The Molecular Mechanism Of Anti-mullerian Hormone(AMH) Regulates Stem Cell Factor (SCF) In Granulosa Cells

Anti-Müllerian hormone (AMH) is an important regulator of primordial follicleassembly that appears to mediate a stromal-epithelial interaction in the developingovary to inhibit follicle assembly. AMH, also known as Müllerian inhibitorysubstance (MIS), is a member of the transforming growth factor beta family (TGF-)and binds to AMH type II receptors (AMHRII). In vivo and in vitro studies showedAMHRII expression co-localized with AMH in the granulosa and the theca cells.There is lack of AMHRII in oocytes. AMH produced by preantral and small antralfollicles in the postnatal ovary has two sites of action in the postnatal ovary. It inhibitsinitial follicular recruitment, and it inhibits the stimulatory effect of FSH on thegrowth of preantral and small antral follicles. On the basis of the pattern of expressionof the receptors described above, it is much more likely that AMH exerts its effect onovarian follicles via the granulosa and theca cells, but not via the oocyte. AMH alsoinfluences transcription factors in the signaling pathways of granulosa cells, mainlythrough the Smad protein, and then regulate gene transcription of other cytokines tomaintain primordial follicles in their arrested state.Stem cell factor (SCF, also called Steel factor or Kit ligand), a granulosa-derivedgrowth factor, binds to oocyte c-Kit receptor and its signal is transduced through thePI3K pathway; this effect appears to be important for the regulation of early folliculardevelopment and enhancement of the production of oocyte factors, which in turnstimulate the proliferation and differentiation of the surrounding granulosa cells. Themechanism of regulating SCF is still unknown.Eric Nilsson reported AMH down-regulates the expression of stimulatory andinhibits the stimulatory actions of basic fibroblast growth factor (bFGF), SCF andkeratinocyte growth factor (KGF).It is unknown whether AMH exerts an autocrine/ paracrine effects on SCF expression. AMH could inhibit phosphorylation of cAMPresponsive element (CRE)-binding protein (CREB) in the cAMP pathway throughautocrine and paracrine mechanisms, which result in reducing FSH-stimulatedtranscription of estrogen synthase. In addition, several studies investigated thecAMP/PKA responsive element (CRE) that exists on the promoter of the human SCFgene. In the rat seminiferous epithelium, SCF gene expression was up-regulated bycAMP/PKA pathway. Based upon those studies, we speculated that AMH mightreduce the SCF transcription acting through the cAMP/PKA pathway.Within this context, we measured AMH and SCF mRNA and protein in theserum, follicular fluid (FF) and granulosa cells of women undergoing in vitrofertilization (IVF) treatment. Subsequently, SCF mRNA (by real-time RT–PCR) andprotein expression (by immunocytochemistry and immunoblotting) were evaluatedafter pretreatment with rhAMH and cAMP (alone or in combination) for48h in thepresence or absence of a PKA inhibitor (H89) to gain insight into the signalingpathway that might be involved in AMH regulation of SCF.Chapter1To study the expression and correlation between AMH andSCF in patients with different ovarian reserve.Objective:This studywas conducted in15patientswho underwentCOH as part of theirIVFtreatment. The inclusion criteria were female age under35years with infertilitydue tomale or tubalfactors. Patients with endometriosis and polycysticovariansyndrome were excluded. Patients received an intramuscular injection of1.25mg of long-acting Diphereline (Ipsen) during the middle of their luteal phase todownregulate the pituitary and an intramuscular injection of150–450IU/d ