Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.

A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X-100 soluble and insoluble proteins were recovered and separated by SDS-PAGE. The separation lanes were dissected into 96 fractions and analyzed individually by LC-MS(n) . A comprehensive protocol, involving LC-MS/MS analysis eventually down to the ninth most intense peak found in the MS-survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual-oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven-pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate-gated ion channel receptors, transient receptor potential cation channels, and a non-genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC-MS/MS analysis.

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

Proteomic profiling of the mouse spermatozoon has generated a unique and valuable inventory of candidates that can be mined for potential contraceptive targets and to further our understanding of the PTMs that regulate the functionality of this highly specialized cell. Here we report the identification of 858 proteins derived from mouse spermatozoa, 23 of which demonstrated testis only expression. The list contained many proteins that are known constituents of murine spermatozoa including Izumo, Spaca 1, 3, and 5, Spam 1, Zonadhesin, Spesp1, Smcp, Spata 6, 18, and 19, Zp3r, Zpbp 1 and 2, Spa17, Spag 6, 16, and 17, CatSper4, Acr, Cylc2, Odf1 and 2, Acrbp, and Acrv1. Certain protein families were highly represented in the proteome. For example, of the 42 gene products classified as proteases, 26 belonged to the 26S-proteasome. Of the many chaperones identified in this proteome, eight proteins with a TCP-1 domain were found, as were seven Rab guanosine triphosphatases. Finally, our list yielded three putative seven-transmembrane proteins, two of which have no known tissue distribution, an extragenomic progesterone receptor and three unique testis-specific kinases all of which may have some potential in the future regulation of male fertility.

Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three samples in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa.

Proteomics represents a powerful tool for the analysis of mammalian spermatozoa, since these terminally differentiated cells are transcriptionally inactive and exhibit a limited dynamic range of protein expression. Here we report the identification of 5123 peptides, leading to 829 unambiguous and 2215 redundant gene products found to be present within rat spermatozoa derived from the cauda epididymis. Bioinformatics demonstrated that 60 proteins appeared to be specifically expressed in the genitourinary tract, including pyruvate dehydrogenase 1, ropporin, testis-specific serine kinase 4, testis-specific transporter, and retinol dehydrogenase 14. We also identified eight members of the ADAM family, seven of which have previously been detected in spermatozoa (ADAM2, -3, -4, -5, -6, -7, and -30) while ADAM34 has been identified in the sperm proteome for the first time. Approximately 21 gene products were found to possess isomerase activity including peptidylprolyl cis/trans isomerases that are known to be involved in germ cell differentiation and protein disulfide isomerases that have been implicated in sperm-oocyte fusion. Furthermore, 51 gene products clustered into ion-transporter activity. This inventory of gene products, the first ever 2-D LC-MS/MS analysis of rat spermatozoa, will be invaluable in directing future research into the molecular mechanisms that drive these highly specialized cells.

Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross-linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS-PAGE, 1 mm slices were digested and used for LC-MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.