Identification and characterization of long non-coding RNAs in subcutaneous adipose tissue from castrated and intact full-sib pair Huainan male pigs.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate adipose tissue metabolism, however, their function on testosterone deficiency related obesity in humans is less understood. For this research, intact and castrated male pigs are the best model animal because of their similar proportional organ sizes, cardiovascular systems and metabolic features. RESULTS: We identified lncRNAs in subcutaneous adipose tissue by deep RNA-sequencing using the intact and castrated Huainan male pigs. The results showed that castration reduced serum testosterone but increased body fatness-related traits (serum triglyceride levels, backfat thickness, intramuscular fat content, and adipocyte size). Meanwhile, 343 lncRNAs from subcutaneous adipose tissue were identified, including 223 intergenic lncRNAs (lincRNAs), 68 anti-sense lncRNAs, and 52 intronic lncRNAs. It was predicted that there were 416 recognition sites for C/EBPα in the 303 lncRNA promoter region, and 13 adipogenesis-promoting miRNAs and five adipogenesis-depressing miRNAs target these lncRNAs. Eighteen lncRNAs, including nine up- and nine down-regulated had more than 2-fold differential expression between the castrated and intact male pigs (q-value < 0.05). Functional analysis indicated that these 18 lncRNAs and their target genes were involved in fatty acid, insulin, and the adipocytokine signaling pathway. We further analyzed the features of a conserved mouse lncRNA gene ENSMUST00000189966 and found it mainly expressed in the cell nucleus and target the Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) gene. In 3 T3-L1 cells, differentiation down-regulated their expression, but dihydrotestosterone (DHT) significantly up-regulated their expression in a concentration-dependent manner (P < 0.05). CONCLUSIONS: These results suggested that lncRNAs and their target genes might participated in the castration-induced fat deposition and provide a new therapeutic target for combatting testosterone deficiency-related obesity.

Autophagy Contributes to Oxidative Stress-Induced Apoptosis in Porcine Granulosa Cells.

Oxidative stress-induced granulosa cell (GC) death is a major cause of follicular atresia. As the major types of programmed cell death, autophagy and apoptosis have been observed in response to H2O2-mediated oxidative stress and have been demonstrated to be responsible for porcine GC death. To date, however, the cellular reactions linking autophagy to the apoptosis of porcine GC under oxidative stress are still poorly understood. Porcine GC were treated with H2O2, and autophagic flux was examined by western blotting. Cell viability and cell death assays were performed after cotreatment of porcine GC with autophagy activator (rapamycin) or inhibitor (3-methyladenine, 3-MA) together with H2O2. We revealed that short exposure (1-3 h) of porcine GC to H2O2 dramatically increased autophagic flux (1.8- to 2.5-fold over that in the control), whereas 6-12 h prolonged treatment decreased autophagy but elevated the caspase-3 activity and GC apoptotic rate. Furthermore, we showed that pretreatment with rapamycin exacerbated H2O2-mediated cytotoxicity and caspase-3 activation but that 3-MA or siRNAs specific for Beclin 1 and Atg7 genes ameliorated H2O2-mediated GC apoptosis. Together, our results indicate that autophagy plays a pivotal role in H2O2-mediated porcine GC apoptosis. Importantly, we show that the early induction of autophagic flux contributes to oxidative stress-induced apoptosis in porcine GC. The results also suggest that regulating the autophagy response in porcine GC under oxidative stress might be a new strategy for abnormal follicular atresia.

miR-181a promotes porcine granulosa cell apoptosis by targeting TGFBR1 via the activin signaling pathway.

Activin/Smad3 signaling plays a pivotal role in follicle development and atresia. However, the precise mechanisms underlying this process are not yet fully understood. Herein, we identified miR-181a as a central component of activin/Smad3-mediated follicle atresia. miR-181a was strikingly upregulated in porcine atretic follicles, which induced the apoptosis of porcine granulosa cells (GCs) in vitro. Furthermore, the transforming growth factor-ß type 1 receptor (TGFBR1) was confirmed as a direct target of miR-181a by bioinformatics analysis and luciferase assays. Transfection with an miR-181a agomir repressed the TGFBR1 mRNA and protein levels. In addition, TGFBR1 overexpression repressed GC apoptosis, whereas TGFBR1 inhibition promoted GC apoptosis. miR-181a overexpression downregulated the phosphorylation of Smad3 and blocked the activation of TGF-ß signaling. Moreover, activin A downregulated miR-181a expression and upregulated the TGFBR1 and p-Smad3 protein levels. Collectively, these data suggest that miR-181a regulates porcine GC apoptosis by targeting TGFBR1 via the activin signaling pathway.

