Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during 17alpha-methyltestosterone-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides)

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 regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alphamethyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. Conclusions:Our study, for the first time, investigated the transcriptome of the male germ cells from orangespotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

Integration of ATAC-seq and RNA-seq Unravels Chromatin Accessibility during Sex Reversal in Orange-Spotted Grouper (Epinephelus coioides)

“…Prior to our current study, there was no reports about the sexual dimorphism of rergl that implied its potential role in sex regulation. However, unlike other genes, cyp17a1 exhibited an entirely different expression pattern in ovary and testis and its distribution varied in different germ cells [41]. The condition of cyp17a1 hinted at the distinct roles in female and male which is agreed with the results in zebrafish [42].…”

Screening and Validation of p38 MAPK Involved in Ovarian Development of Brachymystax lenok

“…In this study, we concluded that the zf-C2H2 protein was the most abundant type of TF in lenok, and its specific function should be further studied. The ZBTB family refers to a class of proteins containing the N-terminal BTB domain and multiple zinc finger domains at the C-terminal ( 48 ), and more than 60 types of ZBTB proteins have been identified as being involved in development, differentiation, and tumor formation ( 49 ). Recent studies have shown that zbtb16 can regulate spermatogenesis by controlling the self-renewal and repair of spermatogonia ( 50 , 51 ).…”

“…Recent studies have shown that zbtb16 can regulate spermatogenesis by controlling the self-renewal and repair of spermatogonia ( 50 , 51 ). In orange-spotted grouper, zbtb40 is specifically expressed in male germ cells and regulates spermatogenesis through its interaction with cyp17a1 ( 49 ).…”

“…In the cellular component (CC), transcripts were mainly enriched in cells (44,531), cell parts (44,364), and organelles (31,422). Most transcripts enriched in molecular processes (MF) were binding (49,208), catalytic activity (29,970), and transporter activity (4,766). For biological processes (BP), transcripts were mainly enriched in cellular processes (45,934), single-organism processes (37,336), and metabolic processes (33,199).…”

Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides)

“…Germ cell marker genes can be used for exploring PGCs formation and migration, germ cell development, and the mechanism behind gametogenesis. In teleost fish, some germ cell-specific genes have been characterized, including PGC-specific gene Nanos3 [ 4 ], mitotic germ cell-specific gene Ly75 [ 5 ], spermatogonium-specific gene Plzf [ 6 , 7 ], oocyte-specific gene Slbp2 [ 8 ], as well as the well-known germline-specific genes Vasa , Dazl and Piwi [ 9 , 10 ].…”