SOD1 Gene Silencing Promotes Apoptosis and Suppresses Proliferation of Heat-Stressed Bovine Granulosa Cells via Induction of Oxidative Stress.
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ABSTRACT:
SUBMITTER: Khan A
PROVIDER: S-EPMC8708094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Heat stress negatively affects reproduction in cattle by disrupting the normal function of ovarian granulosa cells (GCs), ultimately leading to oxidative damage and cell death via apoptosis. Heme oxygenase-1(HO-1) is a member of the heat shock protein family, which are associated with cellular antioxidant defenses and anti-apoptotic functions. Recent studies demonstrated that HO-1 is upregulated in heat-stressed cells. In the present study, we investigated the expression of HO-1 in bovine GCs transiently exposed to heat stress and characterized the expression and activity of key oxidative stress enzymes and molecules. We show that heat stress induced oxidative stress and apoptosis, and enhanced Nrf2 and HO-1 expression in primary GC cultures. Knocking down HO-1 expression using siRNA exacerbated both oxidative stress and apoptosis, whereas pre-treating GCs with hemin, which induces HO-1 expression, partially prevented these effects. These findings demonstrate that HO-1 attenuates heat stress-induced apoptosis in bovine GCs by decreasing production of reactive oxygen species and activating the antioxidant response.
| S-EPMC6710052 | biostudies-literature
Project description:Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Heat stress inhibits ovarian follicular development leading to diminished reproductive efficiency of dairy cows during summer. Ovarian follicle development is a complex process. During follicle development, granulosa cells (GCs) replicate, secrete hormones, and support the growth of the oocyte. To obtain an overview of the effects of heat stress on GCs, digital gene expression profiling was employed to screen and identify differentially expressed genes (DEGs; false discovery rate (FDR) ≤ 0.001, fold change ≥2) of cultured GCs during heat stress. A total of 1211 DEGs including 175 upregulated and 1036 downregulated ones were identified, of which DEGs can be classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results suggested that heat stress triggers a dramatic and complex program of altered gene expression in GCs. We hypothesized that heat stress could induce the apoptosis and dysfunction of GCs. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of steroidogenic genes (steroidogenic acute regulatory protein (Star), cytochrome P-450 (CYP11A1), CYP19A1, and steroidogenic factor 1 (SF-1)) and apoptosis-related genes (caspase-3, BCL-2, and BAX). Radio immunoassay (RIA) was used to analyze the level of 17β-estradiol (E2) and progesterone (P4). We also assessed the apoptosis of GCs by flow cytometry. Our data suggested that heat stress induced GC apoptosis through the BAX/BCL-2 pathway and reduced the steroidogenic gene messenger RNA (mRNA) expression and E2 synthesis. These results suggest that the decreased function of GCs may cause ovarian dysfunction and offer an improved understanding of the molecular mechanism responsible for the low fertility in cattle in summer.
| S-EPMC4837181 | biostudies-literature
Project description:A high temperature-humidity index during summer has deleterious effects on mitochondrial function, reducing oocyte developmental competence. 5-Aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) are both known to support mitochondrial function and have strong anti-oxidant and anti-apoptotic activities. This study aimed to determine the mechanism of action of 5-ALA/SFC on oocyte quality. Bovine oocytes were collected from medium-sized follicles during summer (July-September, temperature-humidity index:76.6), cultured with 0, 1, 2, 4, and 8 µM 5-ALA with SFC at a molar ratio of 1:0.125, fertilized, and cultured for 10 days. The addition of 8/1 µM 5-ALA/SFC had a deleterious effect on oocyte cleavage rate in comparison with control oocytes, but did not affect the blastocyst rate, while 1/0.125 µM 5-ALA/SFC had a significantly higher increase in blastocyst rate than 8/1 µM 5-ALA/SFC. The addition of 1/0.125 and 2/0.25 µM 5-ALA/SFC improved oocyte quality by increasing the mitochondrial distribution pattern and metaphase-II oocytes, reducing reactive oxygen species and upregulating nuclear factor erythroid-2-related factor 2, heme oxygenase-1, and superoxide dismutase-1 in oocytes, and nuclear factor erythroid-2-related factor 2 and mitochondrial transcription factor A in cumulus cells. These results indicate that 1/0.125 and 2/0.25 µM 5-ALA/SFC may support oocyte quality and developmental competence and provide anti-oxidant actions in cumulus-oocyte complexes.
| S-EPMC10602763 | biostudies-literature
Project description:Heat stress affects granulosa cells and the ovarian follicular microenvironment, ultimately resulting in poor oocyte developmental competence. This study aims to investigate the metabo-lomics response of bovine granulosa cells (bGCs) to in vitro acute heat stress of 43 °C. Heat stress triggers oxidative stress-mediated apoptosis in cultured bGCs. Heat-stressed bGCs exhibited a time-dependent recovery of proliferation potential by 48 h. A total of 119 metabolites were identified through LC-MS/MS-based metabolomics of the spent culture media, out of which, 37 metabolites were determined as differentially involved in metabolic pathways related to bioenergetics support mechanisms and the physical adaptations of bGCs. Multiple analyses of metabolome data identified choline, citric acid, 3-hydroxy-3-methylglutaric acid, glutamine, and glycocyamine as being upregulated, while galactosamine, AICAR, ciliatine, 16-hydroxyhexadecanoic acid, lysine, succinic acid, uridine, xanthine, and uraconic acid were the important downregulated metabolites in acute heat stress. These differential metabolites were implicated in various important metabolic pathways directed towards bioenergetics support mechanisms including glycerophospholipid metabolism, the citrate cycle (TCA cycle), glyoxylate and dicarboxylate metabolism, and serine, threonine, and tyrosine metabolism. Our study presents important metabolites and metabolic pathways involved in the adaptation of bGCs to acute heat stress in vitro.
