Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.