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Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis

ABSTRACT: Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide inter-species differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ cell and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells in vitro or culturing organoids and facilitating the expansion of superior livestock breeding.

ORGANISM(S): Bubalus bubalis

PROVIDER: GSE190477 | GEO | 2022/11/24

REPOSITORIES: GEO

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Project description:Sertoli cells provide nutrients and support for germ cell differentiation and maintain a stable microenvironment for spermatogenesis. Comprehensive identification of Sertoli cellular proteins is important in understanding spermatogenesis. In this study, we performed an integrative analysis of the proteome and phosphoproteome to explore the role of Sertoli cells in spermatogenesis. A total of 2912 and 753 proteins were identified from the proteome and phosphoproteome in Sertoli cells; 438 proteins were common to the proteome and phosphoproteome. In the proteome, ACTG1, ACTB, ACTA2, MYH9 were the most abundant proteins. Gene Ontology (GO) analysis indicated that most of the proteins are involved in the processes of localization, biosynthesis, gene expression, and transport. In addition, part of proteins related to Sertoli cell functions were also enriched. In the phosphoproteome, most of the proteins are involved in gene expression and the RNA metabolic process; the pathways mainly involve the spliceosome, mitogen-activated protein kinase（MAPK） signaling pathway, focal adhesion, and tight junctions. The pleckstrin homology-like domain is the most highly enriched protein domain in phosphoproteins. Cyclin-dependent kinases (CDKs) and protein kinases C (PKCs) were found to be highly active kinases in kinase-substrate network analysis. Ten proteins most closely related to network stability were found in the analysis of network interactions of proteins identified jointly by the phosphoproteome and proteome. Through immunohistochemistry and immunofluorescence verification of vimentin, it was found that there were localization differences between phosphorylated and non-phosphorylated vimentin in testicular tissue. This study is the first in-depth proteomic and phosphoproteomic analysis of the buffalo testicular Sertoli cells. The results provide insight into the role of Sertoli cells in spermatogenesis and provide clues for further study of male reproduction.

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Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis

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