Identifying Proteins Important for Spermatogenesis Using Golga3<repro27> Mice.

Male infertility is responsible for up to half of all cases of infertility and affects one in 20 men in the general population. In this study we were interested in the role of the GOLGA3 in spermatogenesis. Golga3 is a chemically-induced point mutation in the golgin subfamily A member 3 gene which disrupts spermatogenesis during meiosis and leads to complete male infertility. The purpose of this study was to determine why the Golga3 mutation causes a more severe phenotype on the C57BL/6J (B6) inbred strain compared to the C3HeB/FeJ (C3Fe) strain. We are using a proteomics approach to determine the role of genetic background in controlling spermatogenesis. Global differences in protein expression from the two strains are being examined using 2-D gel electrophoresis. Testes were isolated from 14 day old B6-Golga3 and C3Fe- Golga3 mice and pooled into two biological protein samples (n=4/sample) from each strain. Proteins were isolated using the Bio-Rad sequential extraction kit, quantified using the BCA protein assay, lyophilized, and the protein quality checked by SDS-PAGE in preparation for 2-D gel analysis carried out in collaboration with the proteomics lab at the University of New Mexico. Sixteen protein spots were identified as differentially expressed in Golga3 mutant mice compared between the two strains. The spots were excised, subjected to trypsin digestion, and analyzed by MALDI mass spectrometry including peptide mass fingerprinting and MS/MS analysis. Differentially expressed proteins will be validated and further characterized.