BACKGROUND Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. RESULTS In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. CONCLUSIONS Our study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

中文翻译：

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激光捕获的生殖细胞的转录组分析和 zbtb40 在 17α-甲基睾酮诱导的橙斑石斑鱼（Epinephelus coioides）精子发生过程中的功能表征。


背景技术精子发生是一个由精细组织的网络调节的复杂过程。橙点石斑鱼（Epinephelus coioides）是一种先雌雌雄同体鱼类，但其精子发生的调控机制尚不清楚。在本研究中，对从橙斑石斑鱼中分离的雄性生殖细胞进行转录组测序，以探索精子发生的分子机制。结果 在这项研究中，通过植入 17α-甲基睾酮 (MT) 诱导橙斑石斑鱼从雌性变为雄性。在精子发生过程中，通过激光捕获显微切割分离雄性生殖细胞（精原细胞、精母细胞、精子细胞和精子）。对分离的细胞进行转录组分析。四个 cDNA 文库总共生成了 244,984,338 个干净读数。 13个与性别分化和激素代谢相关的基因的实时PCR结果表明转录组数据是可靠的。 RNA-seq数据显示，女性相关基因和参与激素代谢的基因在精原细胞和精子中高表达，表明这些基因参与精子发生。有趣的是，zbtb家族基因的表达在RNA-seq数据中显示出显着变化，并且在精子发生过程中进一步检查了它们的表达模式。对Eczbtb40的细胞定位以及Eczbtb40和Eccyp17a1在不同性腺阶段的共定位的分析表明，Eczbtb40可能在精子发生过程中与Eccyp17a1相互作用。 结论我们的研究首次研究了橙斑石斑鱼雄性生殖细胞的转录组，并鉴定了与精子发生相关的功能基因、GO术语和KEGG通路。此外，Eczbtb40 首次被表征并预测了其在精子发生过程中的作用。这些数据将有助于未来硬骨鱼精子发生分子机制的研究。