The dual role of ATG7: Regulation of autophagy and apoptosis in porcine ovarian follicular granulosa cells.

The regulation of mammalian ovarian development involves the coordinated processes of autophagy and apoptosis. The autophagy-related gene ATG7 plays a pivotal role in mediating crosstalk between these pathways. Despite its recognized importance, the specific function of ATG7 in ovarian follicular granulosa cells remains poorly understood. This study aimed to explore the effects of ATG7 overexpression on apoptosis and autophagy in porcine ovarian follicular granulosa cells and thereby provide insights into the interplay between these fundamental cellular mechanisms. An ATG7 overexpression vector was introduced into cells, followed by assessment of cell proliferation using the CCK-8 assay, quantification of related gene expression via real-time quantitative PCR and western blotting, and evaluation of apoptosis using TUNEL staining. ATG7 exhibited a predominant cytoplasmic localization and additional nuclear expression in porcine ovarian follicular granulosa cells. The transfection efficiency of the vector was initially verified, indicating that its overexpression notably increased expression of ATG7 protein. Further investigations confirmed that overexpression of ATG7 inhibited cell proliferation, stimulated autophagy, and promoted apoptosis in these cells. In summary, overexpression of ATG7 influences the viability of porcine ovarian follicular granulosa cells by regulating the interplay between autophagy and apoptosis. This study not only broadens the understanding of functional regulation of autophagy and apoptosis by ATG7, but also sheds light on the intricate mechanisms governing ovarian follicular atresia.

The protective role of quercetin against copper-induced female reproductive toxicity: Insights from transcriptome analysis.

Quercetin has been shown to mitigate the cytotoxic effects of heavy metals. While copper is an essential trace element for bodily functions, excessive intake has been linked to impaired female reproductive function. Transcriptome analysis was employed to identify genes that are differentially expressed in response to high copper and were validated through qRT-PCR and western blotting. ATP content and Tunel were used to identify the damage of mitochondrial and cell apoptosis. PPI analysis revealed that MKI67, TOPII, ASPM, CASP3, PLK1, and TTK are central proteins within the network. Additionally, exposure to elevated levels of copper resulted in the dysregulation of 86 genes associated with mitochondria. Conversely, treatment with quercetin (QUE) in combination with high copper led to the normalization of 42 mitochondria-related genes previously affected by high copper levels. Furthermore, CuSO4 decreases ATP content and induces cell apoptosis, which can be reversed by QUE. Results suggest that elevated copper levels could lead to oxidative stress and apoptosis by inducing mitochondrial damage, while QUE has the potential to mitigate these effects, ultimately safeguarding granulosa cells and halting the progression of cell death. This study provides novel insights into the molecular pathways involved in female reproductive toxicity caused by excessive copper exposure.

Impact of quercetin on autophagy and apoptosis induced by a high concentration of CuSO4 in porcine ovarian granulosa cells.

Copper is a vital micronutrient necessary for the maintenance of physiological functions. However, excessive amounts can lead to organ damage. Porcine ovarian granulosa cells are damaged by a high concentration of CuSO4, which can reduce the reproductive capacity of sows. Quercetin has shown remarkable efficacy in mitigating the harmful effects of heavy metals. Therefore, the aim of this study was to investigate the effects of a high concentration of CuSO4 on autophagy and apoptosis in porcine ovarian granulosa cells and to explore whether quercetin can counteract these toxic effect. Cell morphology, and the mRNA expression levels of autophagy-related genes (LC3-Ⅰ, ATG5, ATG7, ATG12, Beclin1, mTOR, LC3-Ⅱ and P62) were significantly changed upon treatment with 200 and 400 µM CuSO4. Treatment with 200 µM CuSO4 increased expression of P62 protein (P<0.05), promoted LC3-Ⅰ to LC3-Ⅱ conversion (P<0.05), and reduced PINK1 protein expression and the ATP content (P<0.05). In addition, expression of Caspase3 protein was increased and TUNEL staining indicated that the number of apoptotic cells was increased. However, co-treatment with 10 µM quercetin significantly decreased expression of P62 and conversion of LC3-Ⅰ to LC3-Ⅱ. Furthermore, flow cytometric analysis revealed that addition of 10 µM quercetin significantly reduced apoptosis induced by a high concentration of CuSO4. In summary, the results indicate that a high concentration of CuSO4 can trigger mitochondrial and autophagy dysfunction, activate mitochondrial apoptosis pathway, and exert cytotoxic effects. Quercetin can mitigate autophagy dysfunction, enhance autophagic processes, and alleviate apoptosis.

Quercetin Alleviates Toxicity Induced by High Levels of Copper in Porcine Follicular Granulosa Cells by Scavenging Reactive Oxygen Species and Improving Mitochondrial Function.

