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. 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.

ZBTB16作为转录因子，在小鼠原始生殖细胞（primordial germ cells）的自我更新与分化过程中发挥关键作用。然而，ZBTB16在猪睾丸中的亚细胞定位，以及其调控猪未成熟生殖细胞自我更新的分子机制，目前仍不明晰。本研究通过免疫荧光共染色（immunofluorescence co-staining）技术，检测了7、30、70和90日龄猪睾丸中ZBTB16的亚细胞定位，以及其与猪未成熟生殖细胞分子标志物（DBA、ZBTB16、UCHL1）和增殖标志物Ki67的共定位情况。结果显示，四个日龄组猪睾丸中的ZBTB16阳性细胞与SALL4/UCHL1阳性细胞高度共定位，与DBA阳性细胞部分共定位，表明ZBTB16主要表达于猪未成熟生殖细胞，包括猪原始生殖细胞、精原干细胞及未分化精原细胞。此外，ZBTB16与Ki67的免疫荧光共染色结果显示，不同发育阶段猪睾丸中的ZBTB16阳性细胞均具有增殖活性，但该活性存在显著波动。通过RNA测序（RNA-seq）分析，在ZBTB16敲低的猪未成熟生殖细胞中鉴定出5931个差异表达基因；而通过CUT&Tag分析，共鉴定出5409个ZBTB16靶基因，其中2084个与上述差异表达基因重合。ZBTB16优先结合基因启动子区域。敲低ZBTB16的表达会抑制未成熟生殖细胞自我更新相关靶基因的mRNA水平。此外，本研究发现ZBTB16通过PI3K/AKT/mTOR通路调控猪未成熟生殖细胞的自我更新。上述研究结果揭示了ZBTB16在猪睾丸中的表达模式，及其对未成熟生殖细胞自我更新相关靶基因与通路的调控作用。这有助于理解转录因子ZBTB16在猪生殖细胞发育及精子发生（spermatogenesis）过程中的功能与调控网络，对阐明猪生殖细胞发育与精子生成的分子机制具有重要意义。首先，本研究计划选取7、30、70和90日龄的猪睾丸组织进行石蜡包埋。采用未分化生殖细胞的分子标志物（如ZBTB16、SALL4、UCHL1及DBA）进行免疫荧光双染色，以探究ZBTB16在不同日龄猪睾丸中的表达与定位情况，并通过定性与定量分析明确ZBTB16与其他生殖细胞分子标志物的共定位特征。其次，分离原代猪未成熟精原细胞，使用ZBTB16抗体及CUT&Tag技术构建猪未成熟精原细胞中ZBTB16结合靶基因的文库。通过对测序结果的系统性分析，鉴定猪未成熟精原细胞中ZBTB16结合的靶基因，并利用生物信息学软件预测其相关通路。随后，设计并合成靶向猪Zbtb16基因的小干扰RNA（siRNA）序列。将其转染永生化猪未成熟精原细胞系，通过实时荧光定量聚合酶链反应（qPCR）评估Zbtb16的敲低效率，筛选敲低效率最高的siRNA。将设计的寡核苷酸序列包装为慢病毒（lentivirus），通过慢病毒转导将Zbtb16-shRNA导入原代猪未成熟精原细胞，随后进行RNA-seq分析。最后，将CUT&Tag数据与RNA-seq数据进行整合分析，鉴定并实验验证ZBTB16调控猪未成熟精原细胞自我更新的关键基因与通路。此外，本研究将通过下载小鼠ZBTB16的染色质免疫沉淀测序（ChIP-seq）与RNA-seq数据开展跨物种分析，以鉴定ZBTB16在小鼠与猪未成熟精原细胞自我更新过程中调控的保守及差异靶基因与相关通路。