Breed And Gender Differences Of 3BETA-HSD Gene Expression In Porcine Adrenal Gland And Liver And Molecular Mechanisms Involved

The homeostasis of steroid hormones is necessary for the maintenance of normal life activities. Steroid hormones are systhesized in the adrenal gland, and metabolized in the liver. The enzyme 3β-hydroxysteroid dehydrogenase/Δ5-A4 isomerase (3β-HSD) catalyzes the biosynthesis and degradation of all steroid hormones. The functional loss of the 3P-HSD enzyme blocks the production of glucocorticoid, mineralocorticoid, androgen and estrogen in the adrenal gland and the gonads, and inhibits the metabolism of steroids in the liver. It has been reported that the expression and activity of 3P-HSD can be affected by various factors, including the feedback regulation of steroid hormones and transcription factors regulation, however, mjority of the studies are carried out on human cell lines, little is known about 3P-HSD transcriptional regulation in pigs. Therefore, we aimed to compare the breed- and gender-expression of 3P-HSD in the adrenal gland and liver of pigs and to explore the potential factors regulating 3β-HSD transcription.1. Mechanism study of the breed-specific expression of 30-HSD in the adrenal gland of male weaning pigletsFive purebred Large White (LW) and 6 Erhualian (EHL) weaning male piglets at the age of 25 days (d) were used in the study. Animals were sacrificed, serum and the adrenal gland were collected for the analysis. We aimed to compare the expression of 3P-HSD gene in the adrenal gland between LW and EHL piglets and to explore the potential factors regulating 3β-HSD transcription. The results showed that EHL had significantly higher serum levels of cortisol(P< 0.01) and testosterone (P< 0.01) compared with LW piglets. And the expression of adrenal 3P-HSD mRNA (P< 0.01) abundance and protein content (P< 0.05) was higher in EHL than LW piglets. The 5’flanking region of the porcine 30-HSD gene showed significant sequence variations between the two breeds. Eight TTAT repeats were found in the promoter of EHL piglets, while 9 TTAT repeats were in LW piglets. Moreover,3 point mutations were identified in the promoter sequences of 3β-HSD gene, with the breed-specific distribution pattern. And the sequence of EHL demonstrated an elevated promoter activity (P< 0.05) in luciferase reporter gene assay than LW. Further more, EHL had higher adrenal expression of CCAAT/enhancer binding protein P (C/EBPP) mRNA (P< 0.05) and protein (P< 0.05), androgen receptor (AR) mRNA (P= 0.06) and protein (P< 0.05) than LW piglets, but lower glucocorticoid receptor (GR) mRNA (P< 0.05) and protein (P< 0.05) were detected in EHL. Moreover, the binding of C/EBPβ to 3β-HSD promoter was higher in EHL (P< 0.05), and enriched histone H3 acetylation (H3Ac) (P< 0.05) was detected in EHL. Co-immunoprecipitation (Co-IP) analysis revealed interactions of C/EBPβ with both AR and GR. These results indicate that the C/EBPβ binding to 3β-HSD promoter is responsible, at least in part, for the breed-dependent 3β-HSD expression in adrenal gland of piglets. The sequence variations of 3β-HSD promoter and the interactions of AR and/or GR with C/EBPβ may also participate in the regulation.2. Mechanism study of the breed-specific expression of 3P-HSD in the liver of male weaning pigletsThe enzyme 3β-HSD is responsible for the degradation of steroid hormones in the liver. The molecular mechanisms regulating porcine hepatic 3β-HSD expression in different breeds are still poorly understood. In this study we compared the expression of hepatic 3β-HSD gene between male weaning LW and EHL piglets and explored the potential factors regulating 3β-HSD transcription. The results showed that EHL had significantly lower 3β-HSD mRNA expression (P< 0.01) in the liver, which was negative correlation with the significantly higher serum levels of cortisol (P< 0.01) and testosterone (P< 0.01), compared with LW piglets. And EHL had significantly lower promoter activity in the core promoter of 3β-HSD gene (-341 bp+156 bp) than LW (P< 0.01). Lower adrenal expression of 3β-HSD in EHL was accompanied with lower AR (P= 0.07) and C/EBPβ (P< 0.05) mRNA expression, however higher GR mRNA (P< 0.05), and AR (P< 0.05), GR (P< 0.01) and p-Ser211GR (P< 0.01) nucleoprotein level. ChIP assay showed that the binding levels of GR to 3β-HSD promoter were significantly decreased in EHL piglets (P< 0.05), but no differences of AR and C/EBPβ were detected between the two breeds. Co-IP analysis revealed no interactions of GR with either C/EBPβ or AR. These results indicate that the GR binding to 3β-HSD promoter is responsible for the breed-dependent 3P-HSD expression in the liver of piglets. The sequence variations of 3P-HSD promoter, especially the proximal SNP site in the core promoter may also participate in the regulation.3. Mechanism study of the gender-specific expression of 3P-HSD in the adrenal gland of finished pigsTwenty-eight Duroc x Landrace x Yorkshire crossbred piglets were used in the study. The experimental design included 3 groups. One group consisted of 10 male piglets surgically castrated at 1 week (barrows), a second group consisted of 10 female pigs (gilts) and the remaining 8 male pigs (boars) were kept intact through out the study. All the pigs were weaned at 22 days (d) of age, and raised with the standard ration until slauthter (190 d). The results showed that the boars had a significantly higher testosterone level (P< 0.01) than the barrows and the gilts, while the gilts showed significantly higher estradiol content (P< 0.01) than the boars and the barrows. The boars and barrows had significantly lower expression of adrenal 3β-HSD mRNA (P< 0.05) compared to the gilts. The expression of hepatic AR (P< 0.05) and GR (P< 0.05) mRNA and C/EBPβ (P< 0.05) protein was significantly lower in the boars than gilts. ChIP assay showed that the binding levels of C/EBPβ to 3P-HSD promoter were significantly decreased in the boars and barrows compared with the gilts (P< 0.05). Co-IP analysis revealed no interactions of C/EBPβ with either GR or AR. These results suggest that C/EBPβ binding to 3P-HSD promoter is responsible for the gender-dependent 3P-HSD expression in the adrenal of slaughter pigs.4. Mechanisms study of the gender-specific expression of 3P-HSD in the liver of finished pigsIt has reported that the expression of porcine hepatic 3P-HSD existed gender differences, however the molecular mechanisms of this gender-specific expression of 3β-HSD were still unclear. In this study, we compared the expression of hepatic 3β-HSD gene among the boars, barrows and gilts and explored the potential factors regulating 3β-HSD transcription. The results showed that the expression of 3β-HSD mRNA (P< 0.05) and protein (P< 0.05) in the boars was lower than the barrows and gilts. The expression of AR mRNA (P< 0.05) and protein (P< 0.05) was significantly lower in theboars than the barrows and gilts, while C/EBPβ mRNA expression was higher in the boars than barrows and gilts (P< 0.05), but C/EBPβ protein content was higher in the barrows than the boars (P< 0.05). ChIP assay showed that the binding levels of GR to 3β-HSD promoter were significantly higher in the boars compared with the barrows and gilts (P< 0.05), while increased histone H3 acetylation (P< 0.05) was detected in the barrows and gilts than the boars. Co-IP analysis revealed no interactions of GR with AR. These results suggest that GR binding and the enrichment of H3Ac to 3β-HSD promoter are responsible for the gender-dependent 3β-HSD expression in the liver of pigs.