Orphan Receptor Gcnf In Mouse Epididymis

GCNF(Germ Cell Nuclear Factor) is a member of nuclear receptor superfamily and also an orphan receptor. In previous report, GCNF might involve in sperm differentiation in testis, neuron differentiation in brain, somitogenesis and posterior development in embryo. GCNF expressed two transcripts with size of 2.3 and 7.4kb, and the sequence difference between two transcripts was located in their 3'-UTR. Both transcripts are highly expressed in testis, but the smaller one is restrictedly expressed in some developmental stage of testis, while the larger one could be detected weak in lots of tissues, such as brain, liver, kidney, et al.By improving the sensitivity of Northern blot, three transcripts (2.3kb, 3.1kb and 7.4kb) could be detected in vivo, including one novel transcript with size of 3.1kb. The two larger transcripts expressed spreadly in all the tissues detected, including testis, brain, spleen and epididymis. By RT-PCR and sequence analysis, the sequence of the unique transcript is identifical to that of 7.4kb. And the two transcripts (3.1 and 7.4kb) expressed in epididymis are down-regulated by androgen. Additionally, the transcript (7.4kb) expressed in P19 cell is up-regulated by estrogen and progestrogen.To analysis the proteins coded by three different transcripts in vivo, the polyclonal anti-GCNF antisera was raised. Accidentally, an effective modified immunization method was found, by which to raise an effective polyclonal antisera would only take 35 days. By western blot, a single band with size of 55kD, could be detected in both the nuclear protein extract of testis and whole protein extract of epididymis, which was same as that of deduced ORF. By immunohistochemistry and in situ hybridization, GCNF was regionally expressed in the corpus, with no expression or weak expression in caput and cauda. The regional expression pattern of GCNF in epididymis will contribute to the study the downstream regulated genes by GCNF, which expressed in epididymis.