CuSO4 is the most commonly used feed additive in pig production at present, but long-term ingestion of excessive copper would lead to chronic copper toxicity. High copper could reduce the reproductive efficiency of sows and seriously affect the development of the pig industry. Quercetin (QUE), a powerful antioxidant, reduces toxicity of a number of heavy metals. Porcine granulosa cells (pGCs) are crucial to the fate of follicle development. The present study found that high concentrations of CuSO4 induced ROS production, which resulted in decreased mRNA expression of antioxidant-related genes GPX4, CAT, and SOD2 and increased mRNA expression of SOD1, TRX, and HO-1. The protein expression of antioxidant enzymes SOD2 and HO-1 decreased. Moreover, the concentration of MDA increased, the activity of CAT decreased, and the content of GSH decreased. After high copper treatment, the mitochondrial membrane potential (MMP) was decreased and the morphological structure was changed. However, the combined treatment with Quercetin (QUE) reversed these changes, and the level of cellular oxidative stress decreased. Therefore, we conclude that high copper has oxidative toxicity to pGCs, and QUE could remove the ROS induced by high copper, protect mitochondria from oxidative stress damage, and improve the function of pGCs.

The TGF-ß/SMAD Signaling Pathway Prevents Follicular Atresia by Upregulating MORC2.

In mammals, female fertility is determined by the outcome of follicular development (ovulation or atresia). The TGF-ß/SMAD signaling pathway is an important regulator of this outcome. However, the molecular mechanism by which the TGF-ß/SMAD signaling pathway regulates porcine follicular atresia has not been fully elucidated. Microrchidia family CW-type zinc finger 2 (MORC2) is anovel epigenetic regulatory protein widely expressed in plants, nematodes, and mammals. Our previous studies showed that MORC2 is a potential downstream target gene of the TGF-ß/SMAD signaling pathway. However, the role of MORC2 in porcine follicular atresia is unknown. To investigate this, qRT-PCR, western blotting, and TdT-mediated dUTP nick-end labeling were performed. Additionally, the luciferase activity assay was conductedto confirm that the TGF-ß/SMAD signaling pathway regulates MORC2. Our results demonstrate that MORC2 is animportant anti-apoptotic molecule that prevents porcine follicular atresia via a pathway involving mitochondrial apoptosis, not DNA repair. Notably, this studyrevealsthat the TGF-ß/SMAD signaling pathway inhibits porcine granulosa cell apoptosis by up-regulating MORC2. The transcription factor SMAD4 regulated the expression of MORC2 by binding to its promoter. Our results will help to reveal the mechanism underlying porcine follicular atresia and improve the reproductive efficiency of sows.

Betaine Promotes Fat Accumulation and Reduces Injury in Landes Goose Hepatocytes by Regulating Multiple Lipid Metabolism Pathways.

Betaine is a well-established supplement used in livestock feeding. In our previous study, betaine was shown to result in the redistribution of body fat, a healthier steatosis phenotype, and an increased liver weight and triglyceride storage of the Landes goose liver, which is used for foie-gras production. However, these effects are not found in other species and strains, and the underlying mechanism is unclear. Here, we studied the underpinning molecular mechanisms by developing an in vitro fatty liver cell model using primary Landes goose hepatocytes and a high-glucose culture medium. Oil red-O staining, a mitochondrial membrane potential assay, and a qRT-PCR were used to quantify lipid droplet characteristics, mitochondrial ß-oxidation, and fatty acid metabolism-related gene expression, respectively. Our in vitro model successfully simulated steatosis caused by overfeeding. Betaine supplementation resulted in small, well-distributed lipid droplets, consistent with previous experiments in vivo. In addition, mitochondrial membrane potential was restored, and gene expression of fatty acid synthesis genes (e.g., sterol regulatory-element binding protein, diacylglycerol acyltransferase 1 and 2) was lower after betaine supplementation. By contrast, the expression of lipid hydrolysis transfer genes (mitochondrial transfer protein and lipoprotein lipase) was higher. Overall, the results provide a scientific basis and theoretical support for the use of betaine in animal production.

Unveiling differentially expressed genes upon regulation of transcription factors in sepsis.

In this study, we integrated the gene expression data of sepsis to reveal more precise genome-wide expression signature to shed light on the pathological mechanism of sepsis. Differentially expressed genes via integrating five microarray datasets from the Gene Expression Omnibus database were obtained. The gene function and involved pathways of differentially expressed genes (DEGs) were detected by GeneCodis3. Transcription factors (TFs) targeting top 20 dysregulated DEGs (including up- and downregulated genes) were found based on the TRANSFAC. A total of 1339 DEGs were detected including 788 upregulated and 551 downregulated genes. These genes were mostly involved in DNA-dependent transcription regulation, blood coagulation, and innate immune response, pathogenic escherichia coli infection, epithelial cell signaling in helicobacter pylori infection, and chemokine signaling pathway. TFs bioinformatic analysis of 20 DEGs generated 374 pairs of TF-target gene involving 47 TFs. At last, we found that five top ten upregulated DEGs (S100A8, S100A9, S100A12, PGLYRP1 and MMP9) and three downregulated DEGs (ZNF84, CYB561A3 and BST1) were under the regulation of three hub TFs of Pax-4, POU2F1, and Nkx2-5. The identified eight DEGs may be regarded as the diagnosis marker and drug target for sepsis.