Saga of a sperm fertility biomarker

A decade ago a novel sperm protein associated with the fertility of sperm was discovered by quantifying individual proteins in the sperm membrane proteome of cauda epididymal sperm from rats exposed to epididymal toxicants that compromised the fertility of these sperm. Upon identification, this protein (SP22) was found to a ubiquitous, highly conserved protein never before observed in the male reproductive tract. The expression of SP22 in sperm appears driven by a testis specific mRNA transcript, and the molecule is translocated from the cytoplasmic droplet of rete testis sperm to the equatorial segment of epididymal and ejaculated sperm. The appearance of SP22 mRNA and protein coincide with the formation of pachytene spermatocytes and round spermatids, respectively, and given this testis ontogeny of SP22, we validated its use as a biomarker of fertility by extending our studies to toxicants that target spermiogenesis. Studies of both epididymal and testicular toxicants now have demonstrated that compromised SP22 gene expression is sensitive and correlated with fertility. Importantly, this applies to ejaculated sperm as well as epididymal sperm. With the goal of developing a user-friendly diagnostic assay for SP22 on epididymal and ejaculated sperm, we are attempting to identify exposed, functional domains of the protein. For this, we have generated antibodies to both full length and truncated SP22 recombinants, as well as antibodies to synthetic SP22 peptides. Each antibody has been characterized for its ability to inhibit fertilization both in utero and in vitro. Linear epitope mapping has been done for each antibody, and synthetic peptides corresponding to each epitope have been used in competition experiments designed to elucidate exposure on the sperm surface and function. Most of the linear epitopes identified appear to be exposed although there are relative differences in the degree of their exposure. Interestingly, one of the exposed epitopes does not appear to be functional, at least by itself. Many more domains of the molecule need to be studied, but based on our findings with the epitopes already identified, it seems a combinatorial targeting strategy may be beneficial. If one assumes that the protein's role in fertility resides in a single exposed epitope, or some combination of exposed epitopes, such targeting may also ultimately lead to successful modulation of the fertilizing potential of sperm.