Dual-specificity phosphatase 1 regulates cell cycle progression and apoptosis in cumulus cells by affecting mitochondrial function, oxidative stress, and autophagy.

Dual-specificity phosphatase 1 (DUSP1) is differentially expressed in cumulus cells of different physiological states, but its specific function and mechanism of action remain unclear. In this study, we explored the effects of DUSP1 expression inhibition on cell cycle progression, proliferation, apoptosis, and lactate and cholesterol levels in cumulus cells and examined reactive oxygen species levels, mitochondrial function, autophagy, and the expression of key cytokine genes. The results showed that inhibition of DUSP1 in cumulus cells caused abnormal cell cycle progression, increased cell proliferation, decreased apoptosis rates, increased cholesterol synthesis and lactic acid content, and increased cell expansion. The main reason for these effects was that inhibition of DUSP1 reduced ROS accumulation, increased glutathione level and mitochondrial membrane potential, and reduced autophagy levels in cells. These results indicate that DUSP1 limits the biological function of bovine cumulus cells under normal physiological conditions and will greatly contribute to further explorations of the physiological functions of cumulus cells and the interactions of the cumulus-oocyte complex.

DNA Double-Strand Break-Related Competitive Endogenous RNA Network of Noncoding RNA in Bovine Cumulus Cells.

(1) Background: DNA double strand breaks (DSBs) are the most serious form of DNA damage that affects oocyte maturation and the physiological state of follicles and ovaries. Non-coding RNAs (ncRNAs) play a crucial role in DNA damage and repair. This study aims to analyze and establish the network of ncRNAs when DSB occurs and provide new ideas for next research on the mechanism of cumulus DSB. (2) Methods: Bovine cumulus cells (CCs) were treated with bleomycin (BLM) to construct a DSB model. We detected the changes of the cell cycle, cell viability, and apoptosis to determine the effect of DSBs on cell biology, and further evaluated the relationship between the transcriptome and competitive endogenous RNA (ceRNA) network and DSBs. (3) Results: BLM increased γH2AX positivity in CCs, disrupted the G1/S phase, and decreased cell viability. Totals of 848 mRNAs, 75 long noncoding RNAs (lncRNAs), 68 circular RNAs (circRNAs), and 71 microRNAs (miRNAs) in 78 groups of lncRNA-miRNA-mRNA regulatory networks, 275 groups of circRNA-miRNA-mRNA regulatory networks, and five groups of lncRNA/circRNA-miRNA-mRNA co-expression regulatory networks were related to DSBs. Most differentially expressed ncRNAs were annotated to cell cycle, p53, PI3K-AKT, and WNT signaling pathways. (4) Conclusions: The ceRNA network helps to understand the effects of DNA DSBs activation and remission on the biological function of CCs.

CPEB3 regulates the proliferation and apoptosis of bovine cumulus cells.

Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a member of the Cytoplasmic polyadenylation element-binding family, which has been found to regulate the translation of dormant and masked mRNA in Xenopus oocytes and plays potential roles in regulating biological functions in cells and tissues. However, its role in cumulus cells is not clear. In this study, the mRNA expression of CPEB3 in bovine cumulus cells was inhibited with small interfering RNA. Cell cycle progression, proliferation, and apoptosis were measured after inhibition of CPEB3. Subsequently, changes in intracellular Reactive oxygen species content, mitochondrial membrane potential and expansion-related gene expression were examined. The results showed that after CPEB3 inhibition, cumulus cells had an abnormal cell cycle, the numbers of cells in the S and G2/M phases were significantly increased, cell proliferation was increased and apoptosis rates were decreased. These effects were likely due CPEB3 inhibition-induced decreases in intracellular Reactive oxygen species levels; increases in mitochondrial membrane potential; decreases in apoptosis; downregulation of CCNA, CCND, CCNE, CDK2, CDK4, CDK6, p21, and p27 mRNA expression; and upregulation of CCNB, CDK1, HAS2, PTGS2, PTX3, and CEBPB mRNA expression. Therefore, CPEB3 plays potential roles in regulating the biological and physiological functions of bovine cumulus cell.

COL1A1 affects apoptosis by regulating oxidative stress and autophagy in bovine cumulus cells.

The collagen type I alpha 1 chain (COL1A1), as a major component of extracellular matrix, plays a potential role in the growth and development of bovine follicles. However, its specific role in bovine cumulus cells remains unclear. In this study, we examined apoptosis, the cell cycle and reactive oxygen species after inhibition of COL1A1 expression by siRNA in bovine cumulus cells. Cell proliferation was measured by CCK-8, and mitochondrial membrane potential was detected by fluorescence intensities of JC-1 staining. Moreover, cell autophagy was detected by immunofluorescence, and cell migration was detected by a cell scratch assay. Lactic acid and cholesterol concentration were measured to evaluate the glucose utilization and cholesterol synthesis activity in cumulus cell by optical density detection method. RT-qPCR and Western blot analysis were used to measure changes in key gene expression. The results showed that cumulus cells were found to have an abnormal cell cycle, and the numbers of cells in S phase were significantly reduced, accompanied by decreases in cholesterol synthesis, and cell proliferation ability and an increase in apoptosis rate with siRNA-COL1A1 treatment. These findings were likely due to inhibition of COL1A1 resulting in high levels of ROS in the cells, a decrease in mitochondrial membrane potential, an increase in intracellular autophagy, activation of the apoptotic pathway, and a decrease in lactic acid conversion ability. COL1A1 plays an important role in regulating the physiological and biological functions of bovine cumulus cells.

miR-181a regulate porcine preadipocyte differentiation by targeting TGFBR1.

miRNAs have been shown to regulate a variety of biological process. It has been shown that miR-181a regulates porcine adipogenesis by targeting Tumor Necrosis Factor-α (TNF-α), but the overall functions of miR-181a in porcine preadipocyte differentiation remain unclear. This study aimed to explore the functions of miR-181a in porcine preadipocyte differentiation via the TGFß/Smad pathway. The TargetScan program was used to predict miRNAs targeting TGFBR1, and miR-181a was selected as a candidate. To investigate the functions of miR-181a, miRNA mimics and inhibitors were used to overexpress or knockdown miR-181a, respectively. RT-qPCR and Western blotting were used to measure the expression of aP2, PPARγ, C/EBPα and TGFBR1 in porcine preadipocytes. Lipid accumulation and adipocyte apoptosis were detected using Oil Red O staining and flow cytometry, respectively. Taken together, our results indicated that miR-181a promoted porcine preadipocyte differentiation by directly targeting TGFBR1.