SOD1 Gene Silencing Promotes Apoptosis and Suppresses Proliferation of Heat-Stressed Bovine Granulosa Cells via Induction of Oxidative Stress

Heat stress (HS) compromises dairy cattle reproduction by altering the follicular dynamics, oocyte maturation, and normal physiological function of ovarian granulosa cells (GCs), eventually resulting in oxidative damage and cell apoptosis. To protect the cells from oxidative damage, the Superoxide dismutase-1 (SOD1) degraded the hydrogen peroxide (H2O2) to oxygen (O2) and water. The objective of the current study was to investigate the impact of SOD1 silencing on intracellular ROS accumulation, cell viability, MMP, hormone synthesis (P4, E2), cell proliferation, and apoptosis in GCs under HS. The mechanistic role of SOD1 regulation in the heat-stressed GCs was explored. SOD1 gene was successfully silenced in GCs and confirmed at both transcriptional and translational levels. We found that silencing of SOD1 using siRNA under HS aggravated intracellular accumulation of reactive oxygen species, apoptosis, disrupted the mitochondrial membrane potential (MMP), altered transition of the cell cycle, and impaired synthesis of progesterone (P4) and estrogen (E2) in GCs. The associative apoptotic, steroidogenic, and cell cycle genes (BAX, Caspase-3, STAR, Cyp11A1, HSP70, PCNA, and CyclinB1) were used to confirm the results. These results identify a novel role of SOD1 in the modulation of bovine ovarian GC apoptosis, which provides a target for improving the fertility of heat-stressed dairy cows in summer.


Introduction
Environmental temperature significantly affects animal breeding and reproduction [1]. Heat stress causes infertility in dairy cows, a significant source of economic loss in the cattle industry [2][3][4][5]. Loses related to HS due to decreased milk production and a decline in pregnancy rates cost U.S. producers about one billion dollars annually [6]. The ovarian follicle is composed of oocytes surrounded by several layers of GCs. GCs play an important role in supporting and nurturing the developing oocyte. Subsequently, the matured oocyte further participates in the fertilization and formation of an embryo [7]. Heat stress has long been considered a challenging issue that negatively affects the reproductive functions

Production of siRNA and GCs Transfection
RNA interference was carried out for SOD1 silencing using small interfering RNAs directed against cow SOD1 (siSOD1) (the sense and antisense sequence of siRNA are 5 -CCAUCAGUUUGGAGACAAUTT-3 , 5 -AUUGUCUCCAAACUGAUGGTT-3 , respectively) and negative control (NC) (Gene Pharma, Shanghai, China). Bovine GCs were cultured in six-well plates for 48 h until 60% confluence and then transfected with Lipo-fectamineTM 3000 (Invitrogen, Carlsbad, ON, Canada) following the manufacturer's instructions. Subsequently, cells were immediately suspended in DMEM/F-12 (Gibco, Life Technologies Inc., Grand Island, NY, USA) medium and incubated at 38 • C under 5% CO 2 in humidified air. After 24-48 h of transfection, GCs were collected for protein and RNA extraction to validate the effective reduction of SOD1 expression through western blotting and RT-qPCR. In addition, culture media was taken to estimate hormone levels.

Estimation ROS
ROS was estimated in heat stress GCs following the protocol, previously carried out by [28]. GCs from the treated and control groups ((NC, 40 • C + NC, and siSOD1 + 40 • C) were trypsinized and collected 48 h after culturing to study the net intracellular ROS production using a fluorescence microscope (Olympus, Tokyo, Japan). The GCs were rinsed in PBS after treatment, and 10 mol/L H2DCFDA was added to each well. GCs were washed once in 0.1% PVA/DPBS and inspected under a fluorescence microscope after incubation for 30 min at 38 • C in the dark (Olympus, Tokyo, Japan).

