Spermatogonial stem cells differentiation and testicular lobules formation in a seasonal breeding teleost: The evidence from the heat-induced masculinization of genetically female Japanese flounder (Paralichthys olivaceus)

Germ cells undergo a series of cellular changes including differentiation, mitosis, meiosis and maturation, and eventually develop into a large number of functional gametes. The available data regarding teleost gametogenesis in seasonal batch spawners is limited. In this study, we investigated spermatogenesis with special attention on spermatogonia differentiation using heat-induced masculine juveniles of genetically female Japanese flounder. Meanwhile, the Nanos2 expression had been detected by immunohistochemistry for analysis of spermatogonial stem cells (SSCs) distribution. Spermatogonia began mitosis at 35 dph, and basement membrane firstly appeared and gradually surrounded and separated the spermatogonia (type A) into single and paired status. At this period, the spermatogonia continuously maintained mitotic proliferation. As a result, the number of spermatogonia including isolated and clusters (2-8 spermatogonia) significantly increased in the presumptive testes. From 85 dph to 120 dph, with the mitosis of spermatogonia, germline acinar-clusters formed. In the clusters, type A spermatogonia differentiated into type B, and multi-spermatogonia surrounded by several sertoli cell formed cysts，which represented the formation of lobular precursors. After that, type B spermatogonia began meiosis, which indicated the initiation of spermatogenesis. In adult testes, most type A spermatogonia distributed in the peripheral region and a few clung to a basement membrane in the internal germinal epithelium. Various spermatogenic cysts with germ cells in different developmental stages existed in a testicular lobules, moreover the germ cells in earlier stages resided in the distal termini, and the advanced stages were adjacent to the spermatic duct of testes. Therefore, the testes of Japanese flounder belonged to an intermediated distribution of SSCs, which might contribute greatly to multiple spermiation during breeding season. These findings would improve the understanding the mechanisms of SSCs differentiation and testicular development, and may be of great value in future studies of the spermatogenesis regulation.