Effects And Mechanisms Of Anti-müllerian Hormone On The Steroid Hormone Synthesis Enzyme In Porcine Theca Cells

Anti-müllerian hormone(AMH)is a member of transforming growth factor family,it is secreted by the granulose cells in growing follicles.AMH can inhibit the recruitment of primordial follicles and exert inhibitory effect on follicular sensitivity to follicle stimulating hormone,but its mechanism of how to play such function is unclear.Pig is an ideal animal model in medical experiment,in order to investigate effects of AMH on the steroidogenesis expressed key genes of theca cells cultured in vitro for determinating the mechanisms of AMH in theca cells.Then we carried out the following experiments:Experiment 1: The expression patterns of AMH in different orgass or tissuesThis experiment mainly used the technology of RT-qPCR,analyse the expression patterns of AMH and AMHR2 in different organs and granulose cells in different diameters abtained from pigs;And we also investigated the concentration of AMH in pig's follicular fluid in different diameters.Rresults of qPCR showed that AMH and AMHR2 were expressed dominantly in pig ovary and testis.Real-time quantitative showed the abundance of AMH mRNA was significantly decreased in gramulosa cells from 1~3mm to 3~5mm diameter follicle(P<0.05)while the abundance of AMHR2 mRNA was significantly increased in granulosa cells from 3~5mm to >5mm diameter follicle(P<0.05).Intrafollicular AMH concentrations,as deternimed by ELISA,in antral follicles of different size classes from pigs showed that AMH concentration in1~3mm diameter follicle was significantly higher compared with 3~5mm and >5mm diamter follicle.And the AMH concentration in >5mm diameter follicle was almost undetected.Experiment 2: Isolation and purification of pig's theca cellsThis study used three methods to isolate and purify theca cells.Method A:Scraped granulosa cells layer with 25 G syringe needle,and the remaining cell layer was digested with IV collagenase.Method B: cut granulosa cells-basement membrane-theca cells layer into pieces with surgical scissors,then digested with IV collagenase.After digestion,the cells were purified by Percoll(35% and 44%)gradient centrifugation horizontal centrefugal,the theca cells located between 44%and 35%.Method C: the granulosa cells-basement membrane-theca cells layer was digested directly with IV collagenase,then purified by Percoll(35% and 44%)gradient centrifugation horizontal centrefugal twice to obtain theca cells.Then Threekinds of antibody(Viment?Cytokeratin?Factor ?)were used to identify theca cells.Results: Higher viability and purity of theca cells gained with method A than method B and C(92.00% vs.80.33%,85.00%;95.77% vs.87.87%,90.87%,respectively)(P<0.05);More cells were gained from method B than method A and C(1.06×107 vs.0.74×107and 0.86×107)(P<0.05),but less time was used with method B than method A and C(236.67 min vs.260.00 min,268.33min)(P<0.05).In conclusion,method A can obtain theca cells with higer purity and cell vibility.These results would provide foundation for further investigation of theca cells function.Experiment 3: The effect of AMH on the gene expression of steroid hormone synthase in theca cells of pigThere are no significant effects on the expression of LHCGR?STAR?CYP11A1?CYP17A1 and 3?-HSD m RNA on FSH or AMH and FSH treated for 48 h in theca cells.Treatment with FSH or AMH can singnificantly increase the expression of STAR mRNA in theca cells(P >0.05).It showed that FSH and AMH may have a synergies in promoting steroid synthesis.LH treatment significantly increased LHCGR?CYP11A1 and 3?-HSD mRNA(P<0.05).As compared to control group,treatment of theca cells with 10ng/mL AMH had no effect on LHCGR?STAR?CYP11A1?CYP17A1 and 3?-HSD mRNA(P >0.05);Forskolin can significantly increase the level of LHCGR?CYP11A1?CYP17A1 and3?-HSD mRNA(P<0.05).And when teaated with combined AMH and forskolin,the expression of LHCGR ? CYP11A1 ? CYP17A1 and3?-HSD m RNA and has no difference with treatment with forkolin(P >0.05).To exanime whether AMHR2 is required for the suppressive effects of AMH of LH-induced androstenedione production,specific siRNA for AMHR2 was used to konckdown endogenous AMHR2.The results showed that 3?-HSD m RNA expressed was not significantly different between AMH and LH co-treated group and LH treated group.Transgection with siAMHR2 for 48 h obolished the suppressive effects of AMH on LH-stimulated effect on 3?-HSD mRNA expression and androstenedione production in theca cells.In conclusion,this study showed that the expression patterns of AMH in pigs was similar to other species For the first time we found AMHR2 was expressed in pig's theca cells,and AMH have an effect on LH-induced steroid synthesis by regulate the expression of 3?-HSD.