The regulatory repertoire of ZBTB16 in porcine immature spermatogonia.

Spermatogenesis is a highly productive and intricate process occurring in testes that produces functional haploid sperm capable of fertilization therefore sustaining lifelong male fertility. A cornerstone of spermatogenesis is primitive spermatogonia, including spermatogonial stem cells (SSCs), that are able to self-renew and differentiate. The molecular mechanisms for spermatogonial self-renewal and differentiation in large domestic animals such as pigs, in comparison with their counterparts in mice, are poorly understood. In this study, we explored the expression pattern of ZBTB16 (a key transcription factor also known as PLZF) and its regulatory repertoire in porcine immature spermatogonia. We first co-stained ZBTB16 with spermatogonial/proliferative markers (DBA, SALL4, UCHL1 or Ki67) on testis sections from four ages of boars, demonstrating that ZBTB16 cells in prepubertal porcine testes are a subpopulation of immature spermatogonia. Then, we knocked down ZBTB16 in enriched porcine immature spermatogonia, and the following RNA-sequencing (RNA-seq) analysis showed that ZBTB16 knockdown resulted in the manifest transcriptomic change, characterized by downregulation of genes related to spermatogonial self-renewal as well as upregulation of differentiation genes, corroborating ZBTB16 as a factor crucial to porcine spermatogonial self-renewal. Later, by performing a CUT&Tag analysis, we identified the genomic targets of ZBTB16 in porcine immature spermatogonia, and the final integrative analysis for RNA-seq and CUT&Tag data revealed the correlation of ZBTB16 with GDNF and mTOR signaling that facilitates porcine immature spermatogonial self-renewal. Altogether, our results enhance the understanding of molecular mechanisms for spermatogonial self-renewal in pigs, thereby facilitating the in vitro culture of porcine SSCs.