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.

The development of cell types and regional differences in the rat epididymis was studied in specimens of the initial, middle and terminal segments prepared at intervals between birth and postnatal day 94. The development of the epididymis was divided into three phases: (1) an undifferentiated period; (2) a period of differentiation, and (3) a phase of expansion. During the undifferentiated period, from birth to day 15, the epithelial cells had a uniform appearance. Halo cells, which are believed to be migratory leukocytes, appeared on day 14. The period of differentiation extended from day 16 to day 44. Slender, densely staining cells, termed narrow cells, appeared in the epithelium of all three segments on day 16, constituting the first evidence of differentiation of cell types in the epididymal epithelium per se. In addition to their shape and apical nuclei, the narrow cells were distinguished from other epithelial cells by the presence of cup-shaped apical vacuoles and mitochondria with tubular cristae. Principal cells and basal cells were identified on day 28, which also marked the firsh distinction of differences in epithelial height among the different segments. Narrow cells persisted into the adult in the initial segment. In the middle and terminal segments, however, narrow cells disappeared by day 35, when light cells made their appearance. The major event of the period of expansion, from day 45 to 3 months, was the appearance of sperm in the lumen between days 45 and 52. A model for differentation of cell types in the epididymis is proposed and it is suggested that narrow cells are precursors to light cells in the middle and terminal segments. The development of ultrastructural features of adult cell types preceded the appearance of sperm in the lumen.

The objective of this study was to identify the repertoire of proteins exposed on the surface of ejaculated human spermatozoa. High-resolution two-dimensional gel systems for separation of human sperm and seminal plasma proteins were developed using both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by polyacrylamide gel electrophoresis (IEF/PAGE, NEPHGE/PAGE). Proteins were visualized by silver staining of gels and by electroblotting followed by gold staining. The protein patterns were analyzed by computer after laser or camera scanning. One thousand three hundred ninety-seven sperm proteins with a molecular mass between 5 and 160 kDa and isoelectric points (pI) from 4 to 11 were catalogued from silver-stained gels loaded with approximately 0.25 mg of NP-40/urea extracts of sperm harvested by Percoll density gradient centrifugation, and 1191 proteins were resolved following extraction with SDS/3-[3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate/urea. Analysis of seminal plasma proteins obtained from vasectomized patients revealed over 300 silver-stained proteins, which aided the identification of sperm-coating proteins acquired from secretions of the accessory sex organs. Sperm surface proteins accessible to vectorial labeling with 125I or N-hydroxysuccinimide biotin were identified; 181 protein spots were radiolabeled with 125I, while 228 protein spots were biotinylated, including several groups of protein isoforms. Cytoskeletal and intra-acrosomal control proteins were not iodinated or biotinylated, thus verifying the surface specificity of both labeling methods. Ninety-eight sperm surface proteins were labeled by both iodine and biotin, and 22 sperm surface proteins, representing five groups of protein isoforms, were shown to contain phosphotyrosine. A composite computer image showing the position of the dually vectorially labeled sperm surface proteins was constructed, together with a table of the proteins' molecular weight, pI, and relative concentration. In addition, novel isoforms of actin, beta-tubulin, PH-20, and several phosphotyrosine-containing proteins were identified in human sperm.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.