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Simple Summary: Sex formation of gonads encompasses two ancient and highly conserved biological processes, sex determination and sex differentiation. The processes are strictly regulated by a complex of gene networks. There is increasing evidence that miRNAs play key roles in many biological processes. however, information is limited in their contribution to sex differentiation in animals. In the present study, we identified the novel miRNAs involved in sex-related genes regulation and explored the miRNA–mRNA networks underlying the posttranscriptional regulation during the initial sex differentiation in Zhikong scallop, Chlamys farreri. Our findings provide an important basis for studying the sex differentiation mechanisms, as well as developing sex control techniques in bivalves. Research on expressional regulation of genes at the initial sex differentiation of gonads will help to elucidate the mechanisms of sex determination and differentiation in animals. However, information on initial sex differentiation of gonads is limited in bivalves. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that can regulate the target gene expression at the posttranscription level by degrading the mRNA or repressing the mRNA translation. In the present study, we investigated the small RNAs transcriptome using the testes and ovaries of Zhikong scallop Chlamys farreri juveniles with a shell height of 5.0 mm, a critical stage of initial sex differentiation of gonads. A total of 75 known mature miRNAs and 103 novel miRNAs were identified. By comparing the expression of miRNAs between the ovary and testis, 11 miRNAs were determined to be differentially expressed. GO annotations and KEGG analyses indicated that many putative target genes that matched to these differentially expressed miRNAs participated in the regulation of sex differentiation. Furthermore, two selected miRNAs, cfa-novel_miR65 and cfa-miR-87a-3p_1, were confirmed to downregulate expressions of Foxl2 (a female-critical gene) and Klf4 (a male-critical gene), respectively, using a dual-luciferase reporter analysis. Our findings provided new insights into the initial sex differentiation of gonads regulated by miRNAs in bivalves. [ABSTRACT FROM AUTHOR]

SOX17, a member of SRY-related high-mobility-group box (SOX) family, involves in endoderm formation, angiogenesis and carcinogenesis, and its expression characteristics are different in spermatogenesis among several vertebrates. In this study, we cloned a full-length cDNA sequence of sox17 from Zhikong scallop Chlamys farreri , and determined its expression characteristics in gonad at mRNA and protein levels. The cDNA sequence was 2802 bp in length, predicted to encode a protein of 511 amino acids and contained a conservative HMG-box of SOX family, while lacked the C-terminal region of SOX17 comparing to vertebrates. Semi-quantitative RT-PCR showed that C. farreri sox17 ( Cf-sox17 ) mRNA exhibited a different tissue distribution, and the transcript abundance was the highest in the gonads. In situ hybridization determined that the Cf-sox17 mRNA was located in various germ cells in testis and ovary. Similar result of Cf- SOX17 protein was also observed by immunohistochemical detection. The location in gonad is different from that of mammals and fish in which SOX17 is only located in some specific germ cells. Our finding revealed a different characteristic of sox17 expression in gametogenesis between scallop and vertebrates, which implied that Cf-sox17 may involve in gametogenesis of bivalves and the function may differ from that in vertebrates. [ABSTRACT FROM AUTHOR]

Vitellogenin (VTG), the precursor to yolk protein vitellins (Vn), has been widely studied in oviparous animals. However, information about VTG expression during ontogenesis is limited. In this study, we investigate the developmental expression pattern of the zhikong scallop gene,Chlamys farreri vitellogenin (Cf-vtg), usingin situhybridization and real-time RT-PCR techniques. TheCf-vtgmRNA was detected in unfertilized eggs, fertilized eggs, embryos and early-stage larvae, indicating a maternally suppliedCf-vtg. It was located restrictively in D-shaped larvae, and its concentration declined at the gastrula stage and approximated zero from umbo-larva to juveniles of 4–6 mm shell height. However, it was expressed distinctly again in the hepatopancreas of both males and females with shell heights from 7 to 12 mm and in the ovary during the first gonadal development cycle. This report provides experimental evidence of the expression pattern ofvtgwith the maternal supplication, the hepatopancreas and male expression inC. farreri. [ABSTRACT FROM AUTHOR]

Highlights: [•] Type 8 17β-HSD was cloned and characterized in scallop Chlamys farreri. [•] Recombinant 17β-HSD8 catalyzed steroids metabolism in vitro. [•] Variations of 17β-HSD8 expression levels were correlated with gametogenesis. [•] 17β-HSD8 was located in follicle cells and gametes except spermatozoa. [•] 17β-HSD8 may regulate estradiol-mediated gametogenesis in the gonad. [Copyright &y& Elsevier]

Simple Summary: Foxl2 generally presents a sexually dimorphic expression pattern in animal gonads and is highly expressed in the ovary. However, few studies on the transcriptional regulation of Foxl2 have been documented. To understand the transcriptional regulating of Foxl2 high expression in the ovary, we used the Y1H system, a high throughput approach, for the first time to screen the transcription factors binding to the high transcriptional activity region of Foxl2 promoter in Zhikong scallop (Chlamys farreri) gonads. In the present study, the highly transcriptional activity promoter sequence of Cf-Foxl2 was determined at −1000~−616 bp and 11 candidate factors were verified to involve in Cf-Foxl2 transcriptional regulation. Our findings provided valuable data for better understanding the specific transcriptional regulation mechanism of Foxl2 in the ovary and would further assist in the breeding of aquacultural bivalves. Foxl2 is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamysfarreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 107 CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of C. farreri Foxl2 (Cf-Foxl2) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary. [ABSTRACT FROM AUTHOR]

The regulation of gonadal development remains largely unknown in bivalves, and the Foxl2 gene has been proposed as a key regulator of female development. However, limited information is available regarding its regulatory mechanisms. In our previous study on Chlamys farreri , a yeast one-hybrid assay was performed, and an unreported factor (designated Cf -FA1) that may act as a new transcription factor for activating Cf-Foxl2 transcription was identified. In the present study, we revealed that Cf -FA1 is a hydrophilic unstable protein possessing multiple proline-rich and serine/threonine-rich regions. Cf-FA1 exhibited a significantly biased expression in the ovary detected by semi-quantitative RT-PCR, and its cyto-localization was exhibited in ovarian cells during oogenesis through in situ hybridization and immunohistochemistry, while not in male gametes during spermatogenesis. Western blotting demonstrated that Cf -FA1 protein also exhibited a similar sexually dimorphic expression pattern and was more concentrated in the nucleus along with ovarian maturation. ChIP assays confirmed that Cf -FA1 binds to the Cf-Foxl2 promoter. Furthermore, the interference of Cf-FA1 expression in the ovary by in vivo RNAi led to an obvious Cf-Foxl2 expression decrease, ovary developmental retardation, gonadosomatic index decline, and female gametes reduction. These results suggest that Cf -FA1 acts as a transcription factor to activate Foxl2 transcription and participates in ovarian development and oogenesis regulation in bivalves, providing an important basis for sex control breeding in aquaculture. • Cf -FA1 acts as a new transcription activator of Foxl2 in scallops. • Cf - FA1 specifically expresses in ovarian cells during oogenesis. • Cf -FA1 accumulates in ovarian cell nuclei during gonadal cycle. • The reduction of Cf - FA1 after RNAi led to the decreased expression of Cf - Foxl2. • Cf -FA1 plays an important role in ovarian development and oogenesis. [ABSTRACT FROM AUTHOR]