CHAPTER 1 GENERAL INTRODUCTION

Transcription

1 CHAPTER 1 GENERAL INTRODUCTION

2 1 1.1 Introduction Antoni van Leeuwenhoek in 1677 was the first to observe live human spermatozoa through a primitive microscope and wrote "-----man comes not from an egg but from an animalcule in the masculine seed'. This observation that a human being originates from the spermatozoon (which is only partly true) by Antoni van Leeuwenhoek was accepted till the 19th century when it was demonstrated that the spermatozoon is the male gamete and fuses with the oocyte, the female gamete, to form the zygote. The spermatozoon is Figure 1.1 A schematic representation of the structure of a spermatozoon. Different parts of the spermatozoon namely the head (A), neck (B) mid piece (C). principal piece (D) and end piece (E) are labeled. The head of the spermatozoon is further distinguished in to the acrosome (1) the nucleus (2) and the centriole (3). produced in the male gonad, the testis, and subsequently matures in the extra-testicular organ, the epididymis, during which the immature and immotile testicular spermatozoon is transformed into a mature and motile spermatozoon. The mature spermatozoon is a highly differentiated and a polarized cell consisting of a head, midpiece, principal piece

3 2 and end piece (Figure 1.1 ). These different regions are destined to carry out specific functions. The anterior most region of the spermatozoon is the head, which is the seat of the paternal haploid genetic material confined to a compact nucleus. Above the nucleus is present the Golgi derived membrane bound vesicle, the acrosome, which is unique to spermatozoa, and contains hydrolytic enzymes needed to dissolve the extracellular matrix of egg so as to facilitate fusion with the egg. The midpiece of the spermatozoon houses an array of mitochondria, which generate high-energy phosphates by oxidative respiration, needed for various functions of the spermatozoon. The principal piece of the spermatozoon along with the end piece together constitute the tail piece of the spermatozoon and possess all the structural elements required to make it a motility apparatus to facilitate sperm motility. The principal piece of the spermatozoon is also the seat of various major enzymes of glycolysis involved in generation of energy required for motility and hyperactivated motion. Other unique features of a spermatozoon are that it is transcriptionally inactivate, has sperm-specific genes and possesses unique signal transduction pathways such a camp-dependent protein tyrosine phosphorylation, to accomplish its physiological functions such as motility, hyperactivation and acrosome reaction. Premkumar and Bhargava (1972) were the first to demonstrate that transcription and translation in spermatozoa is confined to the sperm mitochondria. But, a recent study demonstrates, nuclear encoded protein translation in spermatozoa (Gur and Breitbart, 2006).

4 3 1.2 Capacitation The ejaculated mammalian spermatozoa are incapable of fertilizing the oocyte immediately following ejaculation. But, following a finite period of residence in the female reproductive tract, the spermatozoa acquire the capacity to fertilise the oocyte. This residence time that confers on the spermatozoon the ability to fertilise the oocyte and its importance was first discovered independently but simultaneously by Chang (1951; 1952) and Austin (1951) and was termed as "capacitation". Chang (1951; 1952) and Austin ( 1951) also emphasized that capacitation is a necessary event in the life cycle of the male gamete. The importance of capacitation also stems from the fact that a block in capacitation could cause male infertility. During capacitation, spermatozoa undergo two distinct physiological changes namely, hyperactivation, during which spermatozoa gain the momentum to proceed towards the oocyte and acrosome reaction, which facilitates the penetration of the oocyte. Concomitant with these changes, at the molecular level, activation of signal transduction cascade leading to increase in tyrosine phosphorylation of proteins is observed in mammalian spermatozoa (Visconti and Kopf, 1998; Lefievre et a/., 2002; Kulanand and Shivaji, 2001; Jha eta/., 2003; Shivaji eta/., 2006). Presently, capacitation is viewed as a culmination of molecular, cellular and physiological changes that occur in the spermatozoa. But as yet the molecular basis of capacitation is still not clearly elucidated. One of the major difficulties in trying to unravel the molecular basis of capacitation is the fact that this takes place in the female reproductive tract, either in the oviduct or in the vicinity of the egg, which is not easily amenable and also due to the fact that very low numbers of spermatozoa reach the site of capacitation, thus making biochemical measurements very difficult. Once the spermatozoa are deposited in the oviduct, lectins on the sperm surface cross link with specific oligosaccharide present on the mucosal epithelium in oviduct (Demott et a/., 1995; Suarez et a/., 1998) so as to initiate and facilitate the capacitation process.

5 4 Furthermore, because of ethical reasons there are experimental limitations for these studies in vivo. The current knowledge on capacitation is based on in vitro studies using chemically defined media to capacitate mammalian spermatozoa (Dow and Bavister, 1989; Bavister and Yanagimachi, 1977) and simultaneously monitor physiological changes such as hyperactivation motility (Mitra and Shivaji, 2004; Kulanand and Shivaji, 2001; Yanagimachi, 1994) and acrosome reaction (Ward and Storey, 1984; Aitken eta/., 1995; Meizel and Turner, 1991; Curry and Watson, 1995; Mitra and Shivaji, 2004) and biochemical changes such as increase in membrane fluidity (Go and Wolf, 1985; Cross, 1998; Davis et at., 1980), activation of transbilayer signaling events (Go and Wolf, 1985; Visconti et a/., 1998; Flesch et a/., 2001 ), changes in redox status of spermatozoa leading to generation of reactive oxygen species (ROS) (Aitken eta!., 1995; O'Fiaherty eta/., 2005; de Lamirande and Gagnon, 1992) and phosphorylation of proteins (leyton and Saling, 1989; Visconti eta/., 1995; Mitra and Shivaji, 2005). 1.3 Hallmarks of capacitation Hyperactivation Mammalian spermatozoa during the process of capacitation show a distinct change in motility, which could be visually characterized under the microscope as a change in the type of motility pattern and the velocity of movement. Yanagimachi (1969) was the first to report hyperactivated movement of spermatozoa in hamster at the site of fertilization. Subsequent studies clearly established that in most species following hyperactivation the spermatozoa exhibit a change in motility pattern form a progressive type of motility (planar motility pattern) to a non progressive type of motility (circular, wriggling, whiplash type of patterns) (Figure 1.2). Hyperactivation is also considered to be critical to the success of fertilization, as it enhances the ability of the spermatozoa to traverse viscoelastic zones in the female reproductive tract more effectively than non hyperactivated ones and to penetrate mucous substances of the cumulus oophorous and

