| Autophagy is a self-catabolic mechanism ubiquitously existed in eukaryocyte.Under some physiological conditions,cells form double-membrane autophagosomes around its own components including protein polymers,organelles and cell skeleton for delivering these cargos to lysosomes for proteolytic degradation,by which the cell elegantly realizes the dynamic control of its metabolism,physiological functions and cell structures.In mammalian cells,the induction of autophagy is closely associated with series of its physiological activities,including cell death,proliferation,migration,differentiation and hormone secretion.Present researches have already revealed that autophagy is an important mechanism involved in normal cells,the aberrance of autophagy is high related with the rise of several diseases.In mammalian cells,the induction and function of autophagy are regulated by various upstream proteins.In the reproductive system,the level of autophagy could be dominated by various gonadotropins.The present project was designed aim to elucidate the role and underlying mechanisms of autophagy induction in the development of follicles,the regulation of luteal development and regression.Furthermore,the present project explored the role of autophagy in some ovarian pathological condition.The main findings as follows:Firstly,the ovarian follicle development model was developed by PMSG administration to clarify the role and regulation of HIF-la mediated autophagy during follicular development.The expressions of ovarian HIF-1a and autophagic marker protein LC3 were detected and found the expressions of both proteins were obviously increased during follicular development.Further investigation indicated the expression of BNIP3,the downstream protein of HIF-la,was concomitantly increased with the upregulation of HIF-la.After HIF-la inhibition,the expression levels of BNIP3 protein and autophagy were simultaneously compromised.Comparing with the control,inhibition of HIF-1a did not affected ovarian SOD level whereas contributed to the increase of ROS level.Western blot analysis showed that inhibition of HIF-la also led to the increase of apoptosis level in the ovaries.These results suggested that HIF-1a/BNIP3-mediated autophagy enabled the survival of granulosa cell under hypoxia niche during follicle development through eliminating ovarian ROS thereby ensuring the normal development of follicles.Secondly,the pregnant luteal development model was used for delineating the expression and regulatory roles of autophagy during the luteal development.The present study evaluated the expression of autophagy related proteins and the variation of autophagy level during the pregnant luteal development.The results indicated that LC3 was mainly expressed and localized in luteal cells and its expression level was obviously increased during the late luteal phase,accompanied by the increase of cell apoptosis and the accumulation of autophagosome.However,the expression of LAMP-2,functional protein of lysosome,was reversely compromised during the late luteal phase leading to the insufficiency of lysosomal functions.These results indicated that the downregulation of LAMP-2 expression level impaired lysosomal functions contributing to the accumulation of autophagosomes and cell apoptosis in corpus luteum(CL)of late pregnancy thereby involving in the regulation of luteal functions.Thirdly,the regulatory role and underlying mechanisms of HIF-la mediated autophagy were analyzed during the granulosa cell differentiation and early luteal formation by in vitro cell culture and in vivo animal model.The present study found that the upregulation of HIF-1a expression is essential for the expression of StAR,the marker protein of granulosa cell differentiation,by in vivo and vitro experiments.Inhibition of HIF-la resulted to the decrease of StAR levels.Further study unveiled the upregulation of autophagy during granulosa cell differentiation,and inhibition of autophagy also led to the curtailment of StAR level.Knockdown of BNIP3 by siRNA disrupted the effect of HIF-la on granulosa cell differentiation and autophagy induction.Inhibition of HIF-la by Ech,a specific HIF-la inhibitor,contributed to the release of cytochrome C and caspase-3 activation.These results indicated that HIF-1a/BNIP3-mediated autophagy is involved in the differentiation of granulosa cells and the development of early CL.Fourthly,the regulatory role and underlying mechanisms of autophagy in the function maintenance of pregnant CL were further analyzed by in vitro cell culture and in vivo animal experiments.In consideration of progesterone production is the main function of CL,the