| S-EPMC8879866 | biostudies-literature
Project description:PURPOSE:The purpose of this study was to evaluate the capacity of bovine granulosa cells to generate reactive oxygen intermediates in response to lipopolysaccharide. We hypothesized that granulosa cells increase reactive oxygen intermediates in response to Gram-negative lipopolysaccharide in a similar manner to immune cells. METHODS:Bovine peripheral blood mononuclear cells and granulosa cells were cultured in the presence of lipopolysaccharide. Oxidative stress was evaluated using the fluorescent marker dye CellROX, and oxidative stress-related genes were measured using real-time RT-PCR. RESULTS:As expected, peripheral blood mononuclear cells increased oxidative stress in response to lipopolysaccharide as measured by accumulation of the fluorescent marker dye CellROX. While granulosa cells demonstrate the capacity to increase accumulation of CellROX dye in response to a positive control menadione, lipopolysaccharide had no effect on accumulation of CellROX dye. The expression of GSR, SOD1, and SOD2 were variable in peripheral blood mononuclear cells treated with lipopolysaccharide but were consistently upregulated when co-incubated with the antioxidant, N-acetyl cysteine. The expression of oxidative stress-related genes was not altered in granulosa cells, with the exception of elevated SOD1 following lipopolysaccharide exposure in the absence of antioxidant. CONCLUSIONS:Combined, these data suggest that while reactive stress is important in pathogen killing and inflammation in immune cells, granulosa cells do not increase oxidative stress in response to lipopolysaccharide.
| S-EPMC5714823 | biostudies-literature
Project description:Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.
| S-EPMC8235242 | biostudies-literature
Project description:Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.
| S-EPMC7519046 | biostudies-literature
Project description:Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2-) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification.
| S-EPMC9101166 | biostudies-literature
Project description:Inhibin A is well known for its inhibitory properties against follicle-stimulating hormone (FSH), released through a pituitary-gonadal negative feedback loop to regulate follicular development. Ovarian folliculogenesis, hormonal biosynthesis, and gametogenesis are dependent on inhibins, playing vital roles in promoting or inhibiting cell proliferation. The present study explored the physiological and molecular response of bovine granulosa cells (GCs) to different concentrations of inhibin A in vitro. We treated the primary GCs isolated from ovarian follicles (3-6 mm) with different levels of inhibin A (20, 50, and 100 ng/mL) along with the control (0 ng/mL) for 24 h. To evaluate the impact of inhibin A on GCs, several in vitro cellular parameters, including cell apoptosis, viability, cell cycle, and mitochondrial membrane potential (MMP) were detected. Besides, the transcriptional regulation of pro-apoptotic (BAX, Caspase-3) and cell proliferation (PCNA, CyclinB1) genes were also quantified. The results indicated a significant (p < 0.05) increase in the cell viability in a dose-dependent manner of inhibin A. Likewise, MMP was significantly (p < 0.05) enhanced when GCs were treated with high doses (50, 100 ng/mL) of inhibin A. Furthermore, inhibin A dose (100 ng/mL) markedly improved the progression of the G1 phase of the cell cycle and increased the cell number in the S phase, which was supported by the up-regulation of the proliferating cell nuclear antigen PCNA (20, 50, and 100ng/mL) and CyclinB (100 ng/mL) genes. In addition, higher doses of inhibin A (50 and 100 ng/mL) significantly (p < 0.05) decreased the apoptotic rate in GCs, which was manifested by down regulating BAX and Caspase-3 genes. Conclusively, our study presented a worthy strategy for the first time to characterize the cellular adaptation of bovine GCs under different concentrations of inhibin A. Our results conclude that inhibin A is a broad regulatory marker in GCs by regulating apoptosis and cellular progression.
| S-EPMC7071129 | biostudies-literature
Project description:Background:Heat stress is known to affect follicular dynamics, oocyte maturation, and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs). The present study explored the physiological and molecular response of bGCs to different heat stress intensities in-vitro. We exposed the primary bGCs to heat stress (HS) at 39?°C, 40?°C and 41?°C along with control samples (38?°C) for 2 h. To evaluate the impact of heat stress on bGCs, several in vitro cellular parameters including cell apoptosis, intracellular reactive oxygen species (ROS) accumulation and HSP70 kinetics were assessed by flow cytometry, florescence microscopy and western blot, respectively. Furthermore, the ELISA was performed to confirm the 17?-estradiol (E2) and progesterone (P4) levels. In addition, the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments. Results:Our findings revealed that the HS significantly decreased the cell viability, E2 and P4 levels in bGCs, whereas, increased the cellular apoptosis and ROS. Moreover, the RNA-Seq experiments showed that all the treatments (39?°C, 40?°C and 41?°C) significantly regulated many differentially expressed genes (DEGs) i.e. BCL2L1, STAR, CYP11A1, CASP3, SOD2, HSPA13, and MAPK8IP1 and pathways associated with heat stress, apoptosis, steroidogenesis, and oxidative stress. Conclusively, our data demonstrated that the impact of 40?°C treatment was comparatively detrimental for cell viability, apoptosis and ROS accumulation. Notably, a similar trend of gene expression was reported by RT-qPCR for RNA-seq data. Conclusions:Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39, 40 and 41?°C) in-vitro. The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle. Moreover, the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.
| S-EPMC7027041 | biostudies-literature