The development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. However, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. To advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative. In this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse RNA-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. We also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. We detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. Through RT-PCR, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. Of these, we ablated four epididymis-specific genes (Spint3, Spint4, Spint5, and Ces5a) and two testis-specific genes (Pp2d1 and Saxo1) in individual or double knockout mice generated through the CRISPR/Cas9 system. Our results validate a functional requirement for Spint4/5 and Ces5a in male mouse fertility, while demonstrating that Spint3, Pp2d1, and Saxo1 are each individually dispensable for male mouse fertility. Our work provides a plethora of novel testis- and epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives.

中文翻译：

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通过分析 RNA-seq 数据集大规模发现男性生殖道特异性基因。

在过去的几十年里，为男性开发一种安全、有效、可逆的非激素避孕方法一直是一项持续的努力。然而，尽管在阐明与生殖有关的关键蛋白质的功能方面取得了重大进展，但对男性生殖生理学的理解受到生殖组织中表达基因的不完整信息的限制，迄今为止还没有避孕靶点达到临床试验。为了推进产品开发，进一步鉴定导致潜在可药用蛋白质靶标的新型生殖道特异性基因势在必行。在这项研究中，我们扩展了以前的单一组织，通过整合对公开可用的人类和小鼠 RNA-seq 数据集的分析进行单一物种研究，这些数据集最初发表的目的不在于识别雄性生殖道特异性目标。我们还结合了对其他新获得的人类和小鼠睾丸和附睾样本的分析，以增加识别的目标数量。我们检测到总共 1178 个基因，这些基因以前没有注释过男性生殖道特异性表达的证据，其中许多是潜在的药物靶标。通过 RT-PCR，我们确认了 51 个新的直系同源人和小鼠基因的生殖道特异性表达，而没有报道的小鼠模型。其中，我们消融了四个附睾特异性基因（Spint3、Spint4、Spint5、和 Ces5a) 和两个睾丸特异性基因 (Pp2d1 和 Saxo1) 在通过 CRISPR/Cas9 系统产生的单个或双敲除小鼠中。我们的结果验证了 Spint4/5 和 Ces5a 在雄性小鼠生育力中的功能要求，同时证明 Spint3、Pp2d1 和 Saxo1 对雄性小鼠生育力都是可有可无的。我们的工作提供了大量新的睾丸和附睾特异性基因，并阐明了其中几个基因的功能需求，这对于了解男性不育症的病因和男性避孕药的开发至关重要。