Estimation of Apoptotic and Dead GCs
Bovine GCs were detected for apoptosis using the Annexin V-FITC kit (Beyotime Biotechnology, China). GCs were extracted and washed three times with pre-heated PBS after receiving the relevant treatments. Following collection, GCs were incubated with Annexin VFITC for 20 min in the dark and propidium iodide (PI) for 2 min, followed by flow cytometry (BD Biosciences, CA, USA). The apoptotic rate is expressed as the sum of the percentage of early (Annexin V+/PI-) and late (Annexin V+/PI-) apoptosis cells. Furthermore, the number of dead cells was determined under a laser-scanning confocal microscope (TCS SP8, Leica, Germany). FlowJo software (version Win 64-10.4.0) was used to analyze the flow cytometry data (version Win 64-10.4.0).

Analysis of Cell Cycle
The GCs cell cycle was detected using a cell cycle and apoptosis analysis kit (Beyotime, China). From each treated group, the GCs were harvested in a 15 mL Falcon tube (Thermo Fisher Scientific, Germany), followed by centrifugation at 750× g for 7 min and washing twice with 1x PBS. A minimum of 1 × 10 6 cells was fixed in pre-chilled 70% ethanol at 4 • C overnight. Ethanol was then removed using centrifugation, and the GCs pellets were washed twice with 500 µL of 1× PBS. Cellular DNA was stained with 50 µg/mL of PI and 50 µg/mL of RNase, incubated for 30 min at 37 • C in the dark, and instantly processed in FACS Calibur (BD Biosciences, CA, USA). The percentage of cells in each cell division phase (G0-G1, S, and G2-M) was estimated using data received from the FL2-A channel using ModFit LT Version 4.1 software (http://www.vsh.com/products/mflt/index.asp accessed on 12 November 2021).

Assessment of Mitochondrial Membrane Potential
Briefly, to examine the MMP of GCs in all treated groups, the mitochondrial membrane potential assay kit with JC-1 (Beyotime, China) was used according to the manufacturer's instructions. Enzymatic digestion using trypsin was used to harvest the GCs and washed with warm PBS. After collection, the MMP assay kit with JC-1 (Beyotime, China) was used to stain GCs. Flow cytometry was used to count labeled cells using a fluorescence activating cell sorter (FACS) and a Calibur flow cytometer (BD Biosciences, CA, USA). The data was analyzed using FlowJo software (version Win 64-10.4.0).

Cell Viability Assay
The viability of bovine GCs from all treatment groups was determined using the MTT cell proliferation and cytotoxicity test kit (njjcbio, Nanjing, China). In brief, the detached cells were inoculated into 96-well plates, and after the indicated treatments, a 50 µL 1 × MTT solution was added to each well and incubated for 4 h. After 4 h, the solution was replaced with 150 µL DMSO, and the absorbance was measured at 570 nm using ELX microplate reader (BioTek, Winooski, VT, USA).

E2 and P4 Levels Determination
To estimate E2 and P4 levels, the cell culture media from all groups (NC, 40 • C + NC, and siSOD1) were used. In addition, enzyme-linked immunosorbent (ELISA) kits for P4 and E2 (ENZO life sciences, Germany) were used to estimate their concentrations, according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as mean ± SEM. In each group, three biological replicates (n = 3) of GCs were used. SPSS 16.0 and GraphPad Prism5 software were used for statistical analysis (GraphPad Software Inc, SanDiego, CA). A one-way ANOVA was used to compare the differences between the control and treatment groups, followed by a multiple comparisons post hoc test. At p < 0.05, differences were judged statistically significant.

Identification of GCs
A multitude of cells makes up the ovarian follicle (GCs, theca cells etc.). Since our study focuses on bovine GCs, immunofluorescence microscopy was used to separate GCs from the rest of the follicular cells. Propidium iodide (PI) was used to stain the GCs, or an anti-FSHR antibody was used to incubate them. According to our findings, the GCs tested positive for FSHR ( Figure 1).
cell proliferation and cytotoxicity test kit (njjcbio, Nanjing, China). In brief, the detached cells were inoculated into 96-well plates, and after the indicated treatments, a 50 μL 1 × MTT solution was added to each well and incubated for 4 h. After 4 h, the solution was replaced with 150 μL DMSO, and the absorbance was measured at 570 nm using ELX microplate reader (BioTek, Winooski, VT, USA).

