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The Pacific oyster (Crassostrea gigas), as the most productive cultured shellfish in the world, has considerable economic importance and value. Yet research on its sex determination remains lacking. Here, a novel HMG-box-containing homolog of the sox gene was uncovered in C. gigas (named CgSox-like). Alignment with the HMG-box of sox genes from other animals revealed that CgSox-like had a high similarity to the Sox gene of scallops. Among different adult tissues, CgSox-like was mainly expressed in the gonads. qPCR analysis revealed that CgSox-like and CgSox8 , a sox gene family gene mainly expressed in male gonads, shared similar expression patterns, with elevated expression as the male gonads developed. RNA interference and dual luciferase reporter assays demonstrated that CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. In addition, analysis of CgSox-like and CgSox8 methylation patterns indicated that both genes were in hypomethylation state during the gonadal development. Finally, demethylase inhibitors resulted in elevated expression levels of CgSox-like and CgSox8 genes, suggesting that epigenetics was engaged in modulating the expression of sex-related genes in the Pacific oyster. Taken together, the novel gene CgSox-like was implicated in the sex determination or gonadal development process in male oysters. These results will provide valuable information for the investigation of sex determination mechanism and sex control breeding. • CgSox-like is involved in sex determination or gonadal development in oysters. • CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. • DNA methylation participates in the regulation of sex determination in Crassostrea gigas. [ABSTRACT FROM AUTHOR]

Triploid oysters generate fewer gametes than diploid oysters and exhibit varying degrees of gonad development. The expression of related genes, such as those involved in gametogenesis and energy metabolism, may influence reproductive potential. In addition, microRNAs (miRNAs) are important regulators of gene expression. However, the contribution of miRNAs has not yet been assessed in sterile triploid Pacific oysters (Crassostrea gigas). We therefore obtained extensive miRNA and gene expression data to identify potentially critical miRNA-mRNA interactions that are linked to sterility in triploid C. gigas. Here, we found miR-263-x to be significantly downregulated in the gonads of sterile females. The upregulated target genes were associated with lipid droplet formation, which revealed the likely involvement of nutrient accumulation during gonadal development as regulated by miR-263-x in female sterile triploids. In males, the upregulated miRNAs (miR-1992-y and miR-2001-x) were shown to regulate spermatogenesis-related gene expression, including flagellar formation, and cell proliferation and migration. Our results suggest that energy redistribution regulated by miRNAs may be critical for gonad development in females, indicating that sterile triploids undergo a significant reduction in gamete number and show an increased accumulation of nutrients in the gonads. Our findings further indicate that decreased expression of genes related to spermatogenesis regulated by miRNAs is important in gonadal dysgenesis in sterile triploids. These results shed light on the epigenetic regulatory mechanisms governing sterility in triploid bivalves. • miRNAs played a vital role in the impaired gametogenesis of triploid Pacific oysters. • miR-263-x was associated with the regulation of lipogenesis in female triploid gonads. • Spermatogenesis-related genes were negatively regulated by miR-1992-y and miR-2001-x. [ABSTRACT FROM AUTHOR]

One of the most important physiological process in the life cycle of most bivalves is the sexual reproduction required for species propagation. Understanding the molecular regulatory mechanisms of gender determination and gonad development have potential application in bivalve aquaculture, but there is still limited knowledge about this issue. The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide, as well as a representative specie to study the sexual development in bivalves for its complicated sexuality, including dioecy, sex change and rare hermaphroditism. In this study, an integrated analysis of miRNA and mRNA expression profiles of oyster gonads at different gametogenesis stages was performed to explore their possibly regulatory patterns in sexual development of C. gigas. Based on small RNA sequencing, a total of 122 miRNAs were identified, including 53 conserved and 69 novel miRNAs. Six miRNAs exhibited sex-specific pattern that expressed exclusively from either male or female gonads with increasing abundance throughout the gonadal development. Moreover, 287 putative targets of the six sex-biased miRNAs were predicted by combining previous transcriptome data. GO and KEGG enrichment analysis of target genes revealed that these sex-biased miRNAs might participate in the regulation of gametogenesis. Further, potential miRNA-mRNA interactions based on six sex-biased miRNAs were collected. The homolog of a key participator in testis development, meiotic recombination protein SPO11 in C. gigas (CgSPO11), was predicted as a target of female-biased miRNA lgi-miR-96a. This negative miRNA-mRNA expression relationship was validated in additional samples by quantitative real-time PCR (qRT-PCR) and the regulation of CgSPO11 by lgi-miR-96a was preliminarily confirmed by a dual-luciferase reporter assay. This study provides information regarding the gonad miRNAs of C. gigas and leads to the discovery of miRNA-mRNA interactions in gonad development, which will facilitate further investigations in molecular mechanisms of bivalve sex differentiation. • A total of 122 miRNAs were identified during the Pacific oyster gonad development. • Six sex-biased miRNAs might participate in the regulation of oyster gametogenesis. • The CgSPO11 gene is a target of lgi-miR-96a. [ABSTRACT FROM AUTHOR]