Study Of Molecular Mechanism Of Melatonin Regulates The Biological Function And Anti-Pyroptosis In Sheep Theca Cells

Melatonin（MT）is a potent antioxidant found in mammalian follicular fluid.MT limits oxidative stress in granulosa cells and oocytes through its antioxidant effect,which in turn is involved in regulating follicle development and ovulation;it can also effectively treat polycystic ovary syndrome,premature ovarian failure and other diseases.Theca cells（TCs）are important structures that make up the follicle,which can provide nutrients and information exchange for the development of granulosa cells and oocytes.At the same time,follicular development and ovulation are the result of highly synergistic development of the three germ cells,and one of the three is indispensable.There are more studies on granulosa cells,oocytes and MT in these two types of cells,but few studies on theca cells have been reported.In this study,we analyzed follicular membrane tissue development and MT receptor（MTR）expression in sheep during estrus using tissue sectioning techniques.TCs were obtained by an improved isolation method to optimize in vitro culture conditions and to analyze the expression and localization of MTR.To investigate the effect and mechanism of MT on the biological function of TCs by adding MT in vitro culture.Lipopolysaccharide（LPS）was used to construct a pyroptosis model of TCs to investigate the effect and protection mechanism of MT on their pyroptotic damage.The results of the study are as follows:1.Sheep TCs first appear in secondary follicles and increase in thickness at the tertiary follicular stage and differentiate to form internal and external membrane structures.The membrane thickness decreases during the mature follicle stage and increases again when the corpus luteum is formed.Both follicular membranes and corpus luteum tissues expressed MTR.The expression of MTR was significantly lower in secondary,atretic follicular theca interna tissue than in tertiary,mature follicular theca interna tissue and theca luteum tissue（P<0.05）.2.TCs with high purity（98.26±0.73%）were obtained by separation of follicular endothelial tissues with toothed forceps and discontinuous percoll gradient centrifugation.The optimal conditions for in vitro culture of TCs were endosomal tissue digestion time of 30 min,culture serum concentration of 15%,temperature of 38°C,CO₂ concentration of 5%,and suitable culture medium of DMEM/F12 and DMEM high sugar medium.TCs both expressed MT1 and MT2,and MT2 expression was significantly higher than MT1（P<0.05）.MT1 and MT2 expression was localized in the cytoplasm and cell membrane of TCs.3.MT significantly increased TCs Cat and Sod1 m RNA expression and decreased ROS content（P<0.05）.MT significantly increased TCs Bcl-2 m RNA and protein expression and significantly decreased Bax m RNA and protein expression（P<0.05）.Except for 10^-10M（physiological concentration）,MT significantly decreased proliferation viability and Cyclin D1 and CDK4 m RNA expression in TCs（P<0.05）.MT promoted progesterone secretion in TCs（P<0.05）and did not affect androgen secretion（P>0.05）.MT increased St AR m RNA and protein expression in TCs（P<0.05）,but did not affect CYP11A1,CYP17A1 and 3β-HSD m RNA expression without effect（P>0.05）.LY294002（PI3K/AKT pathway inhibitor）inhibited TCs St AR and P-AKT protein expression（P<0.05）.MT increased TCs St AR and P-AKT protein expression（P<0.05）.LY294002,Luzindole（non-specific MT1 and MT2 inhibitor）and 4p-PDOT（specific MT2 inhibitor）all inhibited MT-induced progesterone secretion as well as St AR and P-AKT protein expression in TCs（P<0.05）.MT increased MT1 and MT2m RNA expression in TCs（P<0.05）,and both Luzindole and 4p-PDOT inhibited MT-promoted MT1 and MT2 m RNA expression（P<0.05）.4.LPS increased TNF-α,IL-6,IL-1β,IL-18 and androgen secretion in TCs（P<0.05）and inhibited proliferation,progesterone secretion and MTR m RNA expression（P<0.05）.LPS increased NLRP3,Caspase-1,GSDMD and Caspase-4protein expression and ROS content in TCs（P<0.05）.After MT pretreatment,LPS-induced TNF-α,IL-6,IL-1β,IL-18 and androgen secretion were significantly reduced（P<0.05）and proliferative viability,progesterone secretion and MTR m RNA expression were significantly increased（P<0.05）in TCs.After MT pretreatment,NLRP3,Caspase-1,GSDMD and Caspase-4 protein expression as well as ROS content were significantly reduced in LPS-induced TCs（P<0.05）.5.LPS inhibited SIRT1 and SOD2 protein expression（P<0.05）,MT promoted SIRT1 and SOD2 protein expression（P<0.05）,and after MT pretreatment,SIRT1 and SOD2 protein expression was significantly higher than that of LPS alone（P<0.05）and did not differ from that between controls（P>0.05）.EX527（SRIT1 inhibitor）co-interaction with MT resulted in significantly higher inflammatory factor concentration,ROS content and GSDMD protein expression in TCs than MT co-interaction with LPS（P<0.05）,which was not significantly different from LPS alone（P>0.05）,and significantly lower SIRT1 and SOD2 protein expression than MT co-interaction with LPS and LPS alone（P<0.05）.Resveratrol（SRIT1 activator）co-activated with LPS resulted in no difference in inflammatory factor concentration,ROS content and GSDMD protein expression in TCs compared with controls（P>0.05）,and SIRT1 and SOD2 protein expression were significantly higher than LPS alone（P<0.05）.Luzindole co-interaction with MT resulted in significantly higher inflammatory factor concentration,ROS content and GSDMD protein expression in TCs than MT and LPS co-interaction（P<0.05）,which were not significantly different from LPS alone（P>0.05）,and significantly lower SIRT1 and SOD2 protein expression than MT and LPS co-interaction（P<0.05）,which were not significantly different from LPS alone was not significantly different（P>0.05）.After co-interaction of 4p-PDOT with MT,inflammatory factor concentration,ROS content and GSDMD protein expression of TCs were significantly higher（P<0.05）and significantly lower（P<0.05）than those of MT and LPS co-interaction,and SIRT1 and SOD2 protein expression were significantly lower（P<0.05）and significantly higher than those of MT and LPS co-interaction alone（P<0.05）.The results of this study showed that sheep TCs expressed MTR,and MT inhibited their proliferation and apoptosis and promoted MTR expression and progesterone secretion.Under pathological conditions,MT inhibited androgen overproduction,elevated ROS content and pyroptosis damage in TCs,and restored progesterone secretion and cell viability.This study shows that MT can also play a role in slowing follicular atresia,protecting ovarian reserve and inhibiting pathological ovarian activity through TCs,providing a reference for the use of MT to effectively improve reproductive development and treat inflammatory ovarian diseases in mammals.