Eppin (epididymal protease inhibitor [SPINLW1]) is present in a protein complex on the human sperm surface that contains lactotransferrin, clusterin, and semenogelin (SEMG1). During ejaculation the presence of semenogelin inhibits sperm progressive motility until semenogelin is hydrolyzed by prostate-specific antigen (PSA). Although eppin binds all three components in its protein complex, the binding of semenogelin to eppin appears to be critical for the inhibition of progressive motility. The effect of the originally identified seminal plasma motility inhibitor fragment has not been clearly defined on live spermatozoa. Therefore, we have used recombinant semenogelin (rSEMG1) and its fragments, including a semenogelin mutant in which cysteine 239 was changed to glycine, coupled with a computer assisted sperm analysis assay to study the motility inhibitory properties of semenogelin. Each fragment and the mutant were tested for their effects on motility. Recombinant semenogelin significantly inhibited sperm progressive motility in a dose- and time-dependent manner. The C-terminal semenogelin fragment (amino acids 164–283) containing cysteine 239 significantly inhibited sperm progressive motility, whereas the N-terminal fragment (amino acids 24–163), a short C-terminal fragment (amino acids 172–215) without cysteine 239, and the mutant fragment (amino acids 24–283 with glycine 239) did not inhibit motility. After treatment with recombinant semenogelin, spermatozoa could be washed and treated with PSA, partially reversing the inhibition of progressive motility. Cysteine 239 of rSEMG1 appears to be the critical amino acid for both binding to eppin and inhibiting sperm motility.

Recombinant semenogelin inhibits human sperm motility; the amino acid, CYS 239 is required for inhibition.

In mammalian spermatocytes, cell division cycle protein 2 (CDC2)/cyclin B1 and the chaperone heat shock protein A2 (HSPA2) are required for the G2→M transition in prophase I. Here, we demonstrate that in primary spermatocytes, linker histone chaperone testis/embryo form of nuclear autoantigenic sperm protein (tNASP) binds the heat shock protein HSPA2, which localizes on the synaptonemal complex of spermatocytes. Significantly, the tNASP-HSPA2 complex binds linker histones and CDC2, forming a larger complex. We demonstrate that increasing amounts of tNASP favor tNASP-HSPA2-CDC2 complex formation. Binding of linker histones to tNASP significantly increases HSPA2 ATPase activity and the capacity of tNASP to bind HSPA2 and CDC2, precluding CDC2/cyclin B1 complex formation and, consequently, decreasing CDC2/cyclin B1 kinase activity. Linker histone binding to NASP controls the ability of HSPA2 to activate CDC2 for CDC2/cyclin B1 complex formation; therefore, tNASP's role is to provide the functional link between linker histones and cell cycle progression during meiosis.

Linker histone binding to nuclear autoantigenic sperm protein (NASP) controls the ability of HSPA2 to activate CDC2 for CDC2/cyclin B1 complex formation; therefore, tNASP's role in the synaptonemal complex is to provide a link between DNA repair and cell cycle progression during meiosis.

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Anti-eppin antibodies and recombinant semenogelin inhibit human sperm motility.

Eppin (epididymal protease inhibitor; official symbol SPINLW1) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin (SEMG1) on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight line distance and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in non-progressive spermatozoa; however, approximately 25% of antibody treated spermatozoa could be rescued by the addition of cAMP-AM, indicating that anti-eppin treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block SEMG1 binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to SEMG1. Addition of affinity purified antibodies made to the dominant C-terminal epitope of eppin (S21C) had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a non-steroidal male contraceptive.