Single-cell RNA sequencing (scRNA-seq) is widely adopted for identifying the signature molecular markers or regulators in cells, as this would benefit defining or isolating various types of cells. Likewise, the signature transcriptome profile analysis at the single cell level would well illustrate the key regulators or networks involved in gametogenesis and gonad development in animals; however, there is limited scRNA-seq analysis on gonadal cells in lower vertebrates, especially in the sexual reversal fish species. In this study, we analyzed the molecular signature of several distinct cell populations of Asian seabass adult ovaries through scRNA-seq. We identified five cell types and also successfully validated some specific genes of germ cells and granulosa cells. Likewise, we found some key pathways involved in ovarian development that may concert germline-somatic interactions. Moreover, we compared the transcriptomic profiles across fruit fly, mammals, and fish, and thus uncovered the conservation and divergence in molecular mechanisms that might drive ovarian development. Our results provide a basis for studying the crucial features of germ cells and somatic cells, which will benefit the understandings of the molecular mechanisms behind gametogenesis and gonad development in fish.

Spermatogonial stem cells (SSCs) are unique adult stem cells capable of self-renewal and differentiation into sperm. Grouper is a protogynous hermaphroditic fish farmed widely in the tropical and subtropical seas. In this study, we established an SSC line derived from adult testis of orange-spotted grouper, Epinephelus coioides. In the presence of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), the cells could be maintained with proliferation and self-renewal over 20 months and 120 passages under in vitro culture conditions. The cells exhibited strong alkaline phosphatase activity and the characteristics of SSCs with the expression of germ cell markers, including Vasa, Dazl, and Plzf, as well as the stem cell markers Nanog, Oct4, and Ssea1. Furthermore, the cultured cells could be induced by 11-ketotestosterone treatment to highly express the meiotic markers Rec8, Sycp3, and Dmc1, and produce some spherical cells, and even sperm-like cells with a tail. The findings of this study suggested that the cultured grouper SSC line would serve as an excellent tool to study the molecular mechanisms behind SSCs self-renewal and differentiation, meiosis during spermatogenesis, and sex reversal in hermaphroditic vertebrates. Moreover, this SSC line has great application value in grouper fish aquaculture, such as germ cell transplantation, genetic manipulation, and disease research.