cDNA cloning of a stage-specific gene expressed during HCG-induced spermatogenesis in the Japanese eel

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin betaB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.