) and serine (B) residues. Sperm were obtained by swim-up, incubated with nifetepimine for 1 hr at 37 0 C under 100% 71

12 humidity and 5% CO 2 condition. Proteins extracted from 10 7 spermatozoa were analyzed by western blotting. (C, D) Indirect immunofluorescence localization of phospho-tyrosine and serine proteins in control, solvent control, nifetepimine treated caprine spermatozoa using monoclonal anti-phosphotyro-sine/phosphoserine antibody and FITC-conjugated goat anti-rabbit IgG. The arrows indicate different domains with specific phosphorylation. Pink arrow = middle-piece; yellow arrow = principal-piece; blue arrow = equatorial region; Bar=10 µm in each photomicrograph. The experiments were performed three times and representative images are shown Localization of serine, threonine and tyrosine phosphorylated proteins in caprine spermatozoa: effect of nifetepimine The localization of tyrosine-, serine- and threonine phosphorylated proteins and their modifications according to the functional state of caprine spermatozoa were analyzed by indirect immunofluorescence and confocal microscopy. Phosphoresidues were associated with specific sperm domains, and we were able to distinguish different sperm subpopulations according to their intracellular distribution (Figs. 3,4). Less than 10% of cells were unstained for all three residues and no significant differences were found between different samples. These results were highly specific because samples processed with the secondary antibody only showed no signaling (data not shown) and replacing the antiserum with pre-immune serum abolished protein detection. In control samples, tyrosine phosphoproteins were located predominantly in the midpiece segment, either with or without a faint signal in the tail. Nifetepimine led to a decrease in fluorescence intensity at midpiece segment with complete absence of fluorescence in the tail part (Fig.3C). Distribution of proteins phosphorylated at serine and/or threonine residues was similarly analyzed. Before incubation, phosphoserine proteins were mainly distributed at the midpiece segment, also in whole flagellum, though a weak fluorescene was observed in equatorial region. Nifetepimine treatment led to a change in the intensity of flourescence at midpiece with total diminish of fluorescence at flagellum and equatorial segment (Fig.3D). When antiphosphothreonine antibody was used, a significant number of the cells (43%) in control sample showed fluorescence at the midpiece and equatorial regions. Approximately, 28% of the cells showed positive staining at midpiece as well as epical region. Another 28% cells were stained at midpiece and principal piece of the tail. Once again, the incubation with nifetepimine induced threonine phosphorylation only in the midpiece (Fig. 4). 72

13 Fig. 4. Localization of proteins phosphorylated at threonine residues in caprine spermatozoa: effect of nifetepimine. Indirect immunofluorescence localization of phosphothreonine proteins in control, solvent control, nifetepimine treated, and pre-immune sera treated caprine spermatozoa using monoclonal anti-phosphothreonine antibody and FITC-conjugated goat anti-mouse IgG. The arrows indicate different domains with specific phosphorylation. Pink arrow = Middle-piece; yellow arrow = principal-piece; blue arrow = equatorial region; white arrow = apical segment. Each experiment was repeated four times Determining the binding site for nifetepimine in caprine spermatozoa In order to localize the binding site of nifetepimine on sperm cell, the fixed-permeabilized cells were incubated with RITC-tagged nifetepimine. Cells were viewed under a confocal microscope 73

14 following smear preparation. Results showed that the compound binds primarily to the midpiece and principal piece (Fig.5A). Similar results were obtained when live cells were used for analysis. Fig. 5. Co-localization of RITC-nifetepimine and AKAP, RITC-nifetepimine and PKA in caprine spermatozoa. (A) localization of the binding site of nifetepimine on the fixed-permeabilized sperm cells using RITC-tagged nifetepimine. (B) Localization of AKAP3 on the fixedpermeabilized cells shows it to be present in the midpiece, principal piece of the flagellum and in the anterior, equatorial segments of the acrosome. Staining of the anterior acrosomal segment is less pronounced than other regions. (C) Western blot analysis with whole cell lysate confirmed the presence of AKAP3 in caprine sperm. (D) Double labeling experiments with anti-akap3 antibody and RITC-tagged nifetepimine showing the co-localization of 74

