Abstract
Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the process of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells from orange-spotted grouper was performed to explore the molecular mechanisms underlying spermatogenesis.
Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone implantation. During the artificial spermatogenesis, different cell types from cysts containing spermatogonia, spermatocytes, spermatids, and spermatozoa were isolated by laser capture microdissection. Subsequently, transcriptomic analysis for the isolated cells were performed. A series of genes was used to verify and investigate the expression patterns in spermatogenesis. Furthermore, we also analyzed the expression of the same set of genes involved with steroid metabolism and sex throughout spermatogenesis (early-mid, late, and maturing stages) in the orange-spotted grouper. Several generally female-related genes took significantly changes in sex reversal hinted that the female-related genes in previously recognized may also play vital roles in spermatogenesis and sex reversal. In the transcriptomic data, we focused on zbtb family genes, which may be related to the process of spermatogenesis. Their expression patterns and cellular localization were examined, and the location of Eczbtb40 in different gonadal stages was investigated. We found that Eczbtb40 was expressed throughout spermatogenesis. These preliminary findings suggest that Eczbtb40 is highly conserved during vertebrate evolution and plays roles in spermatogenesis. Besides, the expression of Eczbtb40 and Eccyp17a1a overlapped in male germ cells, especially spermatogonium and spermatocyte, which suggested that Eczbtb40 might interact with Eccyp17a1a participant in spermatogenesis and sex reversal.
Conclusions: The present study first depicted RNA sequencing of the male germ cells from orange-spotted grouper, and identified many important functional genes and pathways involved in spermatogenesis. The Eczbtb40 gene was subjected to molecular characterization and expression pattern analysis. These results will contribute to future studies of the molecular mechanism of spermatogenesis and sex reversal.