present study collected CL samples during the middle to late stages of pregnancy by molecular biological technologies including immunohistochemical staining and western blot analysis,and then analyzed the autophagy level and progesterone production for delineating the role and regulation of autophagy in progesterone production of pregnant CL.These results indicated that autophagy is essential to the formation of lipid droplet in luteal cells.Inhibition of autophagy contributed to the decrease of droplet volume in luteal cells and the curtailment of serum progesterone levels.Inactivation of Akt/mTOR pathway contributed to autophagy induction in luteal cells.The present results of in vitro study also showed inhibition of mTOR prompted the upregulation of autophagy and progesterone levels,while inhibition of autophagy led to the decrease of progesterone production.These results indicated that autophagy plays an important role in luteal function maintenance and progesterone production by regulating the storage of lipid droplets in luteal cells.Fifthly,the pseudopregnant luteal development model was used and then the effect of autophagy on luteal cells apoptosis was studied during the process of CL functional and structural regression.The pseudopregnant luteal development model was developed and then the variation of autophagy and apoptosis levels of luteal cells was detected for unveiling the role and underlying mechanisms of HIF-1a mediated autophagy in luteal regression.The results indicated the expressions of HIF-la and its downstream autophagic regulatory proteins,BNIP3 and NIX,were significantly upregulated during the structural regression of CL.During the late phase of luteal regression,autophagy was induced in a Beclin1-independent manner accompanied by the accumulation of autophagosomes.Furthermore,the level of cell apoptosis was also increased during the late phase of luteal regression.Inhibition of HIF-la by Ech significantly prevented luteal atrophy resulted from cell apoptosis,which was consistent with the changes of BNIP3 and NIX expressions as well as the variation of autophagy and apoptosis levels.These results indicated that HIF-la-mediated autophagy is involved in the regulation of luteal cell apoptosis thereby prompting the process of luteal structural regression.Finally,in order to elucidate the role of HIF-la-mediated autophagy in the pathophysiological conditions of ovaries,the present study utilized DMC-induced ovarian toxicity to evaluate the role and regulation of HIF-la-mediated autophagy in ovarian toxicity by detecting the expression of autophagy related proteins.The results indicated that the middle(0.6g/kg BW)or high dosage(6g/kg BW)of DMC treatment obviously disrupted follicular structures and contributed to the upregulation of apoptosis in granulosa cells.However,the structures of ovarian follicle from low dosage group(0.06g/kg)were roughly maintained as well as the level of apoptosis,except for Bcl-2 levels.The expressions of HIF-la pathway and autophagy related proteins indicated low dosage of DMC treatment activated HIF-la/BNIP3 pathway and upregulated autophagy,while the expression of HIF-la/BNIP3 pathway was obviously inhibited in the middle or high dosage of DMC treated groups accompanied by autophagosome accumulation,which contributed to the apoptosis of granulosa cells and follicular atresia.These results further indicated HIF-1a/BNIP3-mediated autophagy plays a role in the survival of granulosa cells during low DMC-induced ovarian toxicity,whereas also plays a role in the apoptosis of granulosa cells during high DMC-induced ovarian toxicity,which implying the dual mechanism of different effects of autophagy under the diverse pathophysiological conditions.Together,the present results firstly revealed the role of HIF-la-mediated autophagy on the survival of granulosa cells;secondly found HIF-1a-mediated autophagy is involved in granulosa cell differentiation and early luteal formation;thirdly clarified the changes of autophagy during luteal development and then revealed that autophagy influences on progesterone production by modulating lipid droplet formation;fourthly unveiled the role of HIF-la-mediated autophagy in the apoptosis of luteal cells during luteal regression;finally evaluated the role and regulation of HIF-la-mediated autophagy in DMC-induced ovarian toxicity.These findings provided theoretic basis for our further understanding of follicular development,luteal development and regression,and also provided new orientation and theoretic foundation for clinically application of luteal regulation... |
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