The Study On The Mechanism Of Bovine Ovary Granulosa Cells Apoptosis Induced By Heat Stress And Non-esterified Fatty Acid

In the dairy industry,high-temperature stress in summer and the high level of non-esterified fatty acid(NEFA)caused by negative energy balance in the perinatal period are important reasons for the decline of reproductive performance of lactating cows.The low reproduction rate causes great economic loss to dairy production.High-temperature stress in summer can inhibit the follicular development of dairy cows,resulting in the decline of reproductive performance in summer.Granulosa cells play an important role in the development of follicles.They can synthesize estrogen and progesterone.At the same time,they can also play an important role in the development and differentiation of follicles through apoptosis.Therefore,the research on granulosa cells in the development of follicles has always been a hot spot.However,the mechanism of heat stress on granulosa cells apoptosis has not been fully elucidated,and the known molecules involved in the regulation of ovarian granulosa cells apoptosis under high-temperature stress are also poorly understood.The elucidation of the specific action mechanism of related molecules can provide a valuable reference for livestock production to improve livestock fecundity.The high level of NEFA caused by perinatal negative energy balance not only affects postpartum ovulation but also affects the function of embryonic oocytes and follicular granulosa cells by changing the lipid composition of follicular fluid.At present,the effect of NEFA on the reproductive performance of dairy cows is limited to the superficial observation and preliminary research,but the more in-depth mechanism research is less;the research on the oxidative stress injury induced by NEFA,the mechanism of related signaling pathways and the interaction between them is mainly focused on human and mouse,while the research on ruminants is less,especially in perinatal dairy cows The specific mechanism of oxidative stress and apoptosis of ovarian granulosa cells has not been reported.Therefore,exploring and find more molecular mechanisms that affect the survival and apoptosis of ovarian granulosa cells in dairy cows has important theoretical significance for improving the fecundity of livestock and the development of animal husbandry.The study is divided into five parts.The research content and results are as follows:Experiment 1.Effect of heat stress on ROS/Nrf2/HO-1 pathway in bovine ovarian granulosa cellsStudies have shown that heat stress can hurt cattle reproduction by destroying the normal function of ovarian granulosa cells.After the identification of primary cultured granulosa cells,the effects of heat stress on ROS accumulation and the expression and activity of key oxidative stress kinases in GCs were studied by immunofluorescence and spectrophotometry.First,heat stress models were established at different temperatures.The results showed that heat stress-induced ROS accumulation and oxidative homeostasis changes in GCs,suggesting that heat stress-induced oxidative stress in GCs.By further detecting the expression of Nrf2 and HO-1 in the antioxidant pathway,we found that heat stress enhanced the expression of Nrf2 and HO-1 in GCs,and heat stress also promoted the entry of Nrf2 into the nucleus.The results showed that heat stress activated Nrf2/HO-1pathway.Experiment 2.Regulation of HO-1 on heat stress-induced apoptosis of bovine ovarian granulosa cellsThe results of Experiment 1 showed that heat stress enhanced the expression of HO-1in GCs.As HO-1 is involved in cellular antioxidant defense and anti-apoptotic function,how HO-1 exerts anti-apoptotic and cytoprotective effects on GCs still needs further study.This study analyzed the expression and localization of HO-1 in GCs,and further explored the effect of HO-1 on heat stress-induced apoptosis of GCs.Immunofluorescence results showed that HO-1 was expressed in both the nucleus and cytoplasm of GCs,but mainly in the nucleus.The results of flow cytometry and Western blot confirmed that 40 ℃ heat stress-induced GCs apoptosis.To investigate the role of HO-1 in heat stress-induced apoptosis of GCs and its mechanism,the oxidative stress level and apoptosis of GCs were detected by knockdown and overexpression of HO-1,respectively.The results showed that HO-1 knockdown increased the level of oxidative stress,such as the increase of ROS accumulation,the decrease of SOD2 protein expression,the decrease of SOD and GSH-Px activity,and the increase of MDA content;on the contrary,HO-1 overexpression alleviated the oxidative stress induced by heat stress(the decrease of ROS accumulation,the increase of SOD2 protein expression and the increase of SOD activity).Also,GCs apoptosis was detected by Tunnel staining,Western blot,and flow cytometry.The results showed that the inhibition of HO-1 expression by si RNA significantly increased the apoptosis of GCs,mainly in the increase of the number of positive cells in tunnel staining,the increase of Bax/Bcl-2 ratio,and cleaved caspase-3 expression,and the increase of apoptosis rate.However,pretreatment with Hemin could induce HO-1 overexpression,reduce the number of positive cells,Bax/Bcl-2 ratio,and apoptosis rate in tunnel staining,and alleviate the apoptosis of GCs.Therefore,HO-1 attenuates heat stress-induced GCs apoptosis by reducing the production of reactive oxygen species and activating an antioxidant response.Experiment 3.CO,a metabolite of HO-1,mediates heat stress-induced apoptosis of bovine ovarian granulosa cells through the ERK1/2 pathwayIn addition to the anti-apoptotic effect of HO-1,carbon monoxide(CO),one of its metabolites,can also play a cytoprotective role.Studies have shown that CO,like NO,can act as a gas signal molecule to regulate cell function in an autocrine or paracrine manner.The results of ELISA confirmed that heat