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Single-cell RNA sequencing reveals the gonadal dynamic changes of sex differentiation in hermaphroditic protogynous orange-spotted grouper, Epinephelus coioides

ABSTRACT: Sex differentiation is a complex process that requires many factors to regulate proliferation, differentiation, development, and organization of the gonads. In teleosts, the molecular mechanisms of sex differentiation are diverse and unclear, especially in hermaphrodite fish. In the present study, 15 cell types, including germ cells, follicle cells, and theca cells, and so on, were identified using single-cell RNA sequencing (scRNA-seq) in the gonads during sex differentiation in a hermaphrodite fish, orange-spotted grouper (Epinephelus coioides). Two pre-follicle cell types were defined, and the differentiating trajectories of the follicle cells were outlined. Notably, both pre-follicle and follicle cells highly expressed male-related genes synchronously, such as amh, sox9, and dmrt3 in pre-follicle cells, as well as dmrt1 in follicle cells. Oocyte possessed two distinct states, with high expression of oocyte development related genes in one state and spermatogenesis related genes in the other state, respectively. Our results provide novel insights into cell types and lineage tracing in the gonads during sex differentiation in hermaphrodite species.

ORGANISM(S): Epinephelus coioides

PROVIDER: GSE218835 | GEO | 2024/07/26

REPOSITORIES: GEO

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Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish. Embryonic gonads were removed by dissection from known genetic female (XX) and male (XY) rainbow trout (Oncorhynchus mykiss) from a transgenic population where expression of GFP is controlled by vasa-gene regulatory elements (Yoshizaki et al. 2000; Takeuchi et al. 2002). These fish were maintained in incubators containing flowing freshwater (10Â°C) at the Ooizumi Research Station, Yamanashi, Japan. Ten female or male transgenic rainbow trout were randomly selected every 5 days, beginning at 30 days (i.e., 300 degree days = incubation temperature in Â°C x number of days) post fertilization through until 70 days (i.e., 700 degree days) post fertilization. The gonads from each sex, at each sampling time, were pooled and immediately frozen for subsequent total RNA isolation.

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Single-cell RNA sequencing reveals the gonadal dynamic changes of sex differentiation in hermaphroditic protogynous orange-spotted grouper, Epinephelus coioides

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