Abstract
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.
Highlights
Reproducing organisms transmitted their genetic information from one generation to the via gametogenesis, a physiological procedure initiated with the primordial germ-cell (PGC) specification and migration (Kunwar and Lehmann, 2003)
The digestion of tissue pieces was stopped using 10% fetal bovine serum (FBS) (Gibco) in phosphate buffer saline (PBS), and the cells were filtered through the 40 μm cell strainer followed by 10 min centrifugation at 300 × g, and washed 2– 3 times with 1× PBS
To construct comprehensive single-cell atlases, single cells were freshly isolated from Asian seabass ovary and the cells were collected using two-step procedure of enzymatic digestion and physical filtering (Valli et al, 2014), were used for sequencing and for global transcriptome analysis using the Illumina NextSeq 500 platform (Figure 1A)
Summary
Reproducing organisms transmitted their genetic information from one generation to the via gametogenesis, a physiological procedure initiated with the primordial germ-cell (PGC) specification and migration (Kunwar and Lehmann, 2003). In recent years, based on molecular and cellular techniques, several identified germ cell markers such as vasa, dazl, and ziwi are known to play essential roles in germ cell development across the animal kingdom (Kehkooi et al, 2009; Leu and Draper, 2010; Aduma et al, 2019). The identification of germ cells in many species was ambiguous due to the absence of germ cells or somatic marker genes, especially for female germ cells in ovary. Female germ cell development is a complex multifactorial regulated process in that embryonic germ cells are driven to become oogonia stem cells through a series of statuses including cell proliferation, apoptosis, and differentiation, subsequently generate ootid through oogenesis (Sánchez and Smitz, 2012). The female germ cell fate mainly relies on the ovarian environments established by somatic cells rather than the sex chromosomes in germ cells (Evans et al, 1977). It is necessary to precisely define the various cell types in order to well understand the mechanism underlying ovarian development
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