Browsing by Author "Kaydos, Emilie Hayes"

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    Fertility and the sperm membrane biomarker (SP22) are compromised in an additive fashion by priority disinfection by-products of drinking water: a validation of enzyme linked immunosorbant assay (ELISA) for SP22.
    (2004-07-12) Kaydos, Emilie Hayes; William Flowers, Ph.D., Committee Co-Chair; Russell Borski, Ph.D., Committee Co-Chair; Gary Klinefelter, Ph.D., Committee Member; Damian Shea, Ph.D., Committee Chair
    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 demonstrated that BCA compromises both the fertility of cauda epididymal sperm and SP22, a sperm membrane protein highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether levels of SP22 on sperm and fertility were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22 to replace the tedious two-dimensional (2D) gels quantitation. The initial study consisted of 8 treatment groups and a water vehicle control group, on which animals were 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. BCA and DBA were also given as two binary mixtures: 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. Proximal cauda epididymal sperm membrane proteins (30 μg) were resolved following concentration/desalting in 2D gels under denaturing conditions (2D SDS-PAGE) and the SP22 protein was quantified. In addition, SP22 was quantified by enzyme-linked immunosorbant assay (ELISA). Full length rat recombinant SP22 (rSP22) was used to generate a standard curve and affinity purified sheep anti-rSP22 was used as primary antibody. The ED50 for the decrease in SP22 quantified by 2D SDS-PAGE for DBA and BCA was 7.15 and 4.61 mg/kg. The ED50 for the decrease in SP22 quantified by ELISA for DBA and BCA was 8.10 and 5.93 mg/kg. The second 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. In this study proximal cauda sperm were also used for in utero insemination to assess fertility. The ED50 for the decrease in fertility for DBA and BCA was 3.5 and 2.7 mg/kg. Immunostaining for SP22 in the testis revealed staining of both round and elongating spermatids and decreased staining in testes exposed to the DBA + BCA mixture. An evaluation of SP22 in testicular parenchyma was also performed by ELISA and Western blotting. Both evaluations revealed a treatment-related decrease in SP22. For either the 2D SDS-PAGE or ELISA quantitation of SP22 on sperm or the fertility of sperm, additivity or synergy is indicated. Finally, the correlation between SP22 levels measured by ELISA versus fertility was r2=0.74, compared to 0.82 for SP22 levels measured by 2D SDS-PAGE versus fertility, suggesting the ELISA could be used to supplant the time-intensive SDS-PAGE.