E2 and P4 Levels Determination
To estimate E2 and P4 levels, the cell culture media from all groups (NC, 40 °C + NC, and siSOD1) were used. In addition, enzyme-linked immunosorbent (ELISA) kits for P4 and E2 (ENZO life sciences, Germany) were used to estimate their concentrations, according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as mean ± SEM. In each group, three biological replicates (n = 3) of GCs were used. SPSS 16.0 and GraphPad Prism5 software were used for statistical analysis (GraphPad Software Inc, SanDiego, CA). A one-way ANOVA was used to compare the differences between the control and treatment groups, followed by a multiple comparisons post hoc test. At p < 0.05, differences were judged statistically significant.

Identification of GCs
A multitude of cells makes up the ovarian follicle (GCs, theca cells etc.). Since our study focuses on bovine GCs, immunofluorescence microscopy was used to separate GCs from the rest of the follicular cells. Propidium iodide (PI) was used to stain the GCs, or an anti-FSHR antibody was used to incubate them. According to our findings, the GCs tested positive for FSHR ( Figure 1). . More than 90% of the cells in the isolated, cultured cells were granulosa cells depicted that the purity of GCs was above 90%. For magnification, a 100 μm scale bar was used.

Efficacy of SOD1 Transfection
SOD1 was silenced with the help of siSOD1. After successful transfection, the GCs were separated into three groups (NC, 40 °C + NC, and 40 °C + siSOD1). SOD1 expression was measured at both the translational and transcriptional levels in all groups. For the . More than 90% of the cells in the isolated, cultured cells were granulosa cells depicted that the purity of GCs was above 90%. For magnification, a 100 µm scale bar was used.

Efficacy of SOD1 Transfection
SOD1 was silenced with the help of siSOD1. After successful transfection, the GCs were separated into three groups (NC, 40 • C + NC, and 40 • C + siSOD1). SOD1 expression was measured at both the translational and transcriptional levels in all groups. For the GCs transfected with siSOD1, RTqPCR revealed that SOD1 mRNA expression was significantly (p < 0.05) lower ( Figure 2A). Likewise, western blot results showed a similar protein expression depicting that SOD1 was successfully silenced with siSOD1 ( Figure 2B,C).

Silencing of SOD1-Induced Intracellular ROS Accumulation under Heat Stress
The oxidation sensitive probe 6-carboxy-2 ,7 -H2DCF-DA was utilized to examine the intracellular accumulation of ROS in all groups (NC, 40 • C + NC, and 40 • C +siSOD1). The results showed that the intracellular abundance of ROS in the 40 • C + NC group was considerably (p < 0.05) higher than in the NC group. Likewise, compared with 40 • C + NC group, the fluorescence of intracellular ROS production significantly (p < 0.05) increased in 40 • C + siSOD1 group. Furthermore, our results demonstrated that SOD1 silencing induced intracellular ROS production ( Figure 3A-D). GCs transfected with siSOD1, RTqPCR revealed that SOD1 mRNA expression was significantly (p < 0.05) lower ( Figure 2A). Likewise, western blot results showed a similar protein expression depicting that SOD1 was successfully silenced with siSOD1 ( Figure 2B,C).

Silencing of SOD1-Induced Intracellular ROS Accumulation under Heat Stress
The oxidation sensitive probe 6-carboxy-2',7'-H2DCF-DA was utilized to examine the intracellular accumulation of ROS in all groups (NC, 40 °C + NC, and 40 °C+siSOD1). The results showed that the intracellular abundance of ROS in the 40 °C + NC group was considerably (p < 0.05) higher than in the NC group. Likewise, compared with 40 °C + NC group, the fluorescence of intracellular ROS production significantly (p < 0.05) increased in 40 °C + siSOD1 group. Furthermore, our results demonstrated that SOD1 silencing induced intracellular ROS production ( Figure 3A-D).