15 AKAP3 and nifetepimine along the length of the mid and principal piece where the AKAP3 signal (green) and the nifetepimine signal (red) merge into small aggregates of orangeyellow staining. (E) Double labeling experiment showing PKA-RIIα subunit and bound nifetepimine to co-localize in mid piece and principal piece of caprine spermatozoa. Bar=10 µm in each photomicrograph. All experiments were repeated thrice Presence and localization of A kinase anchor protein 3 (AKAP3) in caprine spermatozoa Immunofluorescence staining experiments were performed to determine the subcellular locations of AKAP3 (previously known as AKAP110). Using polyclonal antibody, AKAP3 staining was detected in the midpiece and principal piece of the flagellum and in the anterior and equatorial segments of the acrosomal region of goat caudal spermatozoa (Fig. 5B). Staining of the anterior acrosomal segment was less pronounced than other regions. Western blot analysis with whole cell lysate confirmed its presence in these cells (Fig. 5C) Co-localization of RITC-nifetepimine and AKAP3 in caprine spermatozoa To determine whether or not AKAP3 and nifetepimine co-localize in flagella, double labeling experiments with rabbit anti-akap3 antibody and RITC-tagged nifetepimine were performed. As described above, nifetepimine was found in the mid and principal piece of caprine spermatozoa (Fig. 5D, upper panel, red fluorescence), AKAP3 was found in the principal piece, mid piece and in scattered patches throughout the head region (Fig. 5D, middle panel, green fluorescence). Figure 5D, lower panel demonstrated the co-localization of AKAP3 and nifetepimine along the length of the mid and principal piece where the AKAP3 signal (green) and the nifetepimine signal (red) merge into small aggregates of orange-yellow staining Co-localization of nifetepimine and PKA-RIIα subunit in caprine spermatozoa Nifetepimine and PKA-RIIα subunit were found to co-localize in mid piece and principal piece of caprine spermatozoa (Fig.5E). As the above experiments revealed co-localization of nifetepimine with AKAP3 and PKA-RIIα subunit, the effect of nifetepimine on sperm motility and phosphorylation status may be mediated through these two molecules Phosphorylation status of sperm cells during capacitation: The effect of nifetepimine At the heart of the capacitation process lies a spontaneous increase in protein phosphorylation that appears to be an absolute precondition for the attainment of a capacitated state according to various reports (Visconti et al.,1995; Leyton and Saling, 1989; Aitken et al.,1998). In order to determine whether nifetepimine has any effect on this important capacitation associated phenomena, cells were incubated with the compound for 3 hr under capacitating condition. The localization of tyrosine-serine- and threonine phosphorylated proteins and their modifications (if any) according to the experimental condition were analyzed by indirect 75

16 immunofluorescence and confocal microscopy. Phospho-residues were associated with specific sperm domains, and we were able to distinguish different sperm subpopulations according to their intracellular distribution (Fig. 6). Approximately 8 % of the cells were unstained for all three residues and no significant differences were found between samples. These results were highly specific because samples processed with the secondary antibody only showed no signaling in any case (results not shown). It has already been shown (Fig. 3) that in non-capacitated sperm, tyrosine phosphoproteins are located predominantly in midpiece in majority of the cell. In vitro capacitation promoted phosphorylation in whole flagellum in majority of the cells, with differences in intensity from the anterior midpiece and principal piece to the whole flagellum, midpiece being the most brightly fluorescent. Nifetepimine treatment resulted in a weakly fluorescent midpiece with complete absence of signal in any other region of sperm cell. Treating cell with DMSO (as solvent control) showed a similar fluorescence pattern (Fig. 6A) as the control one. Figure 6D shows the phosphoprotein profile of the capacitated control, solvent control and nifetepimine treated samples. The western blot confirmed that treatment with nifetepimine led to a significant decrease in the content of some bands (molecular weight ~ 44 and 72 kda) phosphorylated on tyrosine. The distribution of proteins phosphorylated at serine and/or threonine residues were similarly analyzed. Under capacitating condition, phosphoserine proteins were mainly distributed throughout the flagellum giving strong signals in control and solvent control samples. Nifetepimine treatment resulted in an overall diminished fluorescence (Fig. 6B). Regarding phosphorylation at serine residues, a single molecular weight region was distinguished in western blots of control samples (Fig. 6E). Two bands in the molecular mass region kda was found to be heavily phosphorylated at serine residue under capacitating condition. Nifetepimine treatment caused a slight decrease in band intensity as confirmed by western blot analysis. When antiphosphothreonine antibody was used, in control capacitated samples, ~ 48% of sperm showed fluorescence only at midpiece and anterior principal piece whereas ~ 18 % of cells showed labeling at equatorial ridge along with midpiece and principal piece. Another ~ 17% of cells exhibited signal at the anterior acrosomal region in addition to that in flagellum (Fig. 6C). Treatment with nifetepimine resulted in diminished signal at anterior acrosomal region with fluorescence only at midpiece and principal piece (~ 53 % ), a slight increase in the number of cells showing labeling at midpiece, principal piece and equatorial region (~26.6%). Only ~ 7% of cells showed labeling at anterior acrosomal region along with flagellum. In this case we could not detect any change in fluorescence intensity but there is a change in distribution pattern of threonine phosphorylated protein (Fig 6C). This was verified by western blot analysis where no change in band pattern and/or intensity upon treatment with nifetepimine was observed (result not shown). 76

