Abstract: In this present study, in order to investigate the effect of GnIH on the apoptosis and testosterone synthesis of mouse Leydig cells, its overexpression vector was constructed. RNA was extracted from the testis tissues of male mice at 6-8 weeks, reverse transcribed into cDNA. The GnIH gene was amplified and purified by PCR, and connected to the PLVX-IRES-ZsGreen1 vector through T4 ligase. Then, the recombinant plasmid was transformed into the competent cells, and extracted the plasmid after identified by PCR, double enzyme digestion and sequencing. Subsequently, the plasmid was transfected into the TM3 cell line for 72 h. The experiment was divided into two groups:the empty plasmid group and the GnIH overexpression group. Cell fluorescence was observed under a microscope. The mRNA level of GnIH, apoptosis gene and testosterone synthase gene in the TM3 cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), apoptotic rate and testosterone secretion level were detected by flow cytometry and ELISA, respectively. The results showed that the GnIH amplified sequence was consistent with the reference sequence. After transfecting the GnIH overexpression plasmid into the TM3 cell line for 72 h, high-intensity green fluorescence was observed, and the expression levels of GnIH was significantly increased in the overexpression group (P<0.01). These results indicated that GnIH overexpression vector was successfully constructed and highly expressed in the TM3 cell line. After 72 h of transfection, compared with the empty plasmid group, the apoptosis rate in the overexpression group was significantly increased (P<0.01), and the expression ratio of apoptosis genes P53 and Bax/Bcl-2 was also significantly increased (P<0.01). Compared with the empty plasmid group, the concentration of testosterone was extremely significantly cut down in the overexpression group (P<0.01). Meanwhile, the expression of the testosterone synthesis-related enzyme genes, P450 scc mRNA expression was extremely significantly decreased (P<0.01); and the expressions of the StAR, 3β-HSD and P450c17 mRNA were obviously reduced (P<0.05). There was no significant difference of 17β-HSD mRNA expression between the two groups (P>0.05). In summary, overexpression of GnIH in TM3 cells could induce apoptosis of TM3 cells, inhibit testosterone secretion, and decrease the gene expression levels of testosterone synthesis-related enzymes. We can infer that GnIH plays a negative regulatory role in the process of testosterone synthesis in mice. This study can provide a scientific basis for revealing the role of GnIH in the reproductive regulation of male mammals.

Key words: GnIH, vector construction, Leydig cell, apoptosis, testosterone, testosterone synthase