Association of colistin residues and manure treatment with the abundance of mcr-1 gene in swine feedlots.

BACKGROUND: The extensive use of colistin in swine production may have contributed to the recent emergence of corresponding mobile resistance gene mcr-1. The use of colistin as a feed additive was banned in China in April 2017. OBJECTIVES: To examine the occurrence of colistin and dissemination of mcr-1 in swine feedlots before and after the colistin ban and effects of different manure treatments. METHODS: Environmental samples were collected from swine feedlots before (December 2016) and after (December 2017) the colistin ban. Colistin concentrations were determined by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The prevalence of mcr-1 were determined by quantitative PCR analysis, while bacterial community composition was investigated by 16S rRNA sequencing. RESULTS: In 2016, colistin was detected in feed and fresh manure samples at 67 mg/kg and 17 mg/kg, respectively, but was absent from all samples in 2017. In 2016, the relative abundance of mcr-1 in fresh manure was lower than that in solid samples after natural drying, while a higher relative abundance was detected in fresh manure samples compared with biogas slurry samples. A strong correlation between colistin concentration and relative abundance of mcr-1 was observed in fresh manure. The samples collected in 2017 showed a lower relative abundance of mcr-1 compared with those collected in 2016. Bacterial community analysis showed that the abundance of Enterobacteriaceae, which act as a vehicle and reservoir of mcr-1, increased with natural dying but decreased with anaerobic digestion. CONCLUSIONS: The presence of colistin exerts direct selection pressure for the accumulation of mcr-1 in manure, while the ban on colistin likely halted the dissemination of mcr-1 on pig farms. Anaerobic digestion is an effective waste treatment process for removing mcr-1, which might be mainly driven by the shift in bacterial community structure.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Castration-induced changes in the expression profiles and promoter methylation of the GHR gene in Huainan male pigs.

Castration plays a regulatory role in growth and carcass traits, particularly in fat deposition, but its molecular mechanisms are still not clear. The present study showed that castration significantly reduced the serum growth hormone and the responses of the growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-I), IGF-IR and peroxisome proliferator-activated receptor gamma (PPARγ) to castration were similar in different adipose tissues. However, the GHR expression trends were opposite between the liver and the adipose tissues; bisulfite sequencing PCR (BSP) showed that its methylation in these two tissues was different. In particular, the GHR methylation rate in the liver of castrated and intact pigs were 93.33% and 0, respectively, which was consistent with its higher expression level in the intact group. It was predicted that there were potential binding sites for 11 transcription factors in the ninth CpG site (which was methylated and demethylated in subcutaneous adipose tissue of the intact and castrated groups, respectively), including androgen receptor (AR), CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPß, all of which are important factors in lipid metabolism. These results indicate that DNA methylation may participate in castration-induced fat deposition.

Chronic Exposure to Diquat Causes Reproductive Toxicity in Female Mice.

Diquat is a bipyridyl herbicide that has been widely used as a model chemical for in vivo studies of oxidative stress due to its generation of superoxide anions, and cytotoxic effects. There is little information regarding the toxic effects of diquat on the female reproductive system, particularly ovarian function. Thus, we investigated the reproductive toxic effects of diquat on female mice. Chronic exposure to diquat reduced ovary weights, induced ovarian oxidative stress, resulted in granulosa cell apoptosis, and disrupted oocyte developmental competence, as shown by reactive oxygen species (ROS) accumulation, decreased polar body extrusion rates and increased apoptosis-related genes expression. Additionally, after diquat treatment, the numbers of fetal mice and litter sizes were significantly reduced compared to those of control mice. Thus, our results indicated that chronic exposure to diquat induced reproductive toxicity in female mice by promoting the ROS production of gruanousa cells and ooctyes, impairing follicle development, inducing apoptosis, and reducing oocyte quality. In conclusion, our findings indicate that diquat can be used as a potent and efficient chemical for in vivo studies of female reproductive toxicity induced by oxidative stress. Moreover, the findings from this study will further enlarge imitative research investigating the effect of ovarian damage induced by oxidative stress on reproductive performance and possible mechanisms of action in large domestic animals.

Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2.

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.