Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermat ozoa. During this process there occurs remodeling of cell structure and los s of most of the cytoplasm and a large fraction of cellular proteins. To ev aluate the role of the ubiquitin proteolytic system in this protein loss, w e measured levels of ubiquitinated proteins and rates of ubiquitin conjugat ion in extracts of testes from rats of different ages. Endogenous ubiquitin -protein conjugates increased till day 30 and then reached a plateau. In pa rallel, there was a progressive increase in the rate of conjugation of ubiq uitin to proteins in testis extracts from these animals. To test the import ance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extract s was carried out and the remaining conjugation activity in supernatants wa s assayed. Depletion of either enzyme family resulted in decreased conjugat ion. However, most of the conjugation activity and, more importantly, the i ncreased conjugation during development were UBC4-dependent. Immunocytochem istry demonstrated a marked increase in expression of UBC4 in spermatids, c onsistent with the UBC4-dependent activation of conjugation seen in vitro. Zn situ hybridization studies evaluated the contribution of various UBC4 is oforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 m RNA was restricted to germ cells with high levels of expression in round an d elongated spermatids. UBC4-testis had previously been shown to be express ed only in spermatids. Our data suggest that induction of various UBC4 isof orms activates overall conjugation and plays an important role in the cellu lar remodeling and protein loss occurring during spermatogenesis. (C) 1999 Academic Press.