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.

[Comparison and analysis of lincRNAs expression profile in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows].

The normal estrus in weaned primiparous sows has a great impact on pig production and abnormal estrus is the main reason for the elimination of primiparous sows. In this study, we studied the long intergenic noncoding RNAs (lincRNAs) in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. These long intergenic noncoding RNAs (lincRNAs) were screened and compared through RNA-seq analysis. The expression profiles of lincRNAs were obtained and their characteristics and functions were preliminarily analyzed. There are 3519 novel lincRNAs identified in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. Compared with estrous primiparous sows, 17 differentially expressed lincRNAs were indentified, including 12 up-regulated lincRNAs and 5 down-regulated lincRNAs (FC≥2, P<0.05). The four lincRNA transcripts obtained through selection were verified by qRT-PCR, which are consistent with the RNA-seq results. The GO, KEGG pathway, and lincRNA-mRNA co-expression network analysis of these 17 lincRNAs revealed that these lincRNAs were mainly involved in reproductive activities, such as oocyte meiosis mature, ovarian cells differentiation and granulosa cells apoptosis. The results enriched the data resources of pig lincRNAs and provided useful information for further research about the reproductive performance of primiparous sows.

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.

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.

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.

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.

Effect of fractionated irradiation on the expression of multidrug resistance genes in the CNE1 human nasopharyngeal carcinoma cell line.

Nasopharyngeal carcinoma (NPC) often develops drug resistance following radiotherapy. The molecular basis of radiotherapy-related multidrug resistance (MDR) remains unclear. In the present study, we investigated the effect of fractionated irradiation on the expression of the MDR-1 gene and the MDR-associated protein P-glycoprotein (P-gp) in CNE1 human NPC cells. CNE1 cells were treated with fractionated X-rays. Drug resistance was determined by MTT assay. The expression levels of MDR-1 and P-gp were analyzed by RT-PCR and western blot analysis, respectively. Differential expression was analyzed by gene chips. The results revealed that low levels of mRNA expression of MDR1 were present in non-irradiated CNE1 cells. Compared with the control, the expression of MDR1 mRNA was gradually increased following fractionated irradiation. On day 21, the expression of MDR1 mRNA was increased 1.59- and 2.19-fold, compared with the control, by treatment with 10 and 20 Gy, respectively. We observed decreased MDR1 expression following treatment with 10 and 20 Gy irradiation on days 28 and 35, compared with day 21. On days 21, 28 and 35, expression was increased 1.37-, 1.40- and 1.15-fold by treatment with 20 Gy compared with 10 Gy. Expression of MDR1 was significantly upregulated by treatment with 50 Gy irradiation compared with the control on days 78 and 106. P-gp expression was consistent with that of MDR1 mRNA expression. The sensitivity of CNE1 cells to cisplatin was reduced following irradiation compared with the control. A total of 26 genes were significantly upregulated and 8 genes were significantly downregulated compared with the control. Results of the present study have shown that MDR1 and P-gp are upregulated in CNE1 cells following irradiation. Multiple genes were involved in the mechanism of radiation-induced drug resistance.