| China is the largest producer of waterfowl in the world and the ducks accounts for more than 70% of the total amount of waterfowl breeding in China.Laying ducks farming is an important industry in China,and more than 3 million tons of duck eggs are produced annually.Duck eggs provide a large number of agricultural and sideline products such as salted eggs,salted egg yolk,egg yolk powder,egg butter,preserved eggs and so on.The increasing demand of duck eggs promotes the modern breeding mode,and cage breeding of laying ducks has become a trend.Poultry are sensitive to temperature,and the increasing high temperature in summer caused severe heat stress to laying ducks out of water,leading to the decline of reproductive performance.Studies have shown that the decline of reproductive performance of poultry is closely related to hormone secretion.Granulosa cells,as the main cells of estradiol and progesterone secretion in ovary,provide appropriate hormone levels for follicular development.The effect of heat stress on granulosa cells are the focus of many researches.Although studies on the effects of heat stress on the proliferation,secretion and death of granulosa cells have been relatively clear,the effects of heat stress on the mitochondrial damage and dysfunction of granulosa cells,and the subsequent effects on cell function and survival are still unclear.Therefore,intensive study on mitochondrial changes of granulosa cells under heat stress is necessary to effectively prevent and control the decline of reproductive performance of poultry caused by heat stress.Researches have shown that heat stress causes mitochondrial damage and oxidative stress in poultry muscle cells.It’s worth noting that mitophagy is an important part of mitochondrial quality control.At present,the effect of mitochondria on poultry follicular granulosa cells under heat stress and the role of mitophagy in cell survival or damage are still unclear.In this study,we hypothesized that PINK1-mediated mitophagy under heat stress alleviated mitochondrial damage and apoptosis in follicular granulosa cells.Therefore,this study verified the effects of acute heat stress on mitophagy and cell apoptosis of granulosa cells in vitro and in vivo,and further studied the effects of inhibition of mitophagy on apoptosis of granulosa cells under heat stress by RNA interference technology.The results provided theoretical basis for alleviating heat stress in laying duck breeding.In this study,the effects of acute heat stress on ovarian function,cell apoptosis and mitophagy in laying duck ovary were studied by animal experiment.The results showed that acute heat treatment at 39℃ caused the damage of ovary,that induced oxidative stress and increased MDA significantly.Serum hormone levels showed a decrease in estradiol synthesis and an increase in progesterone synthesis.Further studies showed that heat stress inhibited the expression of steroid synthase genes and proteins such as St AR,3β-HSD and CYP19A1.Tunel and Western Blot results showed that high temperature environment led to slight apoptosis of ovary,and the apoptosis level increased with prolonged heat treatment time.Further ultrastructural level analysis of granulosa cells showed that heat stress damaged the mitochondrial structure and induced a large number of autophagosome vesicles with bilayer and monolayer membranes,and mitochondrial structures were observed in some vesicles.In order to verify the activation process of mitophagy pathway in granulosa cells under heat stress,we used Western Blot to analyze intracellular expression of key proteins of mitochondrial dynamics and mitophagy.The results showed that heat stress increased the level of mitochondrial fission protein Drp1 and decreased the level of Mfn1/2 and other mitochondrial fusion proteins,which promoted the level of PINK1 and LC3 I /LC3 II transformation.In addition,the degradation of P62 also indicates that heat stress significantly increased the autophagy flow of cells and promote complete mitophagy.Based on the verification of mitophagy induced by acute heat stress in granulosa cells of ovary.In order to further study the role of mitophagy in the adaptation and survival of granulosa cells,we established a research model of mitophagy induced by acute heat stress on granulosa cells in vitro.Granulosa cells were subjected to acute heat treatment at 43℃ for0,60,90,120 and 150 min,respectively,and the results confirmed that heat treatment above120 min significantly increased ROS content.Observing the ultrastructure of cells,increased autophagosomes was found.Further observation of stained mitochondria showed that the morphology of mitochondria in the acute heat treatment group was shortened and round,and the level of fission protein Drp1 increased,while the mitochondrial fusion protein decreased.Compared with the control group,a large number of LC3 puncta gathered near the nuclear of the acute heat treatment group.Meanwhile,Western Blot analysis of whole cell,cytoplasm and mitochondrial proteins showed that acute heat stress activated PINK1-mediated mitophagy.In addition,confocal results also revealed the co-localization of mitochondria and LC3 puncta and the co-localization of lysosomes and mitochondria in the acute heat stress group,indicating the relationship between autophagosomes and mitochondria,lysosomes and mitochondria during autophagy.We also used mitophagy inhibitor Cys A to treat granulosa cells under acute heat stress and found that 20 μM Cys A effectively inhibited the transformation of autophagy marker LC3-I/LC3-II.Next,we used si-RNA to inhibit Drp1 and PINK1 to study the effect of inhibiting mitophagy on apoptosis of granulosa cells under acute heat stress.The results showed that inhibition of Drp1-mediated mitochondrial division significantly reduced PINK1 protein level and LC3I/LC3 II value,inhibited P62 degradation,and reduced mitophagy.Meanwhile,Bax/BCL2 and Caspase3 protein level were increased and apoptosis was enhanced.Subsequently,our results also confirmed that inhibition of PINK1 significantly reduced LC3I/LC3 II transformation and P62 degradation,reduced mitophagy,and significantly increased apoptosis-related protein level.Results of Tunel and Annexin-V showed that inhibition of PINK1 promoted apoptosis induced by heat stress.In conclusion,acute heat stress leads to mitochondrial damage,decrease of steroid synthesis in ovary granulosa cells and the cells were subjected to oxidative stress,the apoptosis level was increased.Meanwhile,the mitophagy level is significantly increased under heat stress.Further studies showed that PINK1-mediated mitophagy induced by heat stress,and mitophagy cleared damaged mitochondria,and reduced apoptosis,which is the body’s self-protection mechanism under acute heat stress.This study provides a scientific basis for further exploring the adaptive survival mechanism of granulosa cells under heat stress. |
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