Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2023 Feb 4;24(4):3127. doi: 10.3390/ijms24043127.
Gonadotropin-regulated testicular RNA helicase (GRTH)/DDX25 is a member of DEAD-box family of RNA helicase essential for the completion of spermatogenesis and male fertility, as evident from GRTH-knockout (KO) mice. In germ cells of male mice, there are two species of GRTH, a 56 kDa non-phosphorylated form and 61 kDa phosphorylated form (pGRTH). GRTH Knock-In (KI) mice with R242H mutation abolished pGRTH and its absence leads to infertility. To understand the role of the GRTH in germ cell development at different stages during spermatogenesis, we performed single-cell RNA-seq analysis of testicular cells from adult WT, KO and KI mice and studied the dynamic changes in gene expression. Pseudotime analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to elongated spermatids in WT mice, while in both KO and KI mice the trajectory was halted at round spermatid stage indicating incomplete spermatogenesis process. The transcriptional profiles of KO and KI mice were significantly altered during round spermatid development. Genes involved in spermatid differentiation, translation process and acrosome vesicle formation were significantly downregulated in the round spermatids of KO and KI mice. Ultrastructure of round spermatids of KO and KI mice revealed several abnormalities in acrosome formation that includes failure of pro-acrosome vesicles to fuse to form a single acrosome vesicle, and fragmentation of acrosome structure. Our findings highlight the crucial role of pGRTH in differentiation of round spermatids into elongated spermatids, acrosome biogenesis and its structural integrity.
促性腺激素调节的睾丸 RNA 解旋酶（GRTH）/DDX25 是 DEAD -box 家族 RNA 解旋酶的成员，对于完成精子发生和雄性生育力至关重要，这从 GRTH 敲除（KO）小鼠中可以明显看出。在雄性小鼠的生殖细胞中，存在两种 GRTH，一种是 56 kDa 的非磷酸化形式，另一种是 61 kDa 的磷酸化形式（pGRTH）。具有 R242H 突变的 GRTH 敲入（KI）小鼠消除了 pGRTH，其缺失导致不育。为了了解 GRTH 在精子发生过程中不同阶段的生殖细胞发育中的作用，我们对成年 WT、KO 和 KI 小鼠的睾丸细胞进行了单细胞 RNA-seq 分析，并研究了基因表达的动态变化。拟时分析显示，WT 小鼠的生殖细胞从精原细胞到伸长的精子经历了一个连续的发育轨迹，而在 KO 和 KI 小鼠中，该轨迹在圆形精子阶段停止，表明精子发生过程不完整。KO 和 KI 小鼠的转录谱在圆形精子发育过程中发生了显著改变。参与精子分化、翻译过程和顶体囊泡形成的基因在 KO 和 KI 小鼠的圆形精子中显著下调。KO 和 KI 小鼠的圆形精子的超微结构显示出顶体形成的几种异常，包括前顶体囊泡未能融合形成单个顶体囊泡，以及顶体结构的碎片化。我们的研究结果强调了 pGRTH 在圆形精子向伸长精子分化、顶体发生和其结构完整性中的关键作用。
