The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) plays an important role in androstenone metabolism in pig liver, and its defective expression is related to the development of boar taint. Early age castration is a common practice in many countries to avoid boar taint, yet whether and how castration affects porcine hepatic 3β-HSD expression are still poorly understood. In this study, we aimed to compare the expression of 3β-HSD between intact (boars) and castrated (barrows) male pigs, and to explore the potential factors regulating 3β-HSD transcription. Compared to barrows, boars showed worse carcass quality. Boars had significantly higher levels of serum androstenone (P < 0.01), testosterone (P < 0.01) and hepatic cortisol (P < 0.05), which were contrary to significantly lower expression of 3β-HSD messenger RNA (P < 0.01) and protein (P < 0.01) in the liver. Significant differences were detected for the hepatic expression of androgen receptor (AR) and CCAAT/enhancer binding protein β (C/EBPβ). Chromatin immunoprecipitation (ChIP) assay demonstrated reduced histone H3 acetylation (P < 0.05) but increased glucocorticoid receptor (GR) binding to 3β-HSD gene promoter in boars (P < 0.05). These results indicate that GR binding to 3β-HSD promoter is involved in the differential hepatic 3β-HSD expression between boars and barrows.

Methyl donor nutrients are critical for embryonic development of brain. Hippocampus is the most susceptible brain region to various factors including prenatal supply of methyl donors. Glucocorticoid receptor (GR) expressed in hippocampus is involved in the regulation of energy homeostasis and stress sensitivity. Hippocampal GR expression is highly susceptible to epigenetic regulation, yet the effect of maternal methyl donor supplementation on epigenetic regulation of GR transcription in offspring hippocampus remains unclear. In this study, we fed sows with betaine (3 g/kg) throughout the gestation and analyzed the hippocampal expression of GR mRNA and its variants, as well as the CpG methylation status of the promoter and the microRNAs predicted to target 3’ UTR of porcine GR gene in neonatal piglets. Total GR mRNA (P<0.01) and its variants GR 1-4 (P<0.05) and 1-9,10 (P<0.01), were significantly higher in the hippocampus of betaine-treated piglets, while the content of GR protein was not significantly changed. The CpGs located in the –1650 ~ –1515 segment of GR gene were hypermethylated (P<0.05). The hippocampal expression of miR-130b (P<0.05), miR-181a (P<0.05) and miR-181d (P<0.01) was significantly up-regulated. The targeting efficacy of miR-130b and miR-181d was validated in vitro using dual-luciferase reporter assay system. Our results demonstrate that maternal betaine supplementation during gestation enhances GR mRNA expression in offspring hippocampus, which involves alterations in miRNAs expression.

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Folic acid (FA) is an extremely important nutrient for brain formation and development. FA deficiency is highly linked to brain degeneration and age-related diseases, which are also associated with autophagic activities and circadian rhythm in hippocampal neurons. However, little is known how autophagy- and circadian-related genes in hippocampal neurons are regulated under FA deficiency. Here, hippocampal neuroncells (HT-22) were employed to determine the effect of FA deprivation (FD) on the expression of relevant genes and to reveal the potential role of glucocorticoid receptor (GR). FD increased autophagic activities in HT-22 cells, associated with significantly (P<0.05) enhanced GR activation indicated by higher ratio of GR phosphorylation. Out of 17 autophagy-related genes determined, 8 was significantly (P<0.05) up-regulated in FD group, which includes ATG2b, ATG3, ATG4c, ATG5, ATG10, ATG12, ATG13 and ATG14. Meanwhile, 4 out of 7 circadian-related genes detected, Clock, Cry1, Cry2 and Per2, were significantly (P<0.05) up-regulated. The protein content of autophagy markers, LC3A and LC3B, was also increased significantly (P<0.05). ChIP assay showed that FD promoted (P<0.05) GR binding to the promoter sequence of ATG3 and Per2. Moreover, MeDIP analysis demonstrated significant (P<0.05) hypomethylation in the promoter sequence of ATG12, ATG13 and Per2 genes. Together, we speculate that FD increases the transcription of autophagy- and circadian-related genes through, at least partly, GR-mediated pathway. Our results provide a basis for future investigations into the intracellular regulatory network in response to folate deficiency.

The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms.

BACKGROUND:Hepatic 3β-hydroxysteroid dehydrogenase (3β-HSD) plays an important role in steroid inactivation and catabolism. Serum concentrations of steroid hormones differ significantly between breeds in pigs, however the molecular mechanism regulating hepatic 3β-HSD expression in different breeds of pigs is poorly understood. In the present study, we used preweaning purebred male Large White (LW) and Erhualian (EHL) piglets as model to investigate the breed difference in the expression and regulation of 3β-HSD gene in porcine liver.RESULTS:The hepatic expression of 3β-HSD mRNA was significantly lower (P < 0.01) in EHL piglets compared to that in LW piglets. Significant breed differences were detected for the hepatic expression of transcription factors such as androgen receptor (AR), glucocorticoid receptor (GR), and CCAAT/enhancer binding protein β (C/EBPβ). The nucleoprotein contents of AR (P < 0.05), GR (P < 0.01) and phospho-Ser(211)GR (P < 0.01) were significantly higher in the liver of EHL piglets. Chromatin immunoprecipitation (ChIP) assay demonstrated significantly lower binding of GR, but not AR or C/EBPβ, to 3β-HSD gene promoter in EHL piglets (P < 0.05). GR was not detected to interact with C/EBPβ or AR in the co-immunoprecipitation analysis.CONCLUSIONS:These results indicate that GR binding to 3β-HSD promoter is involved in the breed-dependent 3β-HSD expression in the liver of piglets.

Maternal malnutrition may disrupt ovarian functions in adult offspring. Steroidogenesis and folliculogenesis in the offspring ovary appear to be the major targets of nutritional programming. Nevertheless, the mechanism by which maternal low-protein diet affects the offspring steroidogenesis and folliculogenesis, and the possible pathway linking these two processes remain unclear. In this study, Landrace×Yorkshire crossbred sows were fed either standard (SP) or low-protein (LP, 50% of the SP) diets throughout gestation and lactation. Female offspring were fed the same diet after weaning until 6 months of age. LP offspring had higher serum 17β-estradiol level (P<0.01), which was accompanied by lower mRNA (P<0.05) but higher protein (P<0.05) expression of cytochrome P450 aromatase (CYP19A1) in the ovary. CYP19A1 protein up-regulation was associated with lower ovarian expression of drosha (P<0.05) and miRNAs targeting CYP19A1 (P<0.05). LP offspring had less graafian follicles with more apoptotic granulosa cells (P<0.05), as well as higher caspase 3 activity (P<0.05) and FasL expression (P<0.05) in the ovary. FasL gene up-regulation was associated with higher ERα protein expression (P<0.05) and binding to FasL gene promoter. These results suggest that a maternal LP diet in pregnancy and lactation elevated serum 17β-estradiol level by activating CYP19A1 through miRNA-mediated mechanism, and induced granulosa apoptosis in graafian follicles through ER-activated Fas/FasL-caspase 3 pathway.