Silencing of SOD1-Induced Intracellular ROS Accumulation under Heat Stress
The oxidation sensitive probe 6-carboxy-2',7'-H2DCF-DA was utilized to examine the intracellular accumulation of ROS in all groups (NC, 40 °C + NC, and 40 °C+siSOD1). The results showed that the intracellular abundance of ROS in the 40 °C + NC group was considerably (p < 0.05) higher than in the NC group. Likewise, compared with 40 °C + NC group, the fluorescence of intracellular ROS production significantly (p < 0.05) increased in 40 °C + siSOD1 group. Furthermore, our results demonstrated that SOD1 silencing induced intracellular ROS production ( Figure 3A-D).

Silencing of SOD1-Altered Viability of GCs under Heat Stress
The viability of GCs was measured through MTT assay. It was documented that the viability of GCs in the 40 • C + NC group was significantly (p < 0.05) lower than that of the NC group. Similarly, a significant decline in cell viability was noted in 40 • C + siSOD1 group than in NC and 40 • C + NC groups ( Figure 3E). As a result of the oxidative stress generated by ROS production under HS, the viability of GCs was affected, and SOD1 silencing worsened the viability of GCs, demonstrating its importance in the regulation of cell viability.

Silencing of SOD1 Promoted Apoptosis and Cell Death in GCs under Heat Stress
The involvement of SOD1 in the regulation of GC apoptosis under HS was explained using the Annexin VFITC kit. The flow cytometry results showed a significant increase in the apoptotic rate in the 40 • C + NC group compared with that in the NC group. Similarly, inhibiting SOD1 in GCs at 40 • C enhanced the apoptotic rate considerably (p < 0.05) compared to 40 • C + NC alone ( Figure 4A,B). The results of Fluorescence microscopy showed that the number of dead cells was significantly (p < 0.05) higher when GCs were exposed to 40 • C + NC and 40 • C +siSOD1 compared to NC group ( Figure 4C-F). In addition, SOD1 gene regulation also altered the mRNA and protein expression of pro-apoptotic genes (BAX and Caspase-3) under HS. As shown in Figure 4G-I, compared with that in the NC group, mRNA expression and protein level of BAX and Caspase-3 were significantly higher in the 40 • C + NC group. Consequently, on transcriptional and translational levels, the siSOD1 + 40 • C group demonstrated a substantial (p < 0.05) increase in BAX and Caspase-3 compared to the 40 • C + NC group. Therefore, we can postulate that SOD1 silencing promoted HS-induced cell apoptosis by activation of BAX and Caspase-3. Our findings depicted that SOD1 has an important role in the regulation of GCs apoptosis.
The viability of GCs was measured through MTT assay. It was documented that the viability of GCs in the 40 °C + NC group was significantly (p < 0.05) lower than that of the NC group. Similarly, a significant decline in cell viability was noted in 40 °C + siSOD1 group than in NC and 40 °C + NC groups ( Figure 3E). As a result of the oxidative stress generated by ROS production under HS, the viability of GCs was affected, and SOD1 silencing worsened the viability of GCs, demonstrating its importance in the regulation of cell viability.

Silencing of SOD1 Promoted Apoptosis and Cell Death in GCs under Heat Stress
The involvement of SOD1 in the regulation of GC apoptosis under HS was explained using the Annexin VFITC kit. The flow cytometry results showed a significant increase in the apoptotic rate in the 40 °C + NC group compared with that in the NC group. Similarly, inhibiting SOD1 in GCs at 40 °C enhanced the apoptotic rate considerably (p < 0.05) compared to 40 °C + NC alone ( Figure 4A,B). The results of Fluorescence microscopy showed that the number of dead cells was significantly (p < 0.05) higher when GCs were exposed to 40 °C + NC and 40 °C+siSOD1 compared to NC group ( Figure 4C-F). In addition, SOD1 gene regulation also altered the mRNA and protein expression of pro-apoptotic genes (BAX and Caspase-3) under HS. As shown in Figure 4G-I, compared with that in the NC group, mRNA expression and protein level of BAX and Caspase-3 were significantly higher in the 40 °C + NC group. Consequently, on transcriptional and translational levels, the siSOD1 + 40 °C group demonstrated a substantial (p < 0.05) increase in BAX and Caspase-3 compared to the 40 °C + NC group. Therefore, we can postulate that SOD1 silencing promoted HS-induced cell apoptosis by activation of BAX and Caspase-3. Our findings depicted that SOD1 has an important role in the regulation of GCs apoptosis.

