Expression of the transcription factor ZBTB16 in porcine testes and molecular mechanisms for its regulation in porcine immature spermatogonia self-renewal [CUT&Tag]

ZBTB16, a transcription factor, plays a critical role in the self-renewal and differentiation of mouse primordial germ cells. However, the subcellular localization of ZBTB16 in the porcine testis and its molecular mechanism in regulating the self-renewal of immature porcine germ cells remain unclear. Using immunofluorescence co-staining, we examined the subcellular localization of ZBTB16 and its co-localization with molecular markers for immature porcine germ cells (DBA, ZBTB16, UCHL1) and the proliferation marker Ki67 in the testes of pigs at 7, 30, 70, and 90 days of age. The results showed that ZBTB16-positive cells in the four age groups of pig testes highly overlapped with SALL4/UCHL1-positive cells and partially overlapped with DBA-positive cells, indicating that ZBTB16 is predominantly expressed in immature porcine germ cells, including pig primordial germ cells, spermatogonial stem cells, and undifferentiated spermatogonia. Furthermore, the immunofluorescence co-staining of ZBTB16 and Ki67 revealed that ZBTB16-positive cells in pig testes at different developmental stages exhibited proliferative activity, but with significant fluctuations. RNA-seq analysis identified 5931 differentially expressed genes in ZBTB16-knockdown immature porcine germ cells, while CUT&Tag analysis identified 5409 ZBTB16 target genes, with 2084 of them overlapping with the differentially expressed genes. ZBTB16 preferentially bound to gene promoters. Knockdown of ZBTB16 expression resulted in the suppression of mRNA levels of target genes involved in the self-renewal of immature germ cells. Additionally, we found that ZBTB16 regulates the self-renewal of immature porcine germ cells through the PI3K/AKT/mTOR pathway. These findings reveal the expression pattern of ZBTB16 in the porcine testis and its regulation of target genes and pathways involved in the self-renewal of immature germ cells. This contributes to the understanding of the function and regulatory network of the transcription factor ZBTB16 in porcine germ cell development and spermatogenesis, and holds significant implications for unraveling the molecular mechanisms underlying porcine germ cell development and sperm production. Overall design: Firstly, we plan to select porcine testicular tissues at 7, 30, 70, and 90 days of age for paraffin embedding. Immunofluorescence double staining will be conducted using molecular markers for undifferentiated germ cells, such as ZBTB16, SALL4, UCHL1, and DBA, to investigate the expression and localization of ZBTB16 in different age groups of porcine testes. Qualitative and quantitative analysis will be performed to determine the co-localization of ZBTB16 with other germ cell molecular markers.Next, primary porcine immature spermatogonia will be collected, and a library of ZBTB16-bound target genes in porcine immature spermatogonia will be established using ZBTB16 antibodies and CUT&Tag technology. Through systematic analysis of the sequencing results, ZBTB16-bound target genes in porcine immature spermatogonia will be identified, and their associated pathways will be predicted using bioinformatics software.Subsequently, siRNA sequences targeting the porcine Zbtb16 gene will be designed and synthesized. The efficiency of Zbtb16 knockdown will be evaluated by transfecting immortalized porcine immature spermatogonia lines and performing qPCR. The siRNA with the highest knockdown efficiency will be selected. The designed oligos will be packaged into lentiviruses, and primary porcine immature spermatogonia will be transduced with Zbtb16-shRNA using lentiviral transduction. RNA-seq analysis will then be performed. Finally, the CUT&Tag data will be combined with the RNA-seq data for integrated analysis. Key genes and pathways related to the regulation of porcine immature spermatogonia self-renewal by ZBTB16 will be identified and experimentally validated. In addition, cross-species analysis will be conducted by downloading mouse ZBTB16 ChIP-seq and RNA-seq data to identify conserved and differential target genes and associated pathways regulated by ZBTB16 in mouse and porcine immature spermatogonia self-renewal.