17 Fig. 6. Phosphorylation status of sperm cells during capacitation: effect of nifetepimine. (A,B,C) Indirect immunofluorescence localization of tyrosine, serine- and threonine phosphorylated proteins and their modifications by nifetepimine under capacitating condition were analyzed. Phospho-residues are found to be associated with specific sperm domains in each case. Nifetepimine treatment caused decrease in intensity of fluorescence corresponding to protein phosphorylation. (D,E) Phosphotyrosine and Phosphoserine profile of the capacitated control, solvent control and nifetepimine treated samples as obtained by western blot analysis.all experiments were repeated thrice PKA activity assay Motility expression appears to depend on the net level of phosphorylation of certain specific proteins, under the positive influence of protein kinases (especially protein kinase A) and the 77

18 negative influence of protein phosphatases. Current knowledge indicates that the system is critically controlled through modulation of PKA activity. This modulation is achieved by alterations in the intracellular concentration of the major sperm messenger molecule, cyclic Adenosine MonoPhosphate (camp). Keeping in mind the importance of PKA activity in sperm cell, we measured this in the nifetepimine treated sperm cell. Sperm PKA activity was measured using a nonradioactive PKA assay kit (V5340, Promega Corp., Madison, WI, USA) following manufacturer s instruction. Result thus obtained, revealed a significant decrease in PKA activity in spermatozoa treated with nifetepimine when compared to control cells (Fig.7B). Fig. 7. Involvement of protein kinase A in regulation of protein phosphorylation by nifetepimine. (A) Modulation of PKA activity by nifetepimine was investigated by incubating washed spermatozoa with the compound at two different time periods. Nifetepimine significantly inhibited the activity of sperm protein kinase A. (B) PKA activity in caprine sperm was found to be decreased by nifetepimine after incubation under appropriate conditions for 1 hr when compared with the control. (C) Western blot using an anti-phospho-pka -substrate antibody that recognizes the consensus PKA-phosphorylated motif ((K/R)(K/R)X(S*/T*) showed that nifetepimine treatment led to a significant decrease in phosphorylated PKA substrate level. (D) camp levels in caprine sperm was found to be decreased by 78

