Abstract

Background The reproductive development of Simmental cattle, a vital breed for global beef production, remains poorly understood at the molecular and cellular levels. A systematic analysis of the regulatory mechanisms governing germ cell fate transitions during testicular development is essential for advancing breeding efficiency and reproductive technologies in cattle. Results Using integrated single-cell RNA sequencing (scRNA-seq) and single-nucleus ATAC sequencing (sNucATAC-seq) on testicular tissues from Simmental cattle across postnatal (PN), prepubertal (PP), and pubertal (PUB) stages, we identified core transcriptional regulators—including E2F1, BCLAF1, and YY1—that govern germ cell fate transitions. Several signaling pathways, such as TGF-β, MAPK, ErbB, and AMPK, were found to participate in spermatogenic processes. Sertoli cells were classified into three functional subtypes: Stage 1 is associated with the transition from spermatogonial stem cells (SSCs) to differentiating spermatogonia (Diff-SPG), Stage 2 correlates with development from Diff-SPG to spermatocytes (SPC), and Stage 3 coincides with the transformation from SPC to spermatids (SPT). Cross-species comparative transcriptomics with humans, pigs, and mice revealed conserved pathways in germ cell development, with E2F1 notably conserved during the SSC-to-Diff-SPG transition. Conclusions This study deciphers the regulatory network controlling germ cell fate transitions during bovine testicular development. The identification of conserved regulators and pathways provides novel insights into spermatogenesis and supports the development of strategies to overcome meiotic barriers in stem cell systems. These findings pave the way for precision breeding and advanced reproductive technologies in mammals. Supplementary Information The online version contains supplementary material available at 10.1186/s40104-026-01406-x.