Silencing of SOD1 Altered Cell Cycle Transition in GCs under Heat Stress
To check whether SOD1 is involved in the mediation of cell proliferation under the influence of HS, flow cytometry was performed to examine the cell-cycle profile. The cell cycle distributions were estimated at the indicated groups (40 • C + NC, 40 • C + siSOD1 and NC). Figure 5A,B demonstrated that after exposure of GCs to 40 • C and silencing of SOD1 (siSOD1) significantly (p < 0.05) decreased the proportion of cells in the G0/G1 phase associated with subsequent decline in the S (DNA synthesis) phase and G2/M phase compared to NC group. The transcriptional and translational regulation of cell proliferation genes (PCNA, CyclinB1, and HSP70) were used to confirm the above findings. The mRNA and protein levels of PCNA and CyclineB1 were significantly down-regulated while HSP70 was up-regulated in 40 • C + NC and 40 • C +siSOD1 groups compared with the NC group ( Figure 5C-E). Collectively, these findings suggested that at the G0/G1 and G2/M phases the GCs were arrested for repairing DNA damage caused by siRNA. Thus, the cell cycle progression was regulated by SOD1 under HS.

Silencing of SOD1 Altered Cell Cycle Transition in GCs under Heat Stress
To check whether SOD1 is involved in the mediation of cell proliferation under the influence of HS, flow cytometry was performed to examine the cell-cycle profile. The cell cycle distributions were estimated at the indicated groups (40 °C + NC, 40 °C+ siSOD1 and NC). Figure 5A,B demonstrated that after exposure of GCs to 40 °C and silencing of SOD1 (siSOD1) significantly (p < 0.05) decreased the proportion of cells in the G0/G1 phase associated with subsequent decline in the S (DNA synthesis) phase and G2/M phase compared to NC group. The transcriptional and translational regulation of cell proliferation genes (PCNA, CyclinB1, and HSP70) were used to confirm the above findings. The mRNA and protein levels of PCNA and CyclineB1 were significantly down-regulated while HSP70 was up-regulated in 40 °C + NC and 40 °C+siSOD1 groups compared with the NC group ( Figure 5C-E). Collectively, these findings suggested that at the G0/G1 and G2/M phases the GCs were arrested for repairing DNA damage caused by siRNA. Thus, the cell cycle progression was regulated by SOD1 under HS.

Disruption of Mitochondrial Membrane Potential of GCs under Heat Stress Caused by Silencing of SOD1
The goal of this study was to see if SOD1 intervention was linked to the control of the mitochondrial pathway, which is implicated in GC apoptosis under HS. Flow cytometry was used to determine the MMP of GCs. We found that The MMP was significantly (p < 0.05) lower in the 40 °C + NC group compared to the NC group. Likewise, the MMP was also significantly (p < 0.05) lower in 40 °C + siSOD1 than 40 °C + NC group ( Figure  6A,B). Therefore, based on these findings, we can argue that under HS, the SOD1 has shown an essential role in the regulation of MMP in GCs.

Disruption of Mitochondrial Membrane Potential of GCs under Heat Stress Caused by Silencing of SOD1
The goal of this study was to see if SOD1 intervention was linked to the control of the mitochondrial pathway, which is implicated in GC apoptosis under HS. Flow cytometry was used to determine the MMP of GCs. We found that The MMP was significantly (p < 0.05) lower in the 40 • C + NC group compared to the NC group. Likewise, the MMP was also significantly (p < 0.05) lower in 40 • C + siSOD1 than 40 • C + NC group ( Figure 6A,B). Therefore, based on these findings, we can argue that under HS, the SOD1 has shown an essential role in the regulation of MMP in GCs.

