This study was to explore the regulation effect of PGAM1 on the proliferation, apoptosis and glycolysis pathway of Tibetan sheep Sertoli cells. In this paper, the reproductive organs of male Tibetan sheep before pre-puberty (3 months old), sexual maturity (1 year old) and adult (3 years old) were used as experimental materials. The complete CDS region sequence of PGAM1 gene was cloned for bioinformatics analysis, and had the closest relationship with Tibetan antelope. QRT-PCR, Western blot and immunohistochemical staining were used to detect the expression and localization of PGAM1 in the testis and epididymis tissues of Tibetan sheep at different growth and development stages at the transcription and translation levels. Then the Tibetan sheep primary Sertoli cells (SCs) were isolated to construct PGAM1 gene overexpression and interference vectors, and to transfect primary SCs so as to promote and inhibit PGAM1 gene expression; CCK-8 and flow cytometry were used to detect the proliferation effect of SCs;qRT-PCR technology was employed to detect the changes in the expression of genes related to cell proliferation and apoptosis. Different kits were used to detect pyruvate, lactic acid, ATP production and LDH activity during glycolysis, and to detect the changes in the expression of downstream genes in the glycolysis pathway. The results showed that the CDS region of Tibetan sheep PGAM1 gene was 765 bp in length, which can encode 254 amino acids; and the expression of PGAM1 protein in the testis and epididymis increased at 1Y group and 3Ygroup compared with 3M group, and that the PGAM1 protein mainly existed in SCs and Leydig cells at different developmental stages. CCK-8 and flow cytometry test results found that compared with the empty vector group (pcDNA3.1(+)), the proliferation rate of the PGAM1 gene overexpression group (pcDNA3.1(+)-PGAM1) decreased. The mRNA expression of the cell proliferation related genes PCNA and Bcl2 was significantly decreased (P<0.05), and the expression of apoptosis-related genes Bax and caspase3 was significantly increased (P<0.05). The expression of downstream genes in the glycolysis pathway was significant increased (P<0.05), pyruvate content, ATP content, lactic acid production and LDH activity increased significantly (P<0.05). Compared with the interference control group (NC), the proliferation rate of the PGAM1 gene interference group (si-PGAM1) was weakened. The mRNA expression of the cell proliferation-related genes PCNA and Bcl2 was significantly increased (P<0.05), and the expression of cell apoptosis related genes Bax and caspase3 was significantly decreased (P<0.05). The expression of downstream genes in the glycolysis pathway was significantly reduced (P<0.05), and the pyruvate content, ATP content, lactic acid production and LDH activity were significantly decreased (P<0.05). The PGAM1 gene might regulate the glycolytic metabolism pathway and regulate the sperm formation and maturation process by affecting the proliferation and apoptosis of SCs. This result provides basic data for the study of the function of PGAM1 in sheep testicular development.

本研究旨在探讨PGAM1对藏羊支持细胞增殖、凋亡及糖酵解途径的调控作用。本文以雄性藏羊在青春期前（3月龄）、性成熟（1岁）和成年（3岁）前的生殖器官为实验材料。克隆了PGAM1基因的完整CDS区序列进行生物信息学分析，与藏羚羊关系最密切。QRT-PCR、Western blot和免疫组化染色检测PGAM1的表达及定位在不同生长发育阶段藏羊睾丸和附睾组织中的转录和翻译水平。然后分离藏羊原代支持细胞(SCs),构建PGAM1基因过表达和干扰载体,转染原代SCs,促进和抑制PGAM1基因表达; 采用CCK-8和流式细胞术检测SCs的增殖作用；采用qRT-PCR技术检测细胞增殖和凋亡相关基因表达的变化。使用不同的试剂盒检测糖酵解过程中丙酮酸、乳酸、ATP产生和LDH活性，以及​​检测糖酵解途径下游基因表达的变化。结果表明，藏羊PGAM1的CDS区基因全长765 bp，可编码254个氨基酸；与3M组相比，1Y组和3Y组睾丸和附睾中PGAM1蛋白表达增加，PGAM1蛋白主要存在于不同发育阶段的SCs和Leydig细胞中。CCK-8和流式细胞术检测结果发现，与空载体组(pcDNA3.1(+))相比，PGAM1基因过表达组(pcDNA3.1(+)-PGAM1)的增殖率降低。细胞增殖相关的基因的mRNA表达PCNA和Bcl2的被显著降低（P <0.05），和细胞凋亡相关基因的表达Bax的和凋亡蛋白酶被显著增加（P <0.05）。糖酵解途径下游基因表达量显着增加（P <0.05），丙酮酸含量、ATP含量、乳酸产量和LDH活性显着增加（P <0.05）。与干扰对照组（NC）相比，PGAM1基因干扰组（si-PGAM1）的增殖速度减弱。细胞增殖相关基因PCNA和Bcl2的mRNA表达显着升高（P <0.05），细胞凋亡相关基因Bax和caspase3的表达显着降低（P<0.05）。糖酵解途径下游基因表达显着降低（P <0.05），丙酮酸含量、ATP含量、乳酸产量和LDH活性显着降低（P <0.05）。该PGAM1基因可能通过调节糖酵解代谢途径，并通过影响雪旺细胞增殖和凋亡的调控精子的形成和成熟的过程。该结果为研究PGAM1在绵羊睾丸发育中的作用提供了基础数据。