Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to antiphosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.After ejaculation, sperm are able to move actively but lack fertilizing competence. They acquire the ability to fertilize in the female genital tract in a time-dependent process called capacitation (1). Capacitation is accompanied by a cAMPprotein kinase-dependent increase in tyrosine phosphorylation of a subset of proteins (2, 3). Because a protein kinase cascade is involved in the regulation of the sperm fertilizing ability, it is important to characterize the proteins that undergo phosphorylation and examine how these changes relate to capacitation.Post-translational protein phosphorylation by protein kinases plays a role in many cellular processes including transduction of extracellular signals, intracellular transport, and cell cycle progression. The use of two-dimensional gel electrophoresis followed by tandem mass spectrometry (MS/MS) 1 provides a comprehensive approach to the analysis of proteins involved in cell signaling (4). Specifically, changes in tyrosine phosphorylation can be monitored using two-dimensional gel electrophoresis followed by Western blot analysis with antiphosphotyrosine (␣-PY) antibodies (5). Proteins that undergo changes in tyrosine phosphorylation during cellular pr...

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy.

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.

CD9 is a tetraspan protein that associates with several ␤1 integrins, including ␣6␤1. Because ␣6␤1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin ␤), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin ␤ disintegrin domain. (Both ligands bind to the egg via ␣6␤1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin ␣6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, ␣6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin ␤ ''knockout'' sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin ␤ ؊͞؊ male mice. Our results suggest that high-avidity binding between fertilin ␤ (ADAM 2) and ␣6␤1 requires cooperation between ␣6␤1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as spermegg interactions.