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THE FERTILITY BIOMARKER (SP22) IS COMPROMISED IN AN ADDITIVE FASHION BY HALOACIDS: COMPARATIVE QUANTITATION BY IMMUNOASSAY AND 2D-GEL ANALYSIS
Citation:
KAYDOS, E., J. D. SUAREZ, N. L. ROBERTS, L. F. STRADER, K. L. BOBSEINE, AND G. R. KLINEFELTER. THE FERTILITY BIOMARKER (SP22) IS COMPROMISED IN AN ADDITIVE FASHION BY HALOACIDS: COMPARATIVE QUANTITATION BY IMMUNOASSAY AND 2D-GEL ANALYSIS. Presented at Society for the Study of Reproduction, Cincinnati, OH, July 19-22, 2003.
Description:
Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water known to produce defects in spermatogenesis and fertility in adult rats. Previous work in our laboratory demonstated a high correlation between fertility of sperm from the cauda epididymidis and the sperm membrane protein SP22. In this study, we administered DBA and BCA, individually and in combination, to determine whether levels of SP22 on sperm were diminished in an additive fashion. Secondly, we wished to establish the use of an immunassay for quantitation of SP22 to replace the more tedious quantitation in 2D gels. The study consisted of 8 treatment groups and a water vehicle control group, each comprised of 8 animals exposed by oral gavage daily for 14 days. BCA was administered alone at 1.6, 4, and 8 mg/kg, and DBA was given in equimolar fashion at 2, 5, and 10 mg/kg. Finally, BCA and DBA were given as two binary mixtures: 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. After two weeks, the males were necropsied. Following in utero insemination to assess fertility, remaining proximal cauda epididymal sperm were washed and detergent extracted. Membrane proteins (30 ug) were resolved following concentration/desalting by two dimensional electrophoresis under denaturing conditions (2D SDS-PAGE) and the SP22
protein was quantified. In addition, SP22 (0.5-20 ug) was quantified by enzyme-linked immunosorbant assay (ELISA). Full length rat recombinant
SP22 (rSP22; 0.5-20 ng) was used to generate a standard curve and affinity purified sheep anti-rSP22 was used as primary antibody. Based on ELISA concentration-response curves, we selected the values at the 10 ug concentration to compare treatments. The ED50 for the decrease in SP22 quantified by SDS-PAGE for DBA and BCA was 0.8 and 2.8 mg/kg, respectively; a relative potency factor (RPF) of 3.5 is indicated. The ED50 for the decrease in SP22 quantified by ELISA for DBA and BCA was 1.5 and 2.8 mg/kg, respectively; an RPF of 1.9 is indicated. Based on either SDS-PAGE or ELISA quantitation, and both low and high dose mixtures, effect additivity is indicated for haloacid induced compromise in the fertility biomarker SP22.