Isoform-resolution single-cell RNA sequencing reveals the transcriptional panorama of adult Baoshan pig testis cells.

BACKGROUND

As the primary organ of the male reproductive system, the testis facilitates spermatogenesis and androgen secretion. Due to the complexity of spermatogenesis, elucidating cellular heterogeneity and gene expression dynamics within the porcine testis is critical for advancing reproductive biology. Nevertheless, the cellular composition and regulatory mechanisms of porcine testes remain insufficiently characterized. In this study, we applied integrated long-read (Nanopore) and short-read (Illumina) scRNA-seq to Baoshan pig testes, establishing a comprehensive transcriptional profile to delineate cellular heterogeneity and molecular regulation.

RESULTS

Through systematic analysis of testicular architecture and the temporal progression of spermatogenesis, we characterized 11,520 single cells and 23,402 genes, delineating germ cell developmental stages: proliferative-phase spermatogonia (SPG), early-stage spermatocytes (Early SPC) and late-stage spermatocytes (Late SPC) during meiosis, and spermiogenic-phase round spermatids (RS) followed by elongating/elongated spermatids (ES), culminating in mature spermatozoa (Sperm). We further identified nine distinct testicular cell types, with germ cells spanning all developmental stages and somatic components comprising Sertoli cells, macrophages, and peritubular myoid cells as microenvironmental constituents, revealing the cellular heterogeneity of testicular tissue and dynamic characteristics of spermatogenesis. We obtained the dynamic expression changes of 16 vital marker genes during spermatogenesis and performed immunofluorescence validation on 7 marker genes. Gene ontology analysis revealed that germ cells at various stages were involved in specific biological processes, while cell communication networks highlighted eight pivotal signaling pathways, including MIF, NRG, WNT, VEGF, BMP, CCL, PARs, and ENHO pathways. Long-read sequencing further captured the full integrity and diversity of RNA transcripts, identifying 60% of the novel annotated isoforms and revealing that FSM isoforms exhibited longer transcript lengths, longer coding sequences, longer open reading frames, and a great number of exons, suggesting the complexity of isoforms within the testicular microenvironment.

CONCLUSIONS

Our results provide insight into the cellular heterogeneity, intercellular communication, and gene expression/transcript diversity in porcine testes, and offer a valuable resource for understanding the molecular mechanisms of porcine spermatogenesis.