19 nifetepimine following incubation in appropriate conditions for 1 hr compared to control. Here, percentage of bound form is inversely related to camp concentration (* p<0.05) Intracellular camp concentration and effect of nifetepimine For decades, cyclic AMP (camp) has been reported to play an important role in both the initiation and maintenance of sperm motility. Keeping in mind the important role of camp in sperm motility and other function, intracellular concentration of camp has been measured using camp Enzyme Immunoassay Kit, Direct obtained from Sigma-aldrich. Result (Fig.7D) obtained from the assay showed that nifetepimine reduced intracellular camp level compared to the control Involvement of protein kinase A in regulation of protein phosphorylation by nifetepimine The involvement of protein kinase A in the capacitation related regulation of protein phosphorylation by nifetepimine was investigated by incubating washed spermatozoa with the compound for two different time periods at 37 C in 5% CO 2. Results obtained from PKA activity assay revealed that following 3 hr incubation under capacitating condition, nifetepimine significantly inhibited the activity of sperm protein kinase A (Fig. 7A). PKA inhibition was also studied through the analysis of specific substrate phosphorylation by western blot using an antiphospho-pkasubstrate antibody that recognizes the consensus PKA-phosphorylated motif ((K/R)(K/R)X(S*/T*). Sperm incubated in the absence of nifetepimine showed significant phosphorylation of PKA substrates at molecular weight region kda (Fig.7C, lane 1,2). Nifetepimine treatment caused a significant decrease in phosphorylated PKA substrate level (Fig.7C, Lane 3,4) 3. Discussion The present study, for the first time, characterizes nifetepimine, a dihydropyrimidone, with respect to its involvement in regulating sperm functions. This compound when incubated with highly motile sperm population exerts inhibition of sperm progressive motility in a dose dependent manner without any toxic effect. This motility inhibiting ability is confirmed with CASA which shows decrease in the motility parameters VAP, VSL and VCL. There is also increase in ROS generation in sperm population compared to control upon treatment with nifetepimine which may play a role in motility inhibition (Fig. 1). But study of apoptosis have shown no positive results (data not shown). The role of cyclic AMP (camp) in regulating sperm kinetic activity and metabolism is well established by Vijayaraghavan et al., (1997). The major downstream effectors of camp in germ cells appear to be PKA. So determining the subcellular distribution of PKA and biochemical characterization of its anchoring proteins is important in understanding the involvement of camp in spermatozoa. The present report has been substantiated by western blot and 79

20 immunocytochemical analysis, the intra-sperm distribution of PKA regulatory and catalytic subunit and the effect of nifetepimine treatment. Immunoblot analysis of sperm proteins with affinitypurified isoform-specific antibodies detects the presence of PKA RIIα regulatory subunit and Cα catalytic subunit in caprine sperm (Figure 2). No significant change is observed in case of PKA RIIα subunit expression although a slight decrease in protein level of PKA Cα catalytic subunit is evident when they are treated with nifetepimine at a specific dose. When immunostained with anti-pka- RIIα antibody, spermatozoa exhibits fluorescence at midpiece of flagellum (Figure 2). This clearly suggests that the PKA-RIIα subunit of PKA functions in flagellar signaling pathways. Nifetepimine treated cells similarly immunostained with the same antibody, shows the similar fluorescence distribution as in control cells, but with poor intensity. Intracellular localization of PKA catalytic subunit with anti PKAα-catalytic subunit antibody reveals its presence at the midpiece and principal piece, here the flagellum exhibits a characteristic gradient of staining intensity, with the midpiece being the most brightly stained and a gradual decline of fluorescence intensity is noted proceeding from the midpiece to the end of the flagellum. Nifetepimine treatment leads to a decrease in fluorescence intensity maintaining the pattern of fluorescence same as the control one. Although the significance of this type of diminished fluorescence upon treatment with nifetepimine is not clear at present, however the study surely indicates that nifetepimine exerts its effect on sperm cell via PKA mediated pathways. Role of PKA and its modulation by nifetepimine in sperm function have been confirmed by activity assay with PKA kit which shows a prominent decrease of PKA activity when challenged with nifetepimine (Figure 7). In order to elucidate the molecular mechanism by which nifetepimine leads to inhibition of sperm motility, we investigated the two main intracellular signaling pathways involved in regulation of sperm motility, namely camp and tyrosine-serine-threonine phosphorylation of sperm proteins (Leclerc et al., 1996; Si and Okuno, 1999; Vijayaraghavan et al., 1997; Luconi et al., 2003; Burton and McKnight, 2007). Our results support the fact that PKA modulation occurs by alterations in the intracellular concentration of camp(figure 7D). The exact mechanism of how camp acts in spermatozoa is not fully known yet. On the basis of the knowledge of camp action in somatic cells, it is believed that changes in serine/threonine phosphorylation, and also protein tyrosine phosphorylation by PKA may mediate the action of this cyclic nucleotide in spermatozoa. When nifetepimine-treated sperm cells are analysed by western blot for protein phosphorylation level, a marked decrease in tyrosine phosphorylation (molecular weight region kda) as well as in serine phosphorylation (molecular weight region 48 kda) but not in threonine phosphorylation (results not shown) have been observed. When we localize the tyrosine-, serineand threonine phosphorylated proteins and characterize their modifications (according to the experimental condition) by confocal microscopy, we find that phosphoresidues are associated with specific sperm domains. In caprine spermatozoa, tyrosine phosphoproteins are located predominantly at the midpiece segment, either without or with a faint signal in the tail. Nifetepimine induces a decrease in fluorescence intensity at midpiece segment with complete absence of fluorescence in the tail part. Phosphoserine proteins are mainly distributed at the midpiece segment, also in whole flagellum, with a weak fluorescene observed at equatorial region. Nifetepimine treatment promotes a change in the intensity of flourescence at midpiece with total 80

