Molecular Mechanism Of Nodal/ALK7 Pathway Mediating Photoperiod Regulation Of Follicle Development In Psora Agrar

Djungarian hamsters(Phodopus sungorus)is one of the main grassland rodents in North China with the irreplaceable values in maintaining the ecosystem balance of the grassland.As a typical seasonal breeding animal,the population abundance of Phodopus sungorus fluctuates with the changes of the seasons.Photoperiod,one of the main environmental factors changing with seasons,regulates follicle development through the hypothalamus-pituitary-ovary(HPO)axis and then affects seasonal reproduction.And it is of great ecological significance to explore the mechanism of photoperiod regulating the follicle development of Phodopus sungorus.Studies have shown that granulosa cells are important part of follicles,and Nodal/ALK7 signal pathway can regulate the apoptosis of granulosa cells,then affect the development of follicles.However,the molecular mechanism of photoperiod regulating the follicle development mediated by Nodal/ALK7 pathway has not been studied.Therefore,in this study,adult female Phodopus sungorus were taken as the research object,and divided into three experimental groups,including,long daylight group(16L:8D,LD),short daylight group(8L:16D,SD)and moderate daylight group(12L:12D,MD).Firstly,the effect of photoperiod regulating the apoptosis state of granulosa cell and the developmental state of the follicle in Phodopus sungorus was explored by observing the variations of the follicular microstructure,TUNEL detection,caspase-3 activity detection and ultrastructural changes of granulosa cells under different photoperiod conditions at the histological level.Secondly,in order to explore the molecular mechanism of photoperiod affecting granulosa cell apoptosis and regulating follicle development through FSH-Nodal/ALK7 pathway,the enzyme-linked immunosorbent assay(ELISA),qRT-PCR,Western Blot and immunofluorescence were used to detect the changes of gonadal hormone concentration,the differential expression of genes and proteins of Nodal/ALK7 signal pathway,and the co-localization of Nodal and ACVR1C under different photoperiod conditions at the molecular level.Finally,the proteins interacting with the core protein XIAP of Nodal/ALK7 pathway were screened by bioinformatics and Yeast twohybrid(Y2H)technologyand,the molecular mechanism involved in photoperiod regulation of Nodal/ALK7 pathway was explained.The main results are as follows:(1)Effects of photoperiod on granulosa cell apoptosis and follicle development.At the histological level,the degree of granulosa cell apoptosis and follicle development of Phodopus sungorus were studied under different photoperiod conditions.There were significant differences(P<0.05)in ovarian weight after different photoperiod treatments,which showed that the ovarian weight decreased in SD group and increased in LD group;Secondly,compared with MD and LD groups,the ovarian degeneration was obvious,the number of atretic follicles was significantly increased(P<0.01),the number of primary follicles,secondary follicles and antral follicles significantly decreased(P<0.01)in SD group,and the development of follicles was inhibited.Compared with MD and SD groups,the number of atretic follicles in LD group decreased significantly(P<0.01),while the number of primary follicles,secondary follicles and antral follicles significantly increased(P<0.01),which promoted follicles development In addition,the degree of apoptosis of granulosa cells increased in SD group,which had typical characteristics of apoptosis in animals,including the increase of 3’-OH fragmentation at the end of DNA,significantly increased of caspase-3 activity(P<0.01),the swelling and vacuolation of mitochondria and sparse cristae of mitochondria.However,the degree of apoptosis of granulosa cells decreased in LD group,which was characterized by the decrease of 3’-OH fragmentation at the end of DNA and a significant decrease in caspase-3 activity(P<0.01),and the ultrastructure of granulosa cells remained normal without obvious change.Therefore,short daylight can promote granulosa cell apoptosis and inhibit follicle development,while long daylight can promote follicle development.