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. lmmunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (lg) and ascites lg 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 lg, and 1 sequence recognized by ascites lg. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites O ur initial work described the diminution of a 22-kilodalton (kd) protein in detergent extracts of cauda epididymal sperm from animals exposed for 4 days to epididymal toxicants such as ethane dimethanesulphonate, chloroethylmethanesulphonate, epichlorohydrin, and the known antiandrogen, hydroxyftutamide (Klinefelter et al, 1997). This protein, known as SP22, was the only silver-stained protein out of 120 proteins resolved by twodimensional (2-D) gel electrophoresis that was quantitatively altered in a dose-related and treatment-related fashThe information in this document has been funded wholly (or in part) by the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.Correspondence to: Gary R. Klinefelter, NHEERL, RTD, MD #72, Research Triangle Park, NC 277 l l (e-mail: klinefelter.gary@epa.gov).Received for publication June l l , 200 1; accepted for publication July 24, 2001. 48antibodies. 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 lg and anti-SP22 lg each recognized the equatorial segment of sperm heads from rat, hamster, bull , rabbit, and human. Ascites lg and affinity-purified anti-rSP22 lg 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-rSP2...

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Guanethidine, a chemical that selectively abolishes peripheral noradrenergic nerves, was used to investigate the role of sympathetic innervation in the maintenance of epididymal sperm quantity and quality. Four groups of 10 adult male rats each were treated daily for 21 days, by i.p. injections, with either 0 (saline vehicle), 6.25, 12.5, or 25 mg/kg guanethidine. Norepinephrine content was reduced to undetectable levels in the cauda epididymidis in all guanethidine groups after 3 wk of treatment and was reduced to 7.4% of the control values after 1 wk of 6.25 mg/kg treatment. While body weight gain was significantly decreased at 12.5 and 25 mg/kg compared to that in controls, there was a significant increase in the weights of the seminal vesicles/coagulating glands in all treated groups. The number of homogenization-resistant spermatids per testis and the daily sperm production per testis remained unchanged. The weight of the epididymis was significantly increased at 6.25 and 12.5 mg/kg. Moreover, the number of cauda epididymal sperm and the transit time were increased significantly at 6.25 mg/kg (10.2 days) compared to values in the control cauda (6.3 days). Neither serum testosterone levels nor LH was affected in a dosage-related manner. There were no effects of guanethidine treatment on cauda epididymal sperm motility or morphology. A quantitative analysis of detergent-extracted cauda epididymal sperm proteins by SDS-PAGE revealed no differences, but there were diminutions in seven proteins in homogenates of caput/corpus tissue. Histologic analysis of testis and epididymis sections revealed no differences between control and denervated animals. In a subsequent experiment the lowest effective dosage (6.25 mg/kg) was given to rats for 1 wk, and an increased number of cauda epididymal sperm and a delay in sperm transit were observed. Our results indicate that low-dosage guanethidine exposure denervates the epididymis within 1 wk, thereby delaying epididymal transit; however, neither 1- nor 3-wk exposure produces qualitative changes in the sperm.