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 ìg) 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.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-04132004-133325 |
| Date | 13 April 2004 |
| Creators | Kaydos, Emilie Hayes |
| Contributors | William Flowers, Ph.D., Russell Borski, Ph.D., Gary Klinefelter, Ph.D., Damian Shea, Ph.D. |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-04132004-133325/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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