Fertilization is a process that generates the first cell of a new organism. In mammals, fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility. A comprehensive review on the perspectives of male reproduction, spermatogenesis, the structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2. Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47- treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific. The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed. In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms that govern sperm function in clinical cases of non-obstructive male infertility in the human.

In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and principle piece regions of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 17 differentially phosphorylated protein spots. Out of the 17 spots, 12 were identified by MALDI-MS/MS analysis. The proteins identified to be differentially phosphorylated, upon inhibition of IGF1R, were PDHE1, ODF-2, Tubulin β 2C chain, PDHE2 and ATP synthase β subunit. The RTKs being present in the membrane level may not be directly involved in the phopshorylation of downstream target proteins associated with the mitochondrial membrane, sperm axonemal structures and outer dense fibers. Therefore, the RTKs may interact directly or indirectly with the downstream NRTKs, which may be involved in the phosphorylation of target sperm proteins. Till date, six different families of NRTKs are shown to be expressed in mammalian spermatozoa. The major family of NRTKs involved in sperm function is the Src family of kinases. However, there is very little information available in the context of sperm capacitation and the associated PYP. Therefore, studies were carried out to understand the role of Src family of NRTKs in sperm capacitation and associated PYP. Presence of active Src signaling was observed by the immunolocalization of activated Src (pY416) in the acrosome, mid piece and the principal piece regions of the sperm flagellum. Inhibition of Src family of kinase with a specific Src family kinase inhibitor PP2, showed inhibition of sperm capacitation and the associated hyperactivation. Inhibition of Src family of kinases with PP2 was associated with decrease in PYP of several proteins (Mr. 45,000-120,000), localized mainly to the mid piece region, followed by the principle piece region of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 38 differentially phosphorylated protein spots. Out of the 38 spots, 16 were identified by MALDIMS/MS analysis and these corresponded to seven proteins which included PDHE1, ODF-2, Tubulin β 2C chain, Tektin-2, GAPDS, PDHE2 and ATP synthase β subunit. Additionally, the biochemical and molecular characteristics of the identified proteins were also studied. Bioinformatic analysis predicted the presence of phosphorylation motifs for several kinases and interestingly, all the proteins identified had a Src kinase motif. Comparing the current observations and the previous work in the laboratory, two proteins ODF-2 and Tektin-2 were found to be regulated by EGFR, IGF1R and Src family of kinases. Therefore, characterization of the capacitation-associated tyrosine phoposphorylated proteins ODF-2 and Tektin-2 was performed. By employing PCR and Northern blotting techniques, the presence of the transcripts of both the proteins was shown. Additionally, the ontogeny of expression of ODF2 and Tektin-2 in hamster testis development was studied and the results indicated that the expression of both the proteins started from week 3 onwards till week 8. To confirm the meiotic stage-associated expression of ODF-2 and Tektin-2, germ cells were sorted based on their DNA content. ODF-2 and Tektin-2 transcripts were first expressed in the meiotic germ cells (pachytene spermatocytes) and their expression was upregulated in the post-meiotic germ cells (round spermatids). Sequential extraction of sperm proteins showed that, Tektin-2 was majorly extracted out in the Triton X-100 and DTT fraction, whereas, ODF-2 was maximally extracted in the presence of urea and DTT. In conclusion, these observations indicate that IGF1R and Src family of tyrosine kinases are critical for mammalian sperm capacitation and associated global PYP. Inhibition of sperm capacitation was associated with hypo-tyrosine-phospohorylation of certain proteins associated with mitochondrial membrane, axonemal structures and outer dense fibers of the sperm flagellum. Future work can be directed towards understanding the role of other RTKs and NRTKs involved in sperm capacitation and the molecular characterization of hypophosphorylated proteins critical for sperm function and its fertilization competence.