Effects Of Follistatin And Activin A On Migration Of Mouse Decidual Stromal Cells And Their Mechanisms

During pregnancy,uterine decidualization is the prerequisite for successful pregnancy.The migration of decidual endometrial stromal cells is regulated by estrogen,progesterone and a variety of cytokines.It is still unclear whether a variety of sex hormone regulatory proteins that induced during pregnancy,such as follistatin(FST)and activin A(Act A),are involved in the regulation of decidual endometrial stromal cells migration.Activin A is a member of transforming growth factor β(TGF-β)superfamily,as a sex hormone regulatory protein,is named for the function of promoting follicle-stimulating hormone(FSH)secretion by anterior pituitary gland cells.FST is named for the ability to inhibit FSH secretion by the pituitary gland,and studies have suggested that FST shows high affinity with activin A,so it is also called activin binding protein.Studies have indicated that activin A and FST are highly expressed in the endometrium during pregnancy,but the regulatory role and mechanism of activin A and FST in the migration of decidualized endometrial stromal cells are still unclear.Therefore,this study explored the effects of activin A and FST on decidual stromal cells migration and the related mechanisms by inducing decidualization of mouse endometrial stromal cells in vitro and primary cultured decidual stromal cells from of pregnant mouse by using microfluidic technology,etc.,which is contribute to elaborate the mechanism of decidualization of pregnancy and also provide theoretical basis for seeking effective drug target that regulate uterine decidualization.I.Methods1.Induce decidualization of mouse endometrial stromal cells in vitroEndometrial stromal cells(mESCs)were isolated and cultured from non-pregnant Balb/c female mice of 8-week,immunocytochemical staining was carried to identify the cells.Estrogen and progesterone were used to induce the decidualization of mESCs in vitro.2.Detection of biological activity of decidual stromal cellsCCK-8 assay were performed to determine the cell proliferation.RTCA were used to determine the cell adhesion.Scratch assay were used to detect the wound healing ability of decidual stromal cells.Transwell chamber assay and microfluidic devices were carried out to detect the migration and invasion ability of mouse decidual stromal cells.RT-PCR and Western blotting were used to detect the expression of extracellular matrix and migration-related proteins.3.Activation mechanism of decidual stromal cellsFlow cytometry was used to determine the level of calcium flux of decidual stromal cells.RT-PCR and Western blotting were used to detect the expression of activin classical SMAD signal pathway,non-SMAD signal pathway(MAPK,PI3K/AKT-m TOR and Rho pathway)and progesterone receptor pathway proteins of decidual cells.4.Detection and mechanism analysis of primary cultured mouse decidual stromal cellsEnzyme digestion were used to isolated mouse decidual stromal cells from 6.5dpc Balb/c mice.The identification,activity and mechanism analysis were the same as that decidualized mouse endometrial stromal cells.5.Expression of activin A and FST in decidual tissue of individual during pregnancyEstablish normal pregnant mouse model and spontaneous abortion mice model.Expressions of activin A and FST in decidua tissue were detected.Clinical sample analysis.Expressions of activin A and FST in decidual tissue of normal women of early pregnancy and patients with fetal arrest were detected.II.Results1.Induced decidualization of cultured mouse endometrial stromal cells successfullyCultured the mESCs in vitro showed positive anti-Vimentin staining,suggesting the cells belong to stromal cells.Cells showed typical decidual-like cell morphology after the stimulate of Estrogen and Progesterone,and the expression of prolactin,a decidualization marker,is significantly induced,suggesting that mESCs differentiate to decidualized mouse endometrial stromal cells(d-MESCs)successfully.2.Effects of activin A and FST on the activity of decidual stromal cells(1)Activin A and FST promoted the proliferation of decidual stromal cells.FST induced decidual stromal cells transform into interstitial cells,while there was no significant morphological change in activin A group.(2)Both activin A and FST inhibited the adhesion of decidual stromal cells,especially FST;both activin A and FST promoted wound healing,induced migration and invasion of decidual stromal cells,and FST induced longer migrated distance,and more obvious directionality.Activin A and FST may regulate the migration of decidual stromal cells by activating the expression of MMP9,FN,Col IV,Vimentin,Ezrin and N-cadherin.3.Mechanism of activin A and FST regulating the activation of decidual stromal cells(1)FST increased the calcium influx of decidual stromal cells.(2)Activin A and FST had no significant effect on the expression of classical SMAD pathway signal molecules in decidual stromal cells.FST upregulated the ratio of p-JNK/JNK,FST may activate JNK signal of the non-SMAD pathway.JNK inhibitor treatment reduced the migration number and distance of mouse decidual stromal cells,and the cell migration was attenuated.(3)FST regulated decidualization by activating Fox O1 and Ihh downstream of PR pathway.4.Expression of activin A and FST in decidual tissue of individual during pregnancy(1)In the normal pregnant mouse model,the levels of activin A and FST in decidual tissue increased with the development of pregnancy.After abortion,the expressions of activin A and FST both decreased,while FST decreased more significantly.(2)The expression of FST in decidual tissue of patients with fetal arrest decreased significantly,while no significant changes in activin A.III.ConclusionIn summary,in activin A-FST system,compared to activin A,FST is a more effective factor on inducing decidual stromal cells migration.FST induces decidual cells migration by activating JNK signals,rather than activin classical SMAD pathways.The decreased expression of FST in decidual tissue during abortion suggests that FST may become an important biomarker for evaluating decidual development in early pregnancy,and FST shows potential application value in the treatment of abortion caused by decidual dysplasia.