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LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22
Citation:
Klinefelter, G R., J. E. WELCH, H. MOORE, K. L. BOBSEINE, J. D. SUAREZ, N. L. ROBERTS, AND R. M. ZUCKER. LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. Presented at International Congress of Andrology, Montreal, Quebec, Canada, June 15-19, 2001.
Description:
LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. GR Klinefelter1, JE Welch*1, HDM Moore*2, K Bobseine*1, J Suarez*1 ,N Roberts*1 ,R Zucker *1 1U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC and 2University of Sheffield, Sheffield, UK.
We previously found that levels of SP22 are highly correlated with fertility. Herein, we sought to: 1) describe localization of SP22 in rat testis, epididymis, and sperm, 2) demonstrate inhibition of fertility of rat sperm in vivo and in vitro, and 3) identify linear epitopes. Polyclonal antiserum was raised in sheep against full-length recombinant SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that rSP22 Ig and ascites recognized denatured and native SP22, respectively. Cytoplasm of pachytene spermatocytes and round spermatids immunostained with both rSP22 Ig and ascites. In stage VIII, staining was concentrated along the spermatid head. Isolated rete testis sperm revealed discrete staining at the base of the head; staining migrated to the equatorial segment of the head as sperm moved to the cauda epididymidis. Interestingly, clear cells were immunostained along the length of the epididymis. Ascites, rSP22 Ig, and rSP22 Fab each inhibited fertilization in vivo and in vitro. For example, rSP22 Ig (1:50) added to cauda epididymal sperm 5 minutes prior to in utero insemination decreased fertility from 74% to 34%, and rSP22 Ig (1:10) added to sperm incubated overnight with eggs decreased fertilization from 81% to 39%. Linear epitope mapping of the 189 amino acid SP22 sequence revealed three distinct sequences recognized by rSP22 Ig and one sequence recognized by ascites. Current work is focused on the potential role of these sequences in sperm-egg interaction.