PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica)

Abstract

Spermatogenesis can be initiated by a single injection of human chorionic gonadotropin (hCG) into the cultivated Japanese eel, which produces only spermatogonia in the testis. To isolate the genes responsible for regulating spermatogenesis, we performed a differential mRNA display using poly (A)+ RNA extracted from the testes at different time points after hCG injection. Among several cDNA clones, the expression of which was initiated before the onset of meiosis, one clone has high homology with the proliferating cell nuclear antigen (PCNA). In this study, we investigated the protein expression of eel PCNA and found for the first time in any species that two forms (32-kDa and 36-kDa) of PCNA are present in the testis. Although the 36-kDa form existed in both the testis and spleen, the 32-kDa form was specifically expressed in the testis. In contrast to the appearance of 36-kDa PCNA 1 day after the hCG treatment, the 32-kDa PCNA appeared only 9 days after the hCG treatment, at which time active spermatogonial proliferation occurred in the testis. Both the 32- and 36-kDa forms were recognized by antibodies raised against different epitopes of PCNA, and their N-terminal amino acid sequences were identical. The 36-kDa form, but not the 32-kDa form, was recognized by antibodies against phosphoamino acids. These results suggest that the two PCNA proteins are the same molecule with different chemical modifications, including phosphorylation. We discuss the roles of these two forms of PCNA in the spermatogenesis of the Japanese eel.