Impairment of the Synthesis of P4 and E2 in GCs under Heat Stress with SOD1 Silencing
We used ELISA to estimate P4 and E2 concentrations to see how SOD1 silencing affected hormonal shifts. Our findings established that E2 concentrations were significantly (p < 0.05) lower in 40 • C + NC group than in the NC group. Likewise, compared with 40 • C + NC group, E2 and P4 levels were significantly lower (p < 0.05) than those in 40 • C + siSOD1 group. However, P4 did not show any significant difference with that in NC and 40 • C + NC groups ( Figure 7A,B). The transcriptional and translational expression of steroidogenesis regulatory genes (STAR and Cyp11A1) further confirmed the above findings. The mRNA and protein levels of STAR and Cyp11A1 were significantly downregulated in 40 • C + NC and 40 • C + siSOD1 groups compared to NC group ( Figure 7C-E). These results confirmed that HS impaired the concentration of P4 and E2 in GCs while SOD1 plays a key role in regulating these hormones.

Impairment of the Synthesis of P4 and E2 in GCs under Heat Stress with SOD1 Silencing
We used ELISA to estimate P4 and E2 concentrations to see how SOD1 silencing affected hormonal shifts. Our findings established that E2 concentrations were significantly (p < 0.05) lower in 40 °C + NC group than in the NC group. Likewise, compared with 40 °C + NC group, E2 and P4 levels were significantly lower (p < 0.05) than those in 40 °C + siSOD1 group. However, P4 did not show any significant difference with that in NC and 40 °C + NC groups ( Figure 7A,B). The transcriptional and translational expression of steroidogenesis regulatory genes (STAR and Cyp11A1) further confirmed the above findings. The mRNA and protein levels of STAR and Cyp11A1 were significantly down-regulated in 40 °C + NC and 40 °C+ siSOD1 groups compared to NC group ( Figure 7C-E). These results confirmed that HS impaired the concentration of P4 and E2 in GCs while SOD1 plays a key role in regulating these hormones.  group (A,B). The STAR and Cyp11A1 protein expressions were analyzed by western blotting (C,D) and RT-qPCR (E). β-Actin and GAPDH were kept as housekeeping genes, respectively. Values are shown as mean ± SEM of n = 3. The bars with completely distinct lettering denote a significant difference (p < 0.05).   A,B). Values are shown as mean ± SEM of n = 3. The bars with completely distinct lettering denote a significant difference (p < 0.05).

Impairment of the Synthesis of P4 and E2 in GCs under Heat Stress with SOD1 Silencing
We used ELISA to estimate P4 and E2 concentrations to see how SOD1 silencing affected hormonal shifts. Our findings established that E2 concentrations were significantly (p < 0.05) lower in 40 °C + NC group than in the NC group. Likewise, compared with 40 °C + NC group, E2 and P4 levels were significantly lower (p < 0.05) than those in 40 °C + siSOD1 group. However, P4 did not show any significant difference with that in NC and 40 °C + NC groups ( Figure 7A,B). The transcriptional and translational expression of steroidogenesis regulatory genes (STAR and Cyp11A1) further confirmed the above findings. The mRNA and protein levels of STAR and Cyp11A1 were significantly down-regulated in 40 °C + NC and 40 °C+ siSOD1 groups compared to NC group ( Figure 7C-E). These results confirmed that HS impaired the concentration of P4 and E2 in GCs while SOD1 plays a key role in regulating these hormones.  (A,B). The STAR and Cyp11A1 protein expressions were analyzed by western blotting (C,D) and RT-qPCR (E). β-Actin and GAPDH were kept as housekeeping genes, respectively. Values are shown as mean ± SEM of n = 3. The bars with completely distinct lettering denote a significant difference (p < 0.05). β-Actin and GAPDH were kept as housekeeping genes, respectively. Values are shown as mean ± SEM of n = 3. The bars with completely distinct lettering denote a significant difference (p < 0.05).