stress and the overexpression of HO-1 induced up-regulation of CO and c GMP expression.To investigate the effect of CO on heat stress-induced apoptosis in GCs,exogenous CO releasing molecule Corm-2 was used to release CO,and the apoptosis of GCs was detected by tunnel staining and Western blot.The results showed that CO alleviated the heat stress-induced apoptosis.The number of Tunnel positive cells,the ratio of Bax/Bcl-2,and the expression level of cleaved caspase-3 in the40 ℃ + Corm-2 group were lower than those in the 40 ℃ heat stress group.Besides,Western blot results showed that CO inhibited the phosphorylation of the ERK1/2 pathway induced by heat stress.To further prove whether CO exerts anti-apoptotic effect through ERK1/2 pathway.U0126,an inhibitor of ERK1/2 pathway,was used in this study.The results of western blot,q RT-PCR,and tunnel staining showed that the inhibition of the ERK1/2 pathway prevented CO from playing an anti-apoptotic effect.Compared with 40 ℃ + Corm-2 group,the ratio of p-ERK1/2 / ERK1/2 decreased,the ratio of Bax/Bcl-2 protein and m RNA increased,and the number of tunnel staining positive cells increased in 40 ℃ + Corm-2 + U0126 group.It is proved that CO plays a protective role through ERK1/2 pathway.Therefore,the results suggest that CO plays a protective role in heat-induced GCS apoptosis,and CO inhibits heat stress-induced GCs apoptosis through ERK1/2 pathway.The results of this study provide valuable clues for improving the fecundity of dairy cows under heat stress in summer.Experiment 4.Evaluation of NEFA level in serum of ketosis dairy cows and effects of different concentrations of NEFA on ROS/Nrf2/HO-1 pathway in ovarian granulosa cells of dairy cowsDairy cows are prone to negative energy balance during the perinatal period,which increases the NEFA content in follicular fluid and hinders the proliferation of GCs,thus endangering the development of oocytes and the fecundity of dairy cows.The serum levels of NEFA and MDA in ketosis cows were significantly higher than those in healthy cows,but the activities of SOD and GSH-Px in ketosis cows were significantly lower than those in healthy cows.Besides,NEFA is known to be toxic to a variety of cell types.However,the effect of NEFA on GCs in vitro remains unclear.In this study,different concentrations of NEFA were given to GCs.Immunofluorescence,spectrophotometry,and Western blot were used to detect the toxicity of different concentrations of NEFA to GCs.Enzyme-linked immunosorbent assay(ELISA)showed that NEFA induced the decrease of SOD and GSH-Px activities and the increase of MDA content in GCs.Western blot showed that the expression of SOD2 protein decreased with the increase of NEFA concentration.This suggests that NEFA induces the imbalance of redox homeostasis in GCs.Meanwhile,immunofluorescence data showed that ROS accumulation in GCs increased with the increase of NEFA concentration.In conclusion,NEFA induced oxidative stress in GCs.Also,Western blot showed that NEFA also promoted the expression of nuclear factor E2(Nrf2)and its downstream gene HO-1,which indicated that NEFA activated the Nrf2/HO-1pathway.Experiment 5.ROS/Nrf2/HO-1 pathway participates in NEFA induced apoptosis of bovine ovarian granulosa cellsIn Experiment 4,the oxidative damage of GCs by NEFA has been confirmed and Nrf2/HO-1 pathway is activated.This study further explored the effect of NEFA on apoptosis and related pathways of GCs.The results of flow cytometry showed that the apoptosis rate of GCs increased with the increase of NEFA concentration.Western blot results showed that the ratio of apoptosis-related protein Bax/Bcl-2 increase with the increase of NEFA concentration,indicating that NEFA induced apoptosis in GCs with a dose-dependent manner.However,it is unclear whether the apoptosis of GCs induced by NEFA is related to oxidative stress.To elucidate the mechanism of NEFA,the role of ROS in the apoptosis of GCs was verified by N-acetyl-L-cysteine(NAC).When GCs was pretreated with NAC,immunofluorescence staining and ELISA showed that NAC significantly inhibited ROS accumulation and MDA content in GCs,and increased SOD activity,indicating that NAC could alleviate oxidative stress induced by NEFA.The results of flow cytometry and Western blot showed that NAC decreased the apoptosis rate of GCs,the ratio of apoptosis-related protein Bax/Bcl-2,and the expression of cleaved caspase-3,indicating that NAC alleviated GCs apoptosis induced by NEFA.It suggested that ROS plays an important role in the regulation of apoptosis induced by NEFA.Besides,NAFA induced Nrf2 was blocked by NAC pretreatment,and HO-1 expression was downregulated.These findings improved understanding of the apoptosis mechanism of GCs induced by NEFA,which may help improve ovarian function.To sum up,in this thesis,we take the ovarian granulosa cells of dairy cows as the research object,and take the common summer heat stress and high-level NEFA in the perinatal period as stress conditions,respectively,to reveal the mechanism of heat stress and high-level NEFA on the apoptosis of bovine ovarian granulosa cells.The results showed that the Nrf2/HO-1 pathway was activated by heat stress.HO-1 regulates the apoptosis induced by heat stress by regulating oxidative stress in GCs.At the same time,CO can also protect GCs apoptosis through the ERK1/2 pathway.Then the mechanism of high-level NEFA on apoptosis of ovarian granulosa cells in dairy cows was studied.The results showed that NEFA activated ROS/Nrf2/HO-1 pathway.The addition of NAC indicated that ROS/Nrf2/HO-1 pathway-mediated apoptosis of GCs induced by NEFA.In conclusion,this study elucidates the molecular mechanism of heat stress and high-level NEFA stress in the apoptosis of ovarian granulosa cells in dairy cows,which provides theoretical support for the development of cow ovary,and provides new strategies and ideas for animal husbandry to improve livestock fertility.