MOUSE PROTAMINE GENES ARE CANDIDATE TARGETS FOR THE NOVEL ORPHAN NUCLEAR RECEPTOR, GERM-CELL NUCLEAR FACTOR

Proper expression of the protamine genes is an important event in the terminal differentiation of the male gametes in mammals. Here we prese nt evidence that the novel orphan member of the nuclear receptor gene superfamily, Germ Cell Nuclear Factor (GCNF), may play a role in the r egulation of these genes. Previously, we have shown that high-level ex pression of GCNF mRNA is restricted to spermatids (stages 1-8) in the adult male mouse, which makes it temporally and spatially available to regulate the mouse protamine genes. Furthermore, we have previously i dentified a sequence to which GCNF can bind, which consists of a direc t repeat of the core half site AGGTCA with zero base pairs spacing the repeats (DRO). We have identified several genes that contain DRO sequ ences in their 5' promoter regions, including the protamines. The mous e protamine 1 and 2 (Prm1 and Prm2) genes therefore are potential targ et genes for GCNF regulation. We show that GCNF binds to one of the tw o DRO sequences in the Prm1 promoter, and to the DRO sequence in the P rm2 promoter in a specific manner. Furthermore, by using antibodies di rected against GCNF, we detect endogenous GCNF in testis nuclear extra cts and elutriated round spermatid nuclear extracts in Western blots. Additionally, by using these antibodies in gel-shift assays, we show t hat this endogenous GCNF can bind to both the Prm1 and Prm2 promoters. This evidence supports the hypothesis that GCNF mediates a novel sign aling pathway, two targets of which may be the Prm1 and Prm2 genes in spermatids. Mel. Reprod. Dev. 50:396-405, 1998.