Discussion
In bovine, the high environmental temperature can elevate the body temperature up to 41 • C [28]. The increase in internal body temperature related to short and long-term HS is responsible for the impaired reproductive performance of dairy cattle [31]. The deleterious effect of HS involves alterations in follicle development, impaired steroidogenesis [32], irregular follicular dynamics that affect GC function [33], disruptive oocyte maturation, fertilization, and preimplantation embryonic development [34,35]. Heat-induced is proteotoxic and results in protein denaturation that may become cytotoxic by boosting ROS production [36]. Thus, the apoptosis caused by heat stress in GCs is one of the most compromising factors affecting dairy cow fertility.
It has been well established that SOD1 enzyme counteracts oxidative stress by converting superoxide anion radicals to O 2 and H 2 O 2 [25]. The present study unravels and sheds light on the noxious effects of HS on GCs viability and functions, depicting that SOD1 plays a vital protective role against oxidative damage and apoptosis. Despite evidence for a regulation of SOD1 under HS triggered by ROS accumulation in cells [31,37], its functional significance in heat-stressed GCs had not been investigated. In the current study, we revealed a key role of SOD1 in regulating apoptosis and proliferation of GCs under HS (40 • C). In general, our findings verified that HS could trigger the expression of SOD1, while silencing or over-expression of SOD1 significantly regulated the production of intracellular ROS, cell proliferation, apoptosis, the MMP and steroidogenesis in GCs. These findings infer that SOD1 this gene is involved in oxidative stress in bovine GCs with a mitigation of HS-induced anomalies (Figure 8). [32], irregular follicular dynamics that affect GC function [33], disruptive oocyte maturation, fertilization, and preimplantation embryonic development [34,35]. Heat-induced is proteotoxic and results in protein denaturation that may become cytotoxic by boosting ROS production [36]. Thus, the apoptosis caused by heat stress in GCs is one of the most compromising factors affecting dairy cow fertility.
It has been well established that SOD1 enzyme counteracts oxidative stress by converting superoxide anion radicals to O2 and H2O2 [25]. The present study unravels and sheds light on the noxious effects of HS on GCs viability and functions, depicting that SOD1 plays a vital protective role against oxidative damage and apoptosis. Despite evidence for a regulation of SOD1 under HS triggered by ROS accumulation in cells [31,37], its functional significance in heat-stressed GCs had not been investigated. In the current study, we revealed a key role of SOD1 in regulating apoptosis and proliferation of GCs under HS (40 °C). In general, our findings verified that HS could trigger the expression of SOD1, while silencing or over-expression of SOD1 significantly regulated the production of intracellular ROS, cell proliferation, apoptosis, the MMP and steroidogenesis in GCs. These findings infer that SOD1 this gene is involved in oxidative stress in bovine GCs with a mitigation of HS-induced anomalies (Figure 8). Our previous study showed that HS (40 °C) increases the intracellular ROS abundance, apoptosis, confirmed by the up-regulation of pro-apoptotic genes, i.e., BAX and Caspase-3 [28]. Moreover, a study had documented that oxidative stress due to the increased intracellular ROS production plays a critical role in HS-induced apoptosis [38]. Consistently, the impairment of MMP function under HS has been found to be involved in mitochondrial pathway which leads to GCs apoptosis, as indicated by increases in both cleaved Caspase-3 expression and the Bax/Bcl-2 ratio [39][40][41]. The mitochondria release Cytochrome c the cytosol when the mitochondrial function is altered. The key apoptotic Our previous study showed that HS (40 • C) increases the intracellular ROS abundance, apoptosis, confirmed by the up-regulation of pro-apoptotic genes, i.e., BAX and Caspase-3 [28]. Moreover, a study had documented that oxidative stress due to the increased intracellular ROS production plays a critical role in HS-induced apoptosis [38]. Consistently, the impairment of MMP function under HS has been found to be involved in mitochondrial pathway which leads to GCs apoptosis, as indicated by increases in both cleaved Caspase-3 expression and the Bax/Bcl-2 ratio [39][40][41]. The mitochondria release Cytochrome c the cytosol