(2)FSH-Nodal/ALK7 pathway mediates photoperiod regulating the apoptosis of the granulosa cell and thus follicle development in Phodopus sungorus.At the molecular level,we investigated that the molecular mechanism of photoperiod regulating follicle development through FSH-Nodal/ALK7 pathway.The difference in follicle development under different photoperiod conditions is related to the role of FSH hormone in regulating Nodal/ALK7 pathway in granulosa cells.Compared with MD and LD groups,the concentration of follicle stimulating hormone(FSH)in SD group significantly decreased(P<0.01),the Nodal/ALK7/SMAD pathway was activated,while the phosphatidylinositol 3-kinase(PI3K)-Akt pathway was inhibited.The expression level of BAX in the mitochondrial apoptosis pathway was significantly increased(P<0.05),while the expression of Bcl-xL was significantly decreased(P<0.01),DIABLO and HtrA2 was released outside the mitochondria,resulting in a significant decrease in XIAP expression(P<0.05),the caspase-3 activity significantly increased(P<0.01)and the degree of granulosa cell apoptosis,which inhibited follicle development.Compared with MD and SD groups,the concentration of FSH in LD group significantly increased(P<0.01),Nodal/ALK7/SMAD pathway was inhibited and PI3K-Akt pathway was activated.In the mitochondrial apoptosis pathway,the expression level of BAX significantly decreased(P<0.05),while the expression level of Bcl-xL significantly increased(P<0.01),which inhibited the release of DIABLO and HtrA2,resulting in a significant increase in XIAP expression level(P<0.05)and a significane decrease in caspase-3 activity(P<0.01),which was beneficial to the survival of granulosa cells and promoted the development of follicles.(3)Screening and verification of the proteins interacting with XIAP in Nodal/ALK7 signal pathway.From the specific acting mechanism of the core protein XIAP,the proteins interacting with XIAP were screened and verified.And,the role of proteins interacting with XIAP in the Nodal/ALK7 pathway regulated by photoperiod verified by Y2H and Western Blot.The coding sequences of XIAP was 1491bp,which encodes 496 amino acids.The amino acid sequence of the core protein XIAP in Nodal/ALK7 signal pathway was highly conserved and had multiple phosphorylation sites and conserved domains.It included 28 serine sites,20 threonine sites,6 tyrosine sites,BIR domains(BIR1,BIR2,BIR3),UBA domains and RING domains.36 proteins interacting with XIAP were screened by Y2H,and six proteins that may be involved in Nodal/ALK7 signal pathway were verified by point-to-point verification.Finally,three proteins interacting with XIAP were screened,including RACK1,COL1A1 and COL1A2.RACK1 is involved in mitochondrial apoptosis pathway,COL1A1 and COL1A2 are involved in transforming growth factor-β(TGF-β)/SMAD signal pathway and PI3K-Akt pathway.In addition,it was found that XIAP interacted with RACK1,COL1A1 and COL1A2 through BIR2,UBA/BIR3 and RING/BIR3 domains,respectively.The expression of RACK1 increased in SD group and decreased in LD group,but no significant difference exists(P>0.05)in RACK1 expression level under different photoperiod.There was a significant difference(P<0.05)in COL1A1 and COL1A2 expression quantity at protein levels between SD group and LD group.Among them,the expression levels of COL1A1 and COL1A2 at protein level significantly increased(P<0.05)in SD group.The results showed.that the expression of RACK1,COL1A1 and COL1A2 were regulated by photoperiod,which participate in the Nodal/ALK7 signal pathway to regulate the follicle development by interacting with XIAP.This study investigated that the molecular mechanism regulating of photoperiod regulating follicle development in Phodopus sungorus mediated by Nodal/ALK7 pathway for the first time.It has been found that short daylight can promote granulosa cell apoptosis and inhibit follicle development,which is caused by promoting the expression of genes and proteins involved in the Nodal/ALK7/SMAD pathway,mitochondrial apoptosis pathway and inhibiting PI3K-Akt pathway.As the core protein of Nodal/ALK7 pathway,XIAP can interact with RACK1,COL1A1 and COL1A2 through its own conserved domains(BIR2,UBA/BIR3,RING/BIR3)to exert its antiapoptosis effect,inhibit granulosa cell apoptosis and promote follicle development.Therefore,XIAP-interacting proteins(RACK1,COL1A1 and COL1A2)are also involved in Nodal/ALK7 pathway to mediate photoperiod regulating of follicle development.This study can not only lay a theoretical foundation for further exploring the molecular mechanism of follicle development,but also provide a theoretical basis for the regulation of seasonal reproduction of Phodopus sungorus,and enrich the knowledge of the molecular ecology.