Table_6_Structural and Functional Characterization of a Testicular Long Non-coding RNA (4930463O16Rik) Identified in the Meiotic Arrest of the Mouse Topaz1–/– Testes.XLSX

Spermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reported Topaz1 as a germ cell-specific gene highly conserved in vertebrates. Topaz1 knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterize Topaz1–/– testes, we used RNA-sequencing analyses at two different developmental stages (P16 and P18). The absence of TOPAZ1 disturbed the expression of genes involved in microtubule and/or cilium mobility, biological processes required for spermatogenesis. Moreover, a quarter of P18 dysregulated genes are long non-coding RNAs (lncRNAs), and three of them are testis-specific and located in spermatocytes, their expression starting between P11 and P15. The suppression of one of them, 4939463O16Rik, did not alter fertility although sperm parameters were disturbed and sperm concentration fell. The transcriptome of P18-4939463O16Rik–/– testes was altered and the molecular pathways affected included microtubule-based processes, the regulation of cilium movement and spermatogenesis. The absence of TOPAZ1 protein or 4930463O16Rik produced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, although Topaz1 is essential for the meiosis in male germ cells and regulate the expression of numerous lncRNAs, these studies have identified a Topaz1 regulated lncRNA (4930463O16Rik) that is key for both sperm production and motility.

精子发生（Spermatogenesis）是一系列协同进行的生物学过程，包括减数分裂（meiosis），最终生成具有功能的配子（gametes）。我们此前曾报道，Topaz1是一种在脊椎动物（vertebrates）中高度保守的生殖细胞特异性基因。Topaz1敲除的雄性个体不育，其睾丸内缺乏单倍体生殖细胞（haploid germ cells），原因是减数分裂I期前期后发生了减数分裂阻滞（meiotic arrest）。为了更深入地表征Topaz1敲除小鼠的睾丸表型，我们在两个不同的发育阶段——出生后第16天（P16）与出生后第18天（P18）——开展了RNA测序（RNA-sequencing）分析。研究结果显示，TOPAZ1的缺失会干扰参与微管与/或纤毛运动的基因的表达，而这两类生物学过程正是精子发生所必需的。此外，在P18阶段的差异表达基因中，有四分之一为长链非编码RNA（long non-coding RNAs，lncRNAs），其中3个为睾丸特异性基因，且表达于生精细胞（spermatocytes），它们的转录起始于P11至P15阶段。对其中名为4939463O16Rik的基因进行抑制后，尽管精子参数出现异常、精子浓度下降，但雄性生育力并未发生改变。对P18阶段的4939463O16Rik敲除小鼠的睾丸进行转录组分析发现，其转录组发生了改变，受影响的分子通路包括基于微管的过程、纤毛运动调控以及精子发生。尽管TOPAZ1蛋白缺失与4930463O16Rik缺失所导致的雄性生育力表型存在显著差异，但二者的突变睾丸均产生了相同的富集簇（enrichment clusters）。综上，尽管Topaz1对于雄性生殖细胞的减数分裂至关重要，且可调控众多长链非编码RNA的表达，但本研究鉴定出了一种受Topaz1调控的长链非编码RNA——4930463O16Rik，其在精子生成与精子运动能力两方面均发挥关键作用。