6 5 zona pellucida of the oocyte (Suarez and Dai, 1992; Suarez et a/., 1991 ). Therefore, there is a need to be able to develop methods or criteria to be able to discriminate objectively hyperactivated from non-hyperactivated spermatozoa. Prior to the advent of Computer Aided semen analyzer (CASA) hyperactivation was analysed subjectively and it was not possible to quantitate the velocity parameters of the spermatozoa. Now, it is possible to acquire atleast seven parameters using the CASA and thus one could identify the particular motility pattern that occurs during hyperactivation and also define the same with respect to the seven parameters. The parameters that are normally acquired are VCL (curvilinear velocity which refers to the total distance that the sperm travels per unit time), VSL (straight line velocity which refers to the straight-line distance between the first and last points of the trajectory and gives the net space gain in unit time), VAP (average path velocity which refers to the distance traveled by the spermatozoon in unit time), ALH (amplitude of lateral head displacement which refers to the width of the lateral movement of sperm head), BCF (beat cross frequency which refers to number of times the sperm head crosses the direction of movement), LIN (linearity which is VSLNCL) and STR (straightness which is VSLNAP) These parameters also differentiate the movement of a capacitated spermatozoon from a non capacitated spermatozoon. Thus, based on these parameters it is now possible to objectively discriminate hyperactivated from non-hyperactivated spermatozoa (Kulanand and Shivaji, 2001; David et a/., 1981; Neill and Olds-Ciarke, 1987; Mortimer and Swan, 1995; Cancel eta/., 2000).

7 6 A c Figure 1.2 Tracing of motility pattern of a single hamster caudal spermatozoon. Motility pattern of hamster caudal spermatozoon showing planar type of motility pattern characteristic of a non hyperactivated spermatozoon and a hyperactivated spermatozoon showing circular (B) helical, (C) hatchet (D) and wriggling (E) motility pattern (Shivaji eta/., 1995). Biochemical events during hyperactivation Stauss et a/., (1995) demonstrated that inhibition of hyperactivation could obliterate the ability of hamster spermatozoa to penetrate the zona pellucida in vitro thus emphasizing the importance of this process for successful fertilization. Thus attempts have been made to understand sperm hyperactivation which is an energy driven process utilizing A TP derived from glycolysis as the energy source as observed in rat (Bone et

8 7 PIP;o -..._ / DAG.,. ' \ 1? l 1 '? ', (t-,,,, ' {.t.) HyperactJva on ATP ' AT P cal P ~ I I, KA HCO I ' sac / A. p,: ADP --. '?" l PhosphoryiEHJon,. I [ Flog llr:~r entmg ~/ Figure 1.3 Hypothetical model for molecular mechanisms regulating hyperactivation during capacitation (Suarez and Ho, 2003). Upon signal stimulation, pathways leading to increases in Ca2+ and camp are turned on. Unknown physiological signals activate phospholipase C (PLC) through a heterotrimeric G protein (Gq/11 )-coupled receptor (R1) and produce IP3. Binding of IP3 to IP3 receptors (IP3R) causes an increase in cytoplasmic Ca2+. Activation of the membrane-associated adenylyl cyclase (AC) through high cytoplasmic Ca2+ and possibly G proteins (G?) and membrane potential increases intracellular camp. Bicarbonate may also cause an increase in camp by activating the soluble form of adenylyl cyclase (sac) directly. The increased camp can bind to cyclic nucleotide-gated channels (CNG) to induce Ca2+ influx. Increased camp activates protein kinase A (PKA) to phosphorylate axonemal or fibrous sheath proteins and results in flagellar beating. High cytoplasmic Ca2+ and Ca2+calmodulin complex are responsible for asymmetrical bending of flagella that is characteristic of hyperactivation.

9 8 a/., 2000) and human spermatozoa (William and Ford, 2001 ). Suarez and Ho, (2003) proposed a molecular model for sperm hyperactivation (Figure 1.3) based on the observations that calcium, camp and bicarbonate are required for hyperactivated motility but this requirement varied depending on the animal species. Intracellular calcium has been demonstrated to be essential for hyperactivation of hamster, mouse and human spermatozoa (Cooper and Woolley, 1982; Kula nand and Shivaji, 2001 ). An increase in intracellular levels of calcium was also observed in hyperactivated mouse spermatozoa (Suarez and Osman, 1987; Suarez and Dai, 1995). Ren et a/., (2001) disrupted CatSper1, a sperm specific calcium channel which is localized in the sperm flagellum (Quill et a/., 2001, 2003; Carlson et a/., 2003, 2005; Ren et a/., 2001 ), in male and female mice and found that CatSper1_,_ males were infertile but not females. However, noticeably absent in CatSper1-l- spermatozoa was a hyperactivated form of motility, normally seen at the time of fertilization (Quill eta/., 2003). The observation that caffeine and pentoxifylline, which inhibit phosphodiesterase activity, increase hyperactivated motility is a clear indication that camp is required for sperm hyperactivation. (Kay eta/., 1993; Fraser, 1979). Aoki eta/., (1999) proposed that in hamster spermatozoa camp regulates optimal levels of calcium and thus facilitates hyperactivation. This observation implied that camp may not be directly required for hyperactivation and camps effect is probably mediated by its ability to mobilize intracellular calcium required for hyperactivation. In those spermatozoa were camp is required for hyperactivation it may be attributed to the activation of calcium gated channels by camp/ cgmp for calcium entry as in bull sperm flagella, which has been referred to as capacitative calcium entry (Wiesner et a/., 1998). Bicarbonate ions also play a major role in regulating hyperactivation of mouse and hamster spermatozoa (Neill and Olds-Ciarke, 1987; Stauss et a/., 1995; Kulanand and Shivaji, 2001) probably mediated by its effect on adenylyl cyclase in the flagellum and thus increasing

