Male Reproductive Tract

Association of Eppin with Semenogelin on Human Spermatozoa

Abstract
Eppin (SPINLW1; GeneID: 57119) is a single copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that in seminal plasma and on human spermatozoa following ejaculation Eppin is bound to semenogelin I (Sg). Six different experimental approaches: 1) Immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) Co-localization in semen and spermatozoa, 3) Incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) Far-Western blotting of Eppin and Sg, 5) Saturation binding of ¹²⁵I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of ¹²⁵I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin^75-133 C-terminal fragment binds the Sg^164-283 fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys²³⁹ blocks binding of ¹²⁵I-rEppin indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide anti-microbial activity for spermatozoa, which has been reported for both Eppin and semenogelin derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Key words: Male Reproductive Tract • Seminal vesicles • Sperm

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