Single-Cell Transcriptomic Profiling of the Mouse Testicular Germ Cells Reveals Important Role of Phosphorylated GRTH/DDX25 in Round Spermatid Differentiation and Acrosome Biogenesis during Spermiogenesis

Abstract

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.