title: Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro.
abstract: We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat 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 affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization.
authors.affiliation: US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North Carolina 27711, USA. klinefelter.gary@epa.gov
authors.forename: Gary R
authors.initials: GR
authors.lastname: Klinefelter
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Welch
authors.forename: Sally D
authors.initials: SD
authors.lastname: Perreault
authors.forename: Harry D
authors.initials: HD
authors.lastname: Moore
authors.forename: Robert M
authors.initials: RM
authors.lastname: Zucker
authors.forename: Juan D
authors.initials: JD
authors.lastname: Suarez
authors.forename: Naomi L
authors.initials: NL
authors.lastname: Roberts
authors.forename: Kathy
authors.initials: K
authors.lastname: Bobseine
authors.forename: Susan
authors.initials: S
authors.lastname: Jeffay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D004822:Epididymis / Q000166:cytology; D018604:Epitope Mapping; D005298:Fertility / Q000276:immunology; D066298:In Vitro Techniques; D008297:Male; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D000071617:Protein Deglycase DJ-1; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity; D013094:Spermatozoa / Q000737:chemistry* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; C404267:PARK7 protein, rat; D000071617:Protein Deglycase DJ-1