when the mitochondrial function is altered. The key apoptotic program effector (Caspase-3) is regulated by the released cytochrome c, promoting the condensation of chromatin and fragmentation of DNA [42,43]. Interestingly, we found that the silencing of SOD1 under HS drastically increased ROS production and apoptosis in GCs. Likewise, the altered MMP and viability in the heat-stressed SOD1 silencing group played an important role in regulating survival and apoptosis in GCs. These findings were further manifested by the up-regulation of BAX and Caspase-3 genes in the heat-stressed SOD1 silencing group. Here, we show that silencing of SOD1 promoted HS-mediated oxidative stress and apoptosis in GCs.
High ambient temperature is considered to have adverse effects on reproductive processes by inhibiting GCs proliferation and ovarian steroidogenesis [44][45][46][47][48]. In swine GCs, PCNA and CyclinB1 are considered key proteins for cell proliferation [49,50]. PCNA has been considered a biological indicator for cell proliferation and is mainly regulated during cell proliferation and mainly regulated during the S phase [51]. In addition, CyclinB1, required for the transition of cells from G2 to M cell cycle phase, is mainly regulated during the G2/M phase of the cell cycle [52]. Biomarkers for proliferation and cell cycle such as PCNA and CyclinB1 were downregulated in swine GCs under in vitro HS [47]. Our results revealed that the silencing of SOD1 under HS to arrest in the G1 cell cycle phase. SOD1 silencing also inhibited the proliferation of GCs, decreased the number of cells in the S phase. These results were further verified by the downregulation of PCNA and CyclinB1 in the heat-stressed SOD1 silencing group both on transcriptional and translational levels. These results suggested that SOD1 is a critical player under HS in controlling the cellular progression in GCs.
Moreover, the regulation of genes associated with hormone synthesis (E2 and P4), such as STAR and Cyp11A1 was negatively affected by HS [28]. Positive regulation of Cyp11A1 in the ovarian follicle stimulates the biosynthesis of E2 [53]. Furthermore, P4 being a steroid hormone, plays a fundamental role in bovine estrous cyclicity, and its production is associated with the positive regulation of STAR and Cyp11A1 [54,55]. Some studies reported an over-secretion of ovarian hormones in porcine ovarian GCs under high temperatures [56]. In addition, it was suggested that E2 boost GCs viability by inhibiting apoptosis [21]. This is in line with the results of our previous study, which showed that HS could significantly decrease the levels of P4 and E2 in the GC culture media as well as down-regulated the expression of STAR and Cyp11A1 [28]. In the current study, we found that the biosynthesis of P4 and E2 in GCs was impaired in the heat-stressed SOD1 silencing group by down-regulating the STAR and Cyp11A1 gene at transcriptional and translational levels. Thus, we can postulate that under HS, SOD1 silencing can further aggravate apoptosis by altering the secretion of E2 and P4 in GCs. Our research can further be extended to understand the mechanism of how SOD1 regulation can influence bovine oocyte and embryo development modulation under HS.

Conclusions
Altogether we concluded that HS induces apoptosis, alters cell proliferation, disrupts mitochondrial membrane potential, and impairs E2 and P4 synthesis in GCs by increasing intracellular ROS accumulation. Interestingly, the SOD1 is widely expressed in bovine ovaries, and it was found to alleviate the apoptosis of GCs triggered by HS. In current study we proved that the silencing of SOD1 under HS further deteriorated the GCs functions by promoting apoptosis and ROS generation and suppressed the proliferation of cells. This was further verified by regulating steroidogenic, associative apoptotic, and cell cycle genes (BAX, Caspase-3, STAR, Cyp11A1, HSP70, PCNA, and CyclinB1). These results identify a novel role of SOD1 in the modulation of bovine ovarian GCs apoptosis, which provides a target for improving the fertility of heat-stressed dairy cows in summer.

Institutional Review Board Statement:
This study protocols for collecting bovine ovaries from experimental animals were reviewed and approved by the institutional animal care and use committee of China Agricultural University Beijing, China (permit number: DK996).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in study design, the decision to publish, and preparation of the manuscript.