Abstract
Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether fertility and levels of SP22 on sperm were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22. In a dose finding study, animals were exposed by oral gavage daily for 14 days to: BCA alone at 1.6, 4, and 8 mg/kg; DBA at equimolar levels of 2, 5, and 10 mg/kg; and two binary mixtures of 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. The ED(50)s for the decrease in SP22 quantified by two-dimensional SDS-PAGE were 7.2 and 4.6 mg/kg for DBA and BCA. The ED(50)s for the decrease in SP22 quantified by ELISA were 8.1 and 5.9 mg/kg for DBA and BCA. The definitive study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. The ED(50)s for decreases in fertility assessed by intrauterine insemination were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA. Immunolocalization of SP22 in spermatocytes and spermatids, as well as on the cytoplasmic droplet and the equatorial segment of luminal sperm, was decreased by the DBA + BCA mixture. The decrease in SP22 in testicular parenchyma was comparable to that observed for sperm extracts. Based on 2D SDS-PAGE, ELISA, or fertility the haloacid-induced decreases in SP22 or fertility were additive or synergistic. The correlation between SP22 levels by ELISA and fertility was r(2) = 0.72 compared to 0.82 for SP22 levels by 2D SDS-PAGE and fertility, validating SP22 quantitation by ELISA.
Highlights
The information in this document has been funded wholly by the U.S Environmental Protection Agency
We demonstrated that bromochloroacetic acid (BCA) compromises the fertility of cauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm
The sperm membrane protein SP22 was quantified in detergent extracts of proximal cauda epididymal sperm by both 2D SDS–PAGE and ELISA
Summary
The information in this document has been funded wholly (or in part) by the U.S Environmental Protection Agency. The SP22 protein has been shown to be present on sperm from all species examined including human, and antibodies to SP22 significantly inhibit fertilization both in vivo and in vitro (Klinefelter et al, 2002b) The SP22 mRNA (Welch et al, 1998) and protein (Klinefelter et al, 2002b) are expressed in post-meiotic germ cells and elongated spermatids. This protein is a potential molecular target for chemical insults that target spermiogenesis. The goals of this study were to: (1) determine whether haloacid-induced alterations in fertility and SP22 were additive; (2) validate the use of an ELISA for quantitation of SP22; and (3) determine whether haloacid-induced diminutions in SP22 were present in the testis as well as on epididymal sperm
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