Tissue expression characteristics of SP1 gene and effects of its overexpression on autophagy and apoptosis in Leydig cells from Congjiang Xiang pigs

【Objective】 To analyze SP1 gene expressionin tissues of Congjiang Xiang pigs of different age and its transcriptional regulation of Leydig cell autophagy and apoptosis, and to provide a theoretical basis for investigating molecular mechanism of SP1 gene regulation of Leydig cell autophagy and improving male reproductive performance of Congjiang Xiang pigs.【Method】 Congjiang Xiang pigs of precocious puberty were selected, the sequence of coding region (CDS) of SP1 gene was obtained by PCR amplification, and the sequence of CDS region was bioinformatically analyzed by applying online software. Fluorescence quantitative PCR(qRT-PCR) was used to detect SP1 expression of in different tissues of Congjiang Xiang pigs at the stage of sexual maturity, and qRT-PCR and Western blotting were used to detect SP1 expression in testes of Congjiang Xiang pigs at different age.【Result】 Bioinformatics analysis showed that the CDS sequence of SP1 gene was 2361 bp, encoding 786 amino acids residues, and the secondary and tertiary structures of the protein were mainly randomly coiled and extended chains, without transmembrane structural domains or signal peptide shear sites, and it was an unstable protein. 174 phosphorylation sites were found in the SP1 amino acid sequence, and amino acid homology analysis revealed that pig SP1 had the closest genetic relationship with sheep and cattle.According to SP1 gene expression test in Congjiang Xiang pig tissues, SP1 gene relative expression was the highest in the spleen; and the highest SP1 protein expression was found in testes of 180 day-old pig and high SP1 gene expression was found in 30 day-old and 180 day-old pigs. SP1 gene overexpression vector was further constructed and transfected into the Leydig cells. The qRT-PCR results showed that SP1 gene relative expression was significantly higher in pEGFP-C1-SP1 group than in pEGFP-C1 group(P<0.05, the same below), while the relative expression of autophagy pathway-related factors genes mTOR, LC3B, Beclin-1 and apoptosis-related factors genes Caspase-3 and Bcl-2 was expressed significantly lower in the overexpression pEGFP-C1-SP1 group than in the pEGFP-C1 group, but no significant difference of autophagy and apoptosis factor ERK1/2 relative expression and apoptosis gene Bax was detected(P>0.05). It was hypothesized that SP1 gene could inhibit the occurrence of apoptosis in Leydig cells by decreasing the expression of LC3B, Beclin-1, and Caspase-3, or promote apoptosis by decreasing the expression of mTOR and anti-apoptotic gene Bcl-2.【Conclusion】 SP1 gene is expressed in tissues of Congjiang Xiang pigs in different testis growth periods and by influencing expression of genes related to autophagy and apoptosis of the pigs, SP1 gene plays an important role physiologically during puberty and sexual maturity of Congjiang Xiang pigs.