Spermatogenesis in testis is an important process for sexual reproduction, and worldwide about 10–15 percent of couples suffer from infertility. It is of importance to study spermatogenesis at single cell level in both of human and model organisms. Currently, single-cell RNA sequencing technologies (scRNA-seq) had been extensively applied to the study of cellular components and its gene regulations in the testes of different species, including human, monkey, mouse, and fly, but not in zebrafish. Zebrafish was a widely used model organism in biology and had been extensively used for the study of spermatogenesis in the previous studies. Therefore, it is also important to profile the transcriptome of zebrafish testis at single cell level. In this study, the transcriptomes of 14, 315 single cells from adult male zebrafish testes were profiled by scRNA-seq, and 10 cell populations were revealed, including Leydig cell, Sertoli cell, spermatogonia cell (SPG), spermatocyte, and spermatids. Notably, thousands of cell-type specific novel marker genes were identified, including sumo3b for SPG, krt18a.1 for Sertoli cells, larp1b and edrf1 for spermatids, which were also validated by RNA in situ hybridization experiments. Interestingly, through Ligand-Receptor (LR) analyses, zebrafish Leydig cells demonstrated stronger paracrine influence on germ cells than Sertoli cells. Overall, this study could be an important resource for the study of spermatogenesis in zebrafish and might also facilitate the study of the genes associated with human infertility through using zebrafish as a model organism.

The sex chromosome complement, XX or XY, determines sexual differentiation of the gonadal primordium into a testis or an ovary, which in turn directs differentiation of the germ cells into sperm and oocytes, respectively, in eutherian mammals. When the X monosomy or XY sex reversal occurs, XO and XY females exhibit subfertility and infertility in the mouse on the C57BL/6J genetic background, suggesting that functional germ cell differentiation requires the proper sex chromosome complement. Using these mouse models, we asked how the sex chromosome complement affects gene transcription in the oocytes during follicular growth. An oocyte accumulates cytoplasmic components such as mRNAs and proteins during follicular growth to support subsequent meiotic progression, fertilization, and early embryonic development without de novo transcription. However, how gene transcription is regulated during oocyte growth is not well understood. Our results revealed that XY oocytes became abnormal in chromatin configuration, mitochondria distribution, and de novo transcription compared to XX or XO oocytes near the end of growth phase. Therefore, we compared transcriptomes by RNA-sequencing among the XX, XO, and XY oocytes of 50–60 µm in diameter, which were still morphologically comparable. The results showed that the X chromosome dosage limited the X-linked and autosomal gene transcript levels in XO oocytes whereas many genes were transcribed from the Y chromosome and made the transcriptome in XY oocytes closer to that in XX oocytes. We then compared the transcript levels of 3 X-linked, 3 Y-linked and 2 autosomal genes in the XX, XO, and XY oocytes during the entire growth phase as well as at the end of growth phase using quantitative RT-PCR. The results indicated that the transcript levels of most genes increased with oocyte growth while largely maintaining the X chromosome dosage dependence. Near the end of growth phase, however, transcript levels of some X-linked genes did not increase in XY oocytes as much as XX or XO oocytes, rendering their levels much lower than those in XX oocytes. Thus, XY oocytes established a distinct transcriptome at the end of growth phase, which may be associated with abnormal chromatin configuration and mitochondria distribution.