Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.

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Mytilus coruscus is a significant economic species in China’s eastern coastal areas. As a dioecious species, it lacks secondary sexual characteristics, which makes it difficult for selective breeding. However, limited research is carried out on the genetic data regarding reproductive development and gender differentiation. In the current study, de novo transcriptome sequencing analyses were used to detect gonad-specific genes and miRNAs in M. coruscus. By comparing testis and ovary, 159,970 unigenes and 300 miRNAs were obtained totally, of which differentially expressed genes and miRNAs were 9,566 and 25, respectively. Analysis of qRT-PCR showed that cyp26a, dmrt4, foxl2, gdf9, 17β-hsd14, sc6a9, zar1, and zp4 were highly expressed in the ovary as compared to the testis, while sox2 showed lower expression in the ovary. Expression of miR-750-3p, novel 1, and miR-193 was higher in the ovary than that in the testis, whereas the expression of miR-9-5p, miR-9-3p, miR-317, novel 124, miR-2d and miR-263b were lower in the ovary. Furthermore, analysis of miR-317 by Targetscan and MiRanda predicted to target dmrt4 and the luciferase reporter was performed to confirm it. Our research provides a molecular basis for understanding sexual development and reproductive regulation. Further research is needed on the mechanism of gonadal maturation and differentiation in M. coruscus.

MicroRNAs (miRNAs) are regarded as key regulators in gonadal development and sex determination in diverse organisms. However, the functions of miRNAs in gonads of Acrossocheilus fasciatus, an economically important freshwater species in the south of China, are still unclear. Here, high-throughput sequencing was performed to investigate the mRNA and miRNAs on gonads of A. fasciatus. In total, 49,447 unigenes were obtained, including 11,635 differentially expressed genes (DEGs), among which 4147 upregulated genes and 7488 downregulated genes in the testis compared to the ovary, while 300 (237 known, and 63 novel) miRNAs with 36 differentially expressed miRNAs (DEMs) were identified, from which 17 upregulated and 19 downregulated DEMs. GO and KEGG enrichment analysis were performed to analyze the potential biological functions of DEGs and DEMs. Using qRT-PCR, 9 sex-related genes and 9 miRNAs were selected to verify the sequencing data. By dual-luciferase reporter assay, miR-22a-5p and miR-22b-5p interaction with piwil1, and miR-10d-5p interaction with piwil2 were identified. These findings could provide a reference for miRNA-regulated sex control of A. fasciatus and may reveal new insights into aquaculture and breeding concepts.