Study On Molecular Mechanism About Androstenone Synthesis Influenced By COUP-TFⅠ

Androstenone(5a-androst-16-en-3-one) is one of steroidal compounds produced by pig testis, also, it is one of the compounds caused boar taint. Its deposition level depends on the levels of its synthesis and metabolism in pigs and it is regulated by multiple genes. Recent studies showed that CYP11Al,CYP17A1,StAR are the main genes in the synthesis pathways of androstenone. COUP-TF(Chicken ovalbumin upstream promoter transcription factor) is a transcription factor related to the synthesis and metabolism of androstenone. It has been demonstrated that COUP-TF I is the negative regulation protein of CYP17gene transcription in mice, cattle and human adrenal cortical cells. Meanwhile, it can inhibit the expression of StAR to limit the formation of the carrier compounds. But by now, there is no relevant studies have shown the relationship between COUP-TF I and related genes of androstenone in pig testis. So, in this study, we use the ways of cell culture, eukaryotic expression vector construction and transient transfection, and make use of the analysis methods of qRT-PCR and ELISA, to explore the relationship between COUP-TF I and CYP11A1, CYP17A1, StAR in the synthesis pathways of androstenone in pig testis and clarify the effect of COUP-TF I on molecular mechanism of androstenone synthesis, so that a theoretical basis can be provide to reduce the boar taint and breeding excellent boars.Physiological functions of pig testis and leydig cells are difference in different stages of ages. Some studies have shown that the androstenone level in low-month-old boars is less than high-month-old boars. Bama miniature pig testis in1month old and6months old were selected in this study. The study contains two main aspects:(1) qRT-PCR was used to analyze the nomalized fold expression of COUP-TF I, CYP11Al, CYP17A1, StAR in different mouths, and B-actin was the reference gene;(2) Bama miniature pig testis in1month and6months old were isolated and cultured in vitro by the method of non-steel net filtration. Eukaryotic expression vector COUP-TF I-neo, containing the screening gene "neo" of COUP-TF I,was constructed. This recombinant plasmid was transferred into the pig leydig cells in1month and6months old by transient transfection methods. At the same time, transfected empty vector pEGFP-C1and non-transfected cells were the control groups. After the transfection, qRT-PCR was used to analyze the nomalized fold expression of COUP-TF I, CYP11A1, CYP17A1, StAR in pig leydig cells and ELISA was used to detect the androstenone level in pig leydig cells.Through the experiments, the results were as follows:(1) COUP-TF I, CYP11A1, CYP17A1, StAR were expressed in1month and6months old testis tissue. Compared with1mouth old, the nomalized fold expression of COUP-TF I,CYP17A1and StAR in6mouths old pig testis tissue had a a highly statistical significant difference (P<0.01), but CYP11A1had statistical significant difference (P<0.05).Overall, the nomalized fold expressions of the four genes in6months old were higher than them in1month old.: the levels of androstenone in1and6mouths old testis respectively were1.6583±0.0133ng/mL and3.3812±0.0214ng/mL;(2) COUP-TF I-neo recombinant plasmid was constructed successfully and it was transferred into the pig leydig cells in1month and6months old. The results of qRT-PCR showed that:After the recombinant plasmid transferred into the cells, the nomalized fold expression of CYP11A1, CYP17A1, StAR gene were lower. For1month and6mouths old leydig cells, compared with the corresponding blank control group and transfected with pEGFP-Cl negative control group, the nomalized fold expression of CYP11A1, CYP17A1, StAR in transfected with COUP-TF I-neo had a statistical highly significant difference (P<0.01). Compared the blank control groups and the groups transfected with COUP-TF I-neo in1month and6months old, the nomalized fold expression of COUP-TF I,CYP11A1, CYP17A1in6months had a statistical highly significant difference (P<0.01) with1mouth old cells, and StAR had a statistical significant difference (P<0.05)with1mouth old cells. And in the blank control groups, the nomalized fold expression of CYP11A1, CYP17A1and StAR in6mouths were higher than1month; In the groups transfected with COUP-TF I-neo, the nomalized fold expression of CYP11A1, CYP17A1, StAR in6months were lower than1month. ELISA results showed that:For1month old cells, compared with the corresponding blank control group and transfected with pEGFP-Cl negative control group, the levels of androstenone in transfected with COUP-TF I-neo had a statistical significant difference (P<0.05). And for the6months old cells, it had a highly statistical significant difference (P<0.01).According to the results, the conclusions were as follows:(1) COUP-TF I, CYP11A1, CYP17A1,StAR were expressed in1month and6months old testis tissue, the nomalized fold expression of the four genes and the levels of androstenone in6month old testis tissue were higher than them in1month old; the nomalized fold expressions of CYP11A1, CYP17A1, StAR were positively correlated with the levels of androstenone;(2) Upregulated the expression of COUP-TF I can restrain the nomalized fold expression of CYP11A1, CYP17A1, StAR and the the androstenone levels in1month and6months old cells. And the inhibition on nomalized fold expressions of CYP11A1, CYP17A1, StAR and the the androstenone levels caused by COUP-TF I in6months old leydig cells were significantly stronger than them in1month old leydig cells.