21 diminish of fluorescence at flagellum and equatorial segment. A significant number of the cells (~43%) in control sample show fluorescence for threonine-phosphoprotein at the midpiece and equatorial regions. Approximately 28% of the cells show positive staining at midpiece as well as epical region. Another 28% cells are stained at midpiece and principal piece of the tail. Nifetepimine induces a change in the pattern of threonine phosphorylation and phosphoproteins are found to be present only in the midpiece segment. All the above data cumulatively suggest that tyrosine, serine and threonine phosphorylated proteins present in sperm flagellum are involved in sperm motility. Nifetepimine treatment induces a change, either in distribution pattern or in the intensity of these proteins which could be one of the reasons for the inhibition of sperm motility. In order to determine the actual binding site of nifetepimine in spermatozoa, we perform immunolabeling experiment with RITC-nifetepimine (Figure 5), the result reveals that nifetepimine specifically binds to midpiece and principal piece of spermatozoa. The PKA holoenzyme, comprising two catalytic (C) and two regulatory (R) subunits, is activated when camp levels rise following stimulation of Gs protein-coupled receptors and adenylyl cyclase. The phosphorylation of specific substrates by the C subunit of PKA is regulated in part by the subcellular localization of the PKA holoenzyme through the binding of its dimerized R subunits to the scaffolding A kinase-anchoring proteins (AKAPs). AKAPs also interact with other signaling proteins, including other kinases, phosphodiesterases, and protein phosphatases to form a signaling complex that imparts spatial localization and controls the timing and substrate specificity of protein phosphorylation. Keeping in mind the importance of AKAP in regulating PKA function, we localize AKAP3 in sperm cell, it also shows staining in the mid piece and principal piece of the flagellum along with anterior and equatorial segments of the acrosomal region. Immunofluorescent double labeling experiments of RITC-nifetepimine and AKAP3 demonstrated that sub-populations of these proteins co-localize in the mid piece and principal piece, and occur independently of each other as well. Again, RITCnifetepimine and PKA-RII subunit are also found to colocalize in mid piece and anterior principal piece of caprine spermatozoa. Results thus obtained strongly suggest that effects of nifetepimine on spermatozoa may involve the modulation of the AKAP and/or PKA-RII, subsequent alteration in PKA activity and phosphorylation status of a number of proteins actively engaged in sperm functions. As the present study suggests nifetepimine to be a potential regulator of sperm motility and phosphorylation events, we were curious to know whether it also affects the capacitation associated protein phosphorylation. Results from confocal microscopic study (Figure 6) reveal that in vitro capacitation in spermatozoa promote phosphorylation in whole flagellum in majority of the cells which become confined to only mid piece upon nifetepimine treatment. Nifetepimine also reduces the intensity of fluorescence clearly indicating a significant decrease in tyrosine phosphorylation in some proteins which is again confirmed by western blot analysis. Proteins of two different molecular mass region (~42 kda and ~72 kda) are found to be significantly modulated. When serine and threonine phosphorylation patterns are similarly analyzed in absence and presence of nifetepimine, similar result for serine as that for tyrosine are obtained but threonine phosphorylated proteins only redistribute themselves as a result of nifetepimine treatment. 81