Single-cell multiomic comparison of mouse and rat spermatogenesis reveal gene regulatory networks conserved for over 20 million years.

Spermatogenesis is driven by dramatic temporal and spatial changes in chromatin regulation, gene transcription, and protein expression. To assess the mechanistic bases for these developmental changes, we utilized multiomic single-cell/single-nuclear RNA-sequencing (sc/snRNA-seq) and transposase-accessible chromatin (snATAC-seq) to identify chromatin changes associated with transcription along with cellular indexing of transcriptomes and epitopes (CITE-seq) to assess post-transcriptional control of expression in adult mouse and rat testes. We characterized similarities and variations between the transcriptomes of both species, including a difference in the expression of Id4 in spermatogonial stem cells. In both mice and rats, promoter accessibility and gene expression showed the greatest association during meiosis. We mapped cross-species conservation in putative regulatory regions for key spermatogenic genes, including Sdc4, Sycp3 and Spam1, and we found that the correlation between Cd9 chromatin states and CD9 protein abundance allowed for the isolation of distinct germ cell types with improved resolution. Using a gene regulatory network (GRN) model, we identified 40 key regulons conserved between mouse and rat germ cells, highlighting the relevance of chromatin-related factors in regulating the transcription and translation of key genes across spermatogenesis. (iPSCs) into primordial germ cell-like cells that self-organize within xenogeneic reconstituted testes (xrTestes) generated from mouse fetal testicular cells. Subsequent transplant of xrTestes into immunodeficient mice resulted in efficient generation of undifferentiated and differentiated spermatogonia as well as preleptotene spermatocytes with striking similarities to their in vivo counterparts. As the fertilization competency of human iPSC-generated germ cells cannot be evaluated due to ethical constraints, we utilized a similar strategy to differentiate rhesus iPSCs through all fetal germ cell stages into spermatogonia-like cells. Together, these newly identified models of human gametogenesis will allow further mechanistic assessment of both germ cell development and genetic causes of infertility.