Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were M(r) 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2-D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the protein's functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of acrosomal exocytosis.

Zonadhesin is a mosaic protein in sperm membrane fractions that binds directly and in a species-specific manner to the extracellular matrix (zona pellucida) of the oocyte. The active form of pig zonadhesin from capacitated, epididymal spermatozoa comprises two covalently associated polypeptide chains of M r 105,000 (p105) and M r 45,000 (p45). Here we report detection and characterization of multiple zonadhesin isoforms in freshly ejaculated cells. Antibodies to the predicted von Willebrand D0-D1, D1, and D3 domains of pig zonadhesin recognized p105, p45, and additional M r 60,000 -90,000 polypeptides in particulate fractions of uncapacitated cells. Although the p105/45 form constituted a minority of all zonadhesin forms in sperm membrane fractions, it was the predominant form capable of binding to the pig zona pellucida. Zonadhesin-binding sites were distributed over the entire zona pellucida. Anion exchange chromatography resolved active, p105/45 zonadhesin from the p60 -90 inactive forms. Without disulfide bond reduction some zonadhesin was M r >300,000, including M r 300,000 and 900,000 proteins comprising in part multimers of p105/45. The multimeric forms did not bind the zona pellucida as avidly as did the p105/45 monomer. Expressed D1 and D3 domain fragments containing the CG(L/V)CG sequence motif spontaneously formed multimers at ؊246 mV E h in vitro. Double Cys 3 Ser mutants of the D1 fragment formed multimers with the same apparent kinetics as the wild type protein. Zonadhesin localized to the apical head of pig spermatozoa. We conclude that a heterogeneous combination of specific proteolysis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pellucida.Adhesion of mammalian spermatozoa to the zona pellucida (ZP) 1 is a complex process mediated by binding of sperm proteins to complementary ligands in the ZP (1, 2). The complexity of this process derives partly from cellular changes that occur during gamete interactions. Spermatozoa undergo physiological changes in the female reproductive tract that are required for fertilization and are collectively called capacitation (1, 3). Although the molecular basis of capacitation is only partly understood, in some if not all species avidity of sperm-ZP adhesion increases as capacitation progresses. After capacitation is completed, the membranes involved in initial adhesion events are lost from the sperm surface during the acrosome reaction, but adhesion is sustained by interaction of newly exposed structures with the ZP (1, 2). Unique adhesion molecule pairs likely function at different times during fertilization, and the activities of these molecules may change as fertilization progresses (2). It is therefore important to assess the biochemical and functional properties of sperm adhesion molecules at each stage in the fertilization process. Several sperm proteins that may mediate adhesion to the ZP have been identified and characterized (2). Among these molecules zonadhesi...

Women are more susceptible to anterior cruciate ligament (ACL) injuries than men performing similar athletic activities. Because tissue remodeling may affect ligament strength, we assessed expression of tissue remodeling effector genes in the human ACL. Specifically, we surveyed ACL for RNAs encoding all known matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) by reverse transcription/polymerase chain reaction (RT-PCR). These experiments revealed that mRNAs encoding nine of sixteen MMPs and all four TIMPs are present in the normal ACL. The nine expressed proteases were MMPs 1-3, 7, 9, 11, 14, and 17 (collagenase 1, gelatinase A, stromelysin 1, matrilysin, gelatinase B, stromelysin 3, and membrane types 1 and 4, respectively), and MMP-18. Genes for MMPs 8, 10, 12, 13, 15, and 16 appeared not to be expressed in ACL, as their mRNAs were not detected using RT-PCR conditions that did yield positive signals from other tissues (testis or bone). We conclude that numerous genes encoding tissue remodeling effector proteins are expressed in the human ACL.

Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 µg/h) into fasted adult male rhesus macaques (6–9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42–57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 ± 1.4 ng/ml control vs. 2.7 ± 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 ± 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 ± 1.0 ng/ml) and the fasted + rrhLep (2.85 ± 1.0 ng/ml) groups compared to controls (16.3 ± 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 ± 0.95 pulses/15 h control vs. 12.8 ± 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 ± 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 ± 0.37 ng/ml in control animals, 0.7 ± 0.2 ng/ml in fasted animals and 10.1 ± 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque.

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To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with M(r) 7500-66,000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2-4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.

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