10 9 intracellular camp levels. Recently a soluble form of mammalian bicarbonate-regulated soluble adenylyl cyclase (sac) has been cloned and sac null mutant mice showed decreased motility of spermatozoa (Chen eta/., 2000; Livera eta/., 2005) (Figure 1.3). In summary, it appears that hyperactivation is an important event associated with capacitation (Mortimer, 1997) and signal transduction pathways leading to increase in free intracellular calcium from external or internal sources is crucial for hyperactivation. Calcium increase may be mediated by camp and bicarbonate. The temporal sequence of hyperactivation in spermatozoa too coincides with the time scale of protein tyrosine phosphorylation, suggesting that these two events may be related or linked. A more detailed role of protein tyrosine phosphorylation, calcium, bicarbonate and camp is presented in sections (1.3.3, 1.5, 1.6 and 1.8) Acrosome reaction Fusion point Figure 1.4 Schematic representation of the various stages in progression of the. acrosome reaction in a spermatozoon (Curry and Watson, 1995)

11 10 Sperm acrosome reaction, another hallmark of capacitation, is a highly specialized exocytotic event observed in capacitated spermatozoa and is a prerequisite for successful fertilization (Figure 1.4 ). During this exocytotic event multiple fusions occur between the outer acrosomal membrane and the plasma membrane leading to the formation of mixed membrane vesicles and the simultaneous release of acrosomal enzymes and exposure of molecules present on the inner acrosomal membrane for fusion with the oocyte (Curry and Watson, 1995). Breitbart developed an in vitro system for elucidating the fusion mechanisms during acrosome reaction (Spungin et a/., 1995). The physiological inducer of the acrosome reaction is the zona pellucida (ZP3) (Wassarman, 1995) the outer most investment of the oocyte. Few other inducers of the acrosome reaction are progesterone (Roldan et a/., 1994 ), follicular fluid (Suarez and Osman 1987), cumulus cell secretions containing prostaglandins and glycosaminoglycans (Yudin et a/., 1988; Lenz et a/., 1983). The physiological significance of acrosome reaction is further highlighted by the fact that males with spermatozoa lacking the acrosome are infertile (Baccetti eta/., 1991). 1;3iochemical events during acrosome reaction Calcium influx has been shown to be an absolute requirement for acrosome reaction in spermatozoa (Publicover and Barrat, 1999; Darszon et a/., 2001; De Bias et a/., 2002) (Figure 1.5) and the mandatory influx of calcium has been confirmed based on the observations that thapsigargin (Blackmore, 1993) and A23187, induced acrosome reaction in capacitated spermatozoa by elevating intracellular levels of calcium (Spungin and Breitbart, 1996). In hamster spermatozoa, calcium has been shown to be required for acrosome reaction during capacitation (Kulanand and Shivaji, 2001 ). The change in intracellular ph also affects the level of intracellular calcium ions via putative voltage dependent calcium channels (Fraire-Zamora and Gonzalez-Martinez, 2004 ).

12 11 Egg Zoru pellucida ZP3 H" CCE Ca ~ Ca I ' c. ATP ;. NJ" P,.ADP Outer acroscmsl membrane camp \ PK>\ t,t \ I \ c c~ ', \ I,... ~ IC 2 H lntro-acroscmsl spao.. ea- ADP P Figure 1.5 Hypothetical model for molecular mechanisms regulating acrosome reaction (Breitbart and Naor, 1999). The glycoprotein ZP3 binds to at least two different receptors in the plasma membrane. (R) is a Gi-coupled receptor that activates phospholipase Cb1 (PLCb1 ). The other is a tyrosine kinase (TK) receptor coupled to PLCg. Binding tor would regulate adenylyl cyclase (AC) leading to increased camp and protein kinase (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane, which releases Ca2+ from the interior of the acrosome to the cytosol. This is the first (1), relatively small, increase in intracellular Ca2+ ([Ca2+]i), which leads to activation of the PLCg. The products of phosphatidylinositol 4, 5- bisphosphate (PIP2) hydrolysis by PLC, diacylglycerol (DAG) and inositol triphosphate (IP3) lead to PKC translocation to the plasma membrane and its activation. Protein kinase C opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II), higher, increase in [Ca2+]i. In addition, the PKA and IP3-dependent Ca2+ channels (I and Ill) of the outer acrosomal membrane will cause acrosomal Ca2+ depletion, leading to the activation of a capacitative Ca2+ entry (CCE) in the plasma membrane. The Gi or TK can also activate a Na+-H+ exchanger in the plasma membrane, leading to the alkalization of the cytosol. The increases in [Ca2+)i and phi will lead to membrane fusion and acrosomal exocytosis.

13 12 ZP3 a physiological inducer of acrosome reaction binds to Galactosyltransferase 1, a sperm surface enzyme and triggers an increase in the influx of calcium, through the transient receptor potential channel (TRPC), a member of the calcium channel family (Darszon et a/., 2001; Primakoff and Myles, 2002), and thus induces acrosome reaction. Spermatozoa possess 4 distinct types of capacitative calcium channels, out of which TRPC2 has been proposed to participate in calcium influx triggered by zona pellucida (Jungnickel et a/., 2001 ). Breitbart and Naor (1999) proposed a model for the calcium dependent acrosome reaction involving the activities of four enzymes namely: (a) Phosphatidyl inositol 2 phosphate (PIP2) specific Phospholipase C (PLC) (Roldan and Harrison, 1989; Spungin eta/., 1995), (b) Protein kinase C (Breitbart eta/., 1992), (c) Phospholipase A2 (PLA2) (Roldan and Harrison, 1992) and (d) Actin depolymerization (Spungin eta/., 1995) (Figure 1.5). Recently, it has been shown that actin polymerizes during capacitation and the polymerized F-actin breaks down just before acrosome reaction (Brener et al., 2003). Intracellular increase in ph is another important factor crucial for sperm acrosome reaction (Cross and Faulkner, 1997; Zeng eta/., 1996; Brook eta/., 1996; Arnoult eta/., 1996) since it has been demonstrated that increase in ph activates calcium dependent calmodulin and phospholipases which are known to be important for acrosome reaction (Tulsiani et at., 1998; Florman et a/., 1998; Darszon et a/., 1996). Thus the signal transduction mechanism involved in acrosome reaction invokes increase in calcium levels and elevation in intracellular ph (Fiorman eta/., 1989).

14 Protein tyrosine phosphorylation Concomitant with hyperactivation and acrosome reaction mammalian spermatozoa exhibit tyrosine phosphorylation in an array of sperm proteins (Visconti et a/., 1995). This array of proteins that get phosphorylated during capacitation vary from species to species (Kulanand and Shivaji, 2001; Baccetti et a/., 1991; Visconti et a/., 1995; Leclerc eta/., 1997; Galantino Homer eta/., 1997; Kalab eta/., 1998; Osheroff et a/., 1999; Flesch eta/., 1999; Pukazhenthi eta/., 1998; Si and Okuno, 1999; Bajpai and Doncel, 2003). Increase in protein tyrosine phosphorylation during capacitation of mouse (Dasgupta eta/., 1993), bovine (Galantino-Holmer eta/., 1997), human (Leclerc eta/., 1996; Osheroff eta/., 1999) and hamster (Visconti eta/., 1995) spermatozoa has been shown to be regulated by a camp-dependent protein kinase A (PKA) (Visconti et a/., 1995). This signaling pathway involving protein tyrosine phosphorylation and camp is unique to sperm and has been confirmed by the fact that inhibitors of PKA are able to inhibit protein tyrosine phosphorylation observed during capacitation. PKA is tetrameric enzyme having two regulatory and two catalytic subunits (Vijayaraghavan eta/., 1997). The binding of camp to the regulatory subunit promotes the activation of the two catalytic subunits, which then phosphorylate the serine residues of their target protein. The target protein is probably a protein tyrosine kinase(s) or protein tyrosine phosphatase(s) or both which ultimately regulate phosphorylation of proteins at the tyrosine residues. PKA is in turn regulated by A Kinase Anchoring Protein (AKAP), which tethers protein kinase and phosphatases in close proximity to their target proteins within specific cell compartments. Bajpai and Doncel, (2003) have identified a cross talk between tyrosine kinase and camp-dependent kinase in the regulation of human sperm motility. Attempts have been made to purify protein tyrosine kinases from boar (Berruti and Martegani, 1989) and hamster spermatozoa (Uma devi et a/., 2000). In addition to tyrosine kinases, the extracellular signal-regulated kinase (ERK) family of mitogenactivated protein kinase (MAPK) has been detected in spermatozoa and has also been

15 14 implicated in sperm phosphorylation (Naz and Ahmed, 1992; Ashizawa et a/., 1997; Luconi et a/., 1998; de Lamirande and Gagnon, 2002). The MAPK kinases are dual specificity kinases capable of phosphorylating both threonine and tyrosine residues and inhibition of it blocks protein tyrosine phosphorylation associated with capacitation. ROS too influences protein phosphorylation by increasing intracellular camp, which leads to activation of tyrosine kinase and inhibition of protein tyrosine phosphatase (Ford, 2004 ). Visconti et a/. (1995) suggested that the increase in protein tyrosine phosphorylation associated with sperm capacitation is species-specific and is dependent on the constituents of the medium such as calcium, BSA and bicarbonate which have different effects in different species (Visconti eta/., 1995). Mouse spermatozoa require calcium, bicarbonate and BSA for capacitation and the associated protein tyrosine phosphorylation (Visconti eta/., 1995), whereas human spermatozoa required BSA and bicarbonate but not calcium (Carrera eta/., 1996; Luconi et a/., 1996). Kulanand and Shivaji, (2001) showed that hamster spermatozoa incubated in media without bicarbonate and calcium or without BSA and PVA showed a decrease in protein tyrosine phosphorylation and a delay in hyperactivation, indicating again a link between the two events. In the same study it was demonstrated that absence of calcium or bicarbonate in the capacitation medium delayed phosphorylation of the proteins in hamster spermatozoa but when both calcium or bicarbonate were absent a decrease in protein tyrosine-phosphorylation was observed in hamster spermatozoa. A number of these proteins, which are tyrosine phosphorylated during capacitation, were localized to the sperm flagellum (Mahony and Gwathmey, 1999; Nassar et a/., 1999; Si and Okuno, 1999; Urner eta/ ;Mitra and Shivaji, 2004, Kumar eta/., 2006). Naz and Rajesh, (2004) proposed a model for sperm capacitation based on the involvement of calcium, bicarbonate, camp, protein kinases, protein phosphatases, and

16 15 H ( ' 1!\a' HC( J O: C\1Y>Kf.\'ES< GADA A.'\uhm::-.; II\ PRuuESTER<!<.'E!:\'SIDE Plooopi~") I I -J '\Ul'dll'alt' /' I( PROH:I;\' TY IWSINEPIIOSPIIORY LATIOJ'\ CAPACITATlOl'\ Identified tyrosine phosphorylated protein AKAP3/ AKAP4 Pro AKAP3 CABYR HSP90 GPX4 DLD PDHA2 Figure 1.6 Schematic representation of signaling events associated with capacitation-dependent tyrosine-phosphorylation of proteins in mammalian spermatozoa (Naz and Rajesh, 2004). According to this model three major signaling pathways operate in spermatozoa during capacitation namely a camp/pka-dependent pathway (pathway 1), receptor tyrosine kinase pathway (pathway II) and non-receptor protein tyrosine kinase pathway (pathway Ill). Pathway I, is exclusive to spermatozoa. But the three pathways may not be mutually exclusive and may include cross-talk among several molecules. Many key molecules and receptors are still to be identified to completely elucidate the molecular mechanism and signal transduction cascade involved in capacitation. G-protein coupled receptor pathway has not been included in this model. The components involved in the proposed model are Cytokines, GABA (Gamma amino butyric acid), Angiotensin, Progesterone, HC03-/ Na+ cotransporter (Sodium bicarbonate co transporter), AKAP (A kinase anchoring protein), PKA (Protein kinase A), sac (Soluble adenylyl cyclase), RAS, RAF, MEK (extracellular signal-regulated kinase), MAPK (mitogen-activated protein kinase), She (Sarcomere homology 2 domain containing, an adapter molecule), EGF (epidermal growth factor), IGF-1 (insulin growth factor 1), p190c-met (a proto-oncogene), c-abl tyrosine kinase, e-yes (cellular-yamaguchi sarcoma viral oncogene) and TK-32 (Thymidine kinase) in regulating protein tyrosine phosphorylation during capacitation. Other signaling molecules involved are ROS (reactive oxygen species), camp (cyclic adenosine monophosphate), ATP (adenosine triphosphate), bicarbonate and calcium. For details see section Protein tyrosine phosphorylation.

17 16 tyrosine phosphorylated proteins (Figure 1.6). According to the model three major signaling pathways namely a camp/pka-dependent pathway (pathway 1), a receptor tyrosine kinase pathway (pathway II), and a non-receptor protein tyrosine kinase pathway (pathway Ill) have been proposed to operate during sperm capacitation. In pathway I, increase in protein tyrosine phosphorylation during sperm capacitation is regulated by a camp-dependent pathway involving a protein kinase A (PKA). PKA in turn is regulated by A Kinase Anchoring Protein (AKAP), which tethers tyrosine kinases in close proximity to their target proteins. These tyrosine kinases are of two types namely the receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (PTKs). The RTKs which are involved in pathway II are transmembrane proteins having an extracellular ligand binding domain and a tyrosine kinase domain located inside the cell. Various RTK's such as e-ras in human spermatozoa (Naz eta/., 1992), epidermal growth factor (EGF) in human, mouse, rabbit, rat and bovine spermatozoa (Naz and Ahmed, 1992; Lax eta/., 1994) and insulin growth factor (IGF-1) (Naz and Padman, 1999) have been identified. These RTK's upon extracellular ligand binding get activated and undergo autophosphorylation. This autophosphorylation event produces new binding sites for intracellular adapter molecules such as She (Sarcomere homology 2 domain containing, an adapter molecule), which brings signal transduction molecules into its close proximity. She recruits Grb2 (Growth factor receptor-bound protein 2, an adapter molecule), which then binds to Sos (Son of sevenless, a GTP/GDP exchange factor). These interactions result in targeting of Ras and Raf to the membrane. The activity of Ras and Raf is increased by phosphorylation, which activates other signaling molecules like MEK (extracellular signal-regulated kinase) and MAPK (mitogen-activated protein kinase) and influences protein tyrosine phosphorylation during capacitation. In contrast, PTKs which are involved in pathway Ill are located in the cytoplasm, nucleus or inner side of the plasma membrane. Various identified PTK's are TK-32 (Thymidine kinase) and e-yes (cellular-yamaguchi sarcoma viral oncogene) in the human sperm head

18 17 (Leclerc and Goupil, 2002). The activity of the e-yes kinase increases in the presence of camp thus leading to. an increase in sperm protein phosphotyrosine content indicating that tyrosine phosphorylation of proteins in spermatozoa involves cross-talk between camp and tyrosine kinase(s). Activation of tyrosine kinase (TK-32) also occurred concomitant with capacitation-dependent tyrosine phosphorylation in porcine spermatozoa (Tardif eta/., 2003). Thus it is obvious that phosphorylation of proteins at the tyrosine residue which is the down stream event in the various signal transduction pathways associated with capacitation is crucial for sperm capacitation and therefore the need to characterize these proteins. A few of these capacitation associated tyrosine phosphorylated proteins have been characterized and identified as signaling molecules, metabolic enzymes and chaperones. AKAP82 (AKAP4), its precursor pro-akap82 (pro-akap4) and AKAP3 were the prominent tyrosine phosphorylated proteins in capacitated human spermatozoa (Carrera eta/., 1996; Mandai eta/., 1999). AKAP4 along with its precursor pro-akap4 were also the first the major tyrosine phosphorylated proteins identified in capacitated hamster spermatozoa (Jha and Shivaji, 2002). AKAP4 of hamster spermatozoa showed a peak intensity of phosphorylation at 3-5 h thus correlating with hyperactivation and acrosome reaction (Kula nand and Shivaji, 2001) thus hypothesizing a role for AKAP in spermatozoal capacitation. Further, localization studies indicated that AKAP4 in hamster spermatozoa was found exclusively in the fibrous sheath and was identified to be spermatid and spermatozoa specific. In contrast, AKAP4 of mouse spermatozoa is phosphorylated at serine residues (Moss et a/., 1999; Johnson et a/., 1997). The significance of AKAP is very obvious since it has been predicted that AKAP tyrosine phosphorylation would expose the docking site of kinase (PKA), and thus facilitate its localized action. Recently, Luconi eta/., (2004) reported that tyrosine phosphorylation of AKAP3 recruits PKA to the sperm flagellum thus causing an increase in sperm motility.

19 18 AKAP isoforms have also been localized to the acrosome region of human spermatozoa (Harrison and Miller, 2000; Vijayaraghavan et a/., 1999). Bajpai et a/., (2006) demonstrated that AKAP3 binds to phosphodiesterase EA4 in bovine spermatozoa and may thus influence capacitation. However, it is difficult to understand as to why AKAP knock out mice show no obvious fertility defects (Burton eta/., 1997) though it could also mean that there are other compensating mechanisms, which overcome the functional disability of AKAP. CABYR is yet another spermatozoa specific 86kDa calcium binding and tyrosine phosphorylation regulated protein, located in the principal piece of human spermatozoa (Naaby-Hansen et a/., 2002). Phosphorylation of calcium-binding isoform of 86 kda CABYR was increased during in vitro sperm capacitation and was abolished by dephosphorylation with alkaline phosphatase, suggesting that CABYR may be involved in the process of capacitation and motility of sperm (Naaby-Hansen et a/., 2002). It is speculated that CABYR establishes a link between tyrosine phoshorylation and calcium release in signal transduction pathway of capacitation (Ho and Suarez, 2001) and is probably involved in the calcium waves that accompany flagellar beating (Suarez et a/., 1993). Four murine CABYR variants orthologous to human CABYR forms, I, Ill, IV and VI have been identified involving two coding regions, CR-A and CR-B similar to human CABYR edna. Of these two isoforms of CABYR namely CABYR (CR-A) and CABYR (CR-B) were localized in the fibrous sheath of mouse spermatozoa and it was further demonstrated that calcium-binding property was associated only with CABYR (CR-A) (Kim eta/., 2005). A motif homologous to the Rll dimerization domain of PKA and seven potential tyrosine phosphorylation were also identified in CABYR (CR-A). Lipoamide dehydrogenase DLD, was identified as a major tyrosine phosphorylated protein in capacitated hamster spermatozoa by Mitra and Shivaji, (2004)

20 19 and it was demonstrated that the activity of DLD correlated with the time course of tyrosine phoshorylation and acrosome reaction in hamster spermatozoa (Mitra et a/., 2005). The forward activity and tyrosine phosphorylation of DLD positively correlated with acrosome reaction Where as the reverse activity correlated positively with tyrosine phosphorylation of DLD in principal piece. Down regulation of the activity of the DLD blocked acrosome reaction completely and hyperactivation partially, confirming the role of dihydrolipoamide dehydrogenase in hamster spermatozoal capacitation. It is worth mentioning that DLD is a component of pyruvate metabolizing complex (PDHc) thus emphasizing an important role for pyruvate in hamster spermatozoa capacitation. Localization studies indicated that hamster spermatozoal DLD along with its host pyruvate dehydrogenase complex, is localized in the acrosome and in the principal piece of the sperm flagellum (Mitra et a/., 2005). Therefore, it is likely that the other components of PDHc are also involved in sperm capacitation. This indeed is so, because recently Kumar eta/. (2006) demonstrated that PDHA2, a component of PDHc, 'is yet another protein, which gets tyrosine phosphorylated in hamster spermatozoa during capacitation. PDHA2, which exhibits capacitation-dependent tyrosine phosphorylation in hamster spermatozoa, is a testis specific isoform and exists as three distinct proteins with identical molecular weights but vary in their isoelectric point (Kumar et a/., 2006). It was also observed that the activity of PDHA correlated positively with hyperactivation. Further, localization studies indicated that hamster spermatozoal PDHA2 exhibits extramitochondrial non-canonical localization in hamster, cat and human spermatozoa (Kumar et a/., 2006). Capacitation-dependent tyrosine phosphorylation kinetics of PDHA2 peaked between 3-5 h, hypothesizing its role in hyperactivation and acrosome reaction of hamster spermatozoa. Vijayaraghavan eta/., (1997) reported a 55 kda tyrosine phosphorylated protein associated with bovine sperm motility. This protein is tyrosine phosphorylated to a much

21 20 higher degree in motile caudal than in immotile caput epididymal spermatozoa. Motility inhibition of caudal epididymal sperm by inhibiting protein kinase A (PKA) or by ionomycin-induced calcium led to the disappearance of tyrosine phosphorylation of this protein. Conversely, treatment of spermatozoa with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. It was suggested that motility regulation of this phosphorylated protein might also involve cross talk between PKA, calcium, and tyrosine kinase pathways. Aitken eta/. (1995) and Leclerc eta/. (1997) demonstrated two proteins namely p105/p81 in mouse spermatozoa which exhibited time dependent tyrosine phosphorylation during capacitation. The phosphorylation of these proteins was inhibited by antioxidants (SOD and catalase) and stimulated by exogenous ROS. These two tyrosine phosphorylated proteins were localized to the fibrous sheath of sperm flagellum (Leclerc et a/., 1997) and were antigenetically related to mouse A kinases anchoring proteins (AKAP's) which sequester protein kinases A (PKA) through its regulatory subunits (Carrera et a/., 1996). It was also hypothesized that phosphorylation of p105/p81 may be related to acquisition of hyperactivated motility (Leclerc eta/., 1997). Heat shock protein 90 (HSP90), a conserved molecular chaperone, also exhibits tyrosine phosphorylation in human and mouse sperm during capacitation (Ecroyd et a/., 2003). This was the first study establishing the role of molecular chaperones in sperm capacitation. In this study it was also clearly demonstrated that geldanamycin an ansamycin antibiotic, which disrupts ATP-dependent chaperoning function of HSP90 did not inhibit motility, hyperactivation, tyrosine-phosphorylation, or ability of spermatozoa to bind to zona pellucida suggesting that activity of HSP90 in mouse spermatozoa is not dependent on its A TPases activity but probably it is dependent on some other activity associated with HSP90. HSP90 being a chaperone is necessary component of a variety of signaling pathways, where it acts in concert with other proteins, such as Cdc37/p50, to

22 21 ensure the correct folding and, therefore, activity of target proteins. The signaling pathway leading to tyrosine phosphorylation is mediated via camp and PKA suggesting that HSP90 has the potential to mediate serine/threonine and tyrosine kinase signaling in the pathway, which leads to the global increase in protein tyrosine phosphorylation (Aitken eta/., 1998; Brugge eta/., 1981). In mice, multiple isoforms of type 1 hexokinase are transcribed during spermatogenesis, including at least three that are presumably germ cell specific: HK1- sa, HK1-sb, and HK1-sc. Among them the germ cell specific hexokinase (HK1-sc), is the only tyrosine phosphorylated protein (116/95k0a) and localized to head, midpiece and principal piece (Travis et a/., 1998). Presence of HK1-sc in various regions of spermatozoa suggested that it might be supplying glucose 6-phosphate to a pathway other than glycolysis, such as the pentose phosphate pathway (PPP), for generating extramitochondrial energy sources in these regions. Naz, (1996) also identified a hexokinase in human spermatozoa that was different from the mice hexokinase in that it was not phosphorylated at tyrosine residues; however, antibodies against hexokinase caused agglutination of spermatozoa and a concentration-dependent inhibition of fertilizing capacity. Albarracin eta/. (2004) compared hexokinase activity in dog and boar spermatozoa and found dog spermatozoal hexokinase more active compared to the activity in boar spermatozoa. This increased activity of hexokinase in dog spermatozoa correlated with overall protein tyrosine phosphorylation during capacitation. This glucokinase-like protein was distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal piece of dog spermatozoa. NagDas et a/. (2005) identified capacitation-dependent tyrosine phosphorylation of phospholipid hydroperoxide glutathione peroxidase (GPX4 ), the major structural protein of the sperm mitochondrial capsule and proposed that it may represent an

23 22 important event in the signaling pathway(s) associated with capacitation and thus could potentially affect mitochondrial function. Tyrosine phosphorylation contributed to the generation of multiple isoforms of GPX4. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological mid piece alterations, were also linked to insufficient GPX4 (Foresta et a/., 2002). Due to lack of GPX4 excess hydroperoxides generated from mitochondria were not reduced which oxidatively damaged spermatozoa. However further studies are in progress to establish role of GPX4 in spermatozoal capacitation. A global phosphoproteome analysis of human spermatozoa (Ficarra eta/., 2003), led to the identification of a set of eighteen proteins which get tyrosine phosphorylated during sperm capacitation which includes signaling molecules, chaperones, ion channels, metabolic enzymes, structural proteins etc. They identified VCP (valosin containing protein) also known as p97, a member of the AAA family (ATPases associated with various cellular activities), N-ethylmaleimide sensitive factor (NSF), golgi t-snare syntaxin 5 which mediates the fusion of golgi membranes, TRAP-1 (tumor necrosis factor type 1 receptor-associated protein), pyruvate dehydrogenase J3 (PDHJ3, a metabolic enzyme), voltage-dependent anion select channel 2 (VDAC2, an ion channel), HSP-90a and GPX4. Further they confirmed tyrosine-phosphorylation of AKAP-3 and AKAP-4 during human sperm capacitation and mapped eight phosphorylation sites of these proteins using a combination of Fe 3 + -immobilized metal affinity chromatography (IMAC) and MS/MS. 1.4 Role of cholesterol in spermatozoal capacitation Freshly ejaculated mammalian spermatozoa gain the ability to undergo acrosome reaction by removal of cholesterol from the plasma membrane as one of the modifications of capacitation. This cholesterol efflux induces plasma membrane lipid

24 23 reorganization, ultimately increasing membrane permeability to several crucial ions needed for capacitation (Visconti et a/., 1998). Cholesterol is a key lipid and major sterol in mammalian spermatozoa [exceptions are hamster and mouse spermatozoa, which have "'40% desmosterol] (Awano et a/., 1993) in plasma membrane with higher concentrations overlying the head region. Cholesterol efflux is important for many events occurring during sperm capacitation, such as increase in tyrosine phosphorylation via camp/ PKA pathway (Osheroff et a/., 1999; Visconti et a/., 1999) and lipid scrambling in the plasma membrane (Flesch et a/., 2001 ). Cholesterol depleting agent like p Cyclodextrin increased protein tyrosine phosphorylation through the camp/pka pathway (Visconti eta/., 1999). Bovine Seminal Plasma proteins (BSPs) which have been widely characterized in bovine species are deposited on the surface of spermatozoa and bind to the cholesterol effluxing agents like heparin and high density lipoproteins (HDL) hypothesizing that their binding to spermatozoa increases the binding site of these cholesterol effluxing agents in the female tract thus providing the trigger for capacitation. Besides, the increase in sperm membrane fluidity has also been correlated with lateral reorganization of the membrane proteins (Rochwerger and Cuasnicu, 1992; Cross and Overstreet, 1987), leading to formation of specialized plasma membrane microdomains known as lipid rafts. Several proteins associated with metabolism are found in lipid raft fractions as HK1, testis-specific LDH, glucose transporter Glut3, and pantophysin. The presence of metabolic enzymes in sperm lipid rafts suggests that cholesterol removal mediates some of the changes in energy metabolism observed during capacitation. 1.5 Role of calcium in spermatozoal capacitation Calcium is known to influence various functions of the spermatozoa such as motility, capacitation and fertilizing ability. Intracellular calcium surge in spermatozoa occurs at two stages, first from the internal stores and later from the extracellular medium (Spungin and Breitbart, 1996). Elevation of calcium in the flagellum of

25 24 spermatozoa drives hyperactivation in many species (Ho and Suarez, 2001) and this action of calcium could be at the level of the sperm flagella as revealed by experiments with demembranated rat (Lindemann and Goltz, 1988) and bull (Lindemann et a/., 1991) spermatozoa. In bull spermatozoa the redundant nuclear envelope (RNE's) in the neck and mitochondria of the spermatozoa have been recognized as the major sites for calcium storage (Ho and Suarez, 2003). Knock out male mice of Catsper(s), which are sperm specific calcium channels localized to the sperm flagellum (Quill et a/., 2001, 2003; Carlson et a/., 2003, 2005; Ren et a/., 2001) have been found to be defective in hyperactivation and are thus infertile. Calcium is also indispensable for acrosome reaction. Acrosome itself is thought to be a calcium store, which is proposed to release calcium during acrosome reaction by activating PKA and IP3 dependant calcium channels in the outer acrosomal membrane (Breitbart and Naor, 1999). Calcium signaling is mediated through a number of calcium binding proteins, of which calmodulin is the most ubiquitous. The calcium dependent CaMK2 and CaMKIV are widely distributed, multifunctional serine-threonine kinase that have been implicated in the regulation of flagellar and ciliary motility (Smith, 2002; Marin Briggiler eta/., 2005) and acrosome reaction (Fournier eta/., 2003). The role of calcium in the capacitation-dependant tyrosine phosphorylation event is quite controversial and discussed in section (1.3.3). In human spermatozoa, calcium negatively modulated tyrosine phosphorylation due to its inhibitory effect on kinases (Luconi et a/., 1996) or by decreasing the availability of A TP by activating calcium dependant A TPases (Baker et a/., 2004 ). In hamster, calcium is not required for tyrosine-phosphorylation during capacitation (Kulanand and Shivaji, 2001 ). The change in intracellular ph is also known to affect the levels of intracellular calcium ions via putative voltage dependent calcium channels (Fraire-Zamora and Gonzalez-Martinez, 2004 ).

26 Role of bicarbonate in spermatozoal capacitation Bicarbonate is a major component of the uterine and oviductal fluid. Lee and Storey {1986) unequivocally established bicarbonate ion as a first messenger in the process of capacitation based on the observation that replacement of the bicarbonate buffer in the in vitro fertilization medium with HEPES buffer effectively inhibited fertilization. Numerous studies have indicated that capacitation is a bicarbonate dependent process {Lee and Storey, 1986; Neill and Olds-Ciarke, 1987; Visconti eta/., 1995). DIDS and SITS, anion channel inhibitors, prevented capacitation-associated processes like hyperactivation and tyrosine phosphorylation; the effect on hyperactivation being recently quantified {Wennemuth et a/., 2003). Absence of bicarbonate ions could be overcome by camp analogues during capacitation in mouse {Visconti et a/., 1995) and hamster spermatozoa {Visconti et a/., 1999) suggesting strongly that camp is the second messenger for bicarbonate ions in capacitation. Bicarbonate activates spermatozoa by directly switching on the sac/camp/pkasignaling. Other effects of bicarbonate are induction of hyperpolarizing current during capacitation {Demarco et a/., 2003); ability to increase the sensitivity of voltage dependant calcium channels {Wennemuth et a/., 2000); to remodel the membrane lipid architecture probably through a bicarbonate phospholipid 'scramblase' {Gadella and Harrison, 2000; 2002) and increasing the intracellular alkalinity, which is a very essential step early in capacitation {Zeng eta/., 1996). The migration of CRISP-1 to the equatorial segment during capacitation is bicarbonate ion dependant in rat spermatozoa {Da Ros et a/., 2004 ). The manifold importance of bicarbonate ion in sperm capacitation in vivo became evident in a very interesting report wherein the investigators postulate that the low female fertility in cystic fibrosis patients could be due to hampered uterine secretion of bicarbonate ions through defective cystic fibrosis transmembrane receptor {CFTR) in the patients thus, causing insufficient sperm capacitation (Wang et a/., 2003). Mutant mice deficient in expression of either Catsper1 or 2 are infertile due to the lack of

27 26 capacitation induced hyperactivated motility in response to bicarbonate ions (Carlson et a/., 2003 and Quill eta/., 2003) Role of reactive oxygen species in spermatozoal capacitation The superoxide anion and hydrogen peroxide have been implicated in hyperactivation (de Lamirande and Gagnon, 1993), acrosome reaction (Size eta/., 1991; Aitken et a/., 1996; Rivlin et a/., 2004) and protein tyrosine phosphorylation, which are associated with capacitation of spermatozoa (de Lamirande eta/., 1997; Aitken eta/., 1995; Leclerc eta/., 1997; O'Fiaherty eta/., 2005) based on the observations that sperm capacitation can be induced by the addition of superoxide and peroxide ions and decreased by antioxidants. At the molecular level ROS acts on adenylyl cyclase to increase intracellular levels of camp, which further activates PKA (Aitken, 1997; Rivlin et a/. 2004) (Figure 1.6) and the subsequent down stream events. However, the involvement of ROS in signal transduction through camp is controversial, as some authors claim it to be upstream of the cascade (Aitken eta/., 1998) while others believe ROS to be acting downstream of camp (Leclerc eta/., 1998). In addition to superoxide and peroxide ions reactive nitrogen species (RNS), like nitric oxide, at low concentrations also increase mouse, hamster and bull sperm acrosome reaction and zona pellucida binding ability in human spermatozoa (Herrero eta/., 1997; Kameshwari eta/., 2003; Sengoku eta/., 1998). Recently, it was suggested that the LDH-C4 (Sperm isoenzyme) which is responsible for cytosolic lactate generation and supply of NADH (O'Fiaherty eta/., 2005) might participate in the regulation of the redox status required to achieve the acrosome reaction in bovine spermatozoa. Aitken and his colleagues (1995, 1997) reported that NADPH but not NADH, when used at lower concentrations stimulates superoxide generation and capacitation. O'Fiaherty eta/., (2005), evaluated the phosphorylation of phospho-mek (mitogen-activated protein kinase or extracellular

28 27 signal-regulated kinase) like proteins and showed that they are modulated by ROS and influence capacitation associated protein tyrosine phosphorylation. Leclerc eta/., (1998) proposed that calcium is required for induction of superoxide generation and once initiated calcium is not required for superoxide production. Sperm superoxide generation is also regulated by intracellular ph. The stimulation of sperm capacitation and superoxide generation by inhibitors of phosphodiesterases (IBMX) and Calyculin A suggests that increase in camp and serine/ threonine phosphorylation is prerequisite for sperm oxidase activation (Leclerc eta/., 1998). 1.8 Role of cyclic AMP in spermatozoal capacitation Cyclic adenosine monophosphate (camp) has been known to participate in sperm functions namely motility, capacitation (Visconti eta/., 1995; Harrison, 2003) and the acrosome reaction (DeJonge eta/., 1991; Garde and Roldan, 2000). There are several ligands, which bind to the specific receptors and act as "first messengers" and effect the production of camp, which further acts as a "secondary messengers". Membrane permeable analogue of camp like dbcamp (but not cgmp) regulate capacitation, acquisition of hyperactive motility and acrosome reaction (Lefievre et a/., 2002). Cyclic AMP concentrations are regulated by modulating its synthesis by adenylyl cyclases (AC's) and/or its degradation by phosphodiesterases (POE's). There are two classes of AC's in mammalian cells: G protein-regulated transmembrane adenylyl cyclases (tmacs) and bicarbonate- and calcium-regulated sac's. Buck et a/., 1999 purified a truncated form of a soluble adenylyl cyclase from rat testis (Harrison, 2003; Buck et a/., 1999) and found that the soluble enzyme was highly responsive to bicarbonate stimulation (and not to forskolin) and is substantially homologous to the cyanobacterial enzyme. A number of POE isoforms have been detected in mammalian sperm using type-specific POE inhibitors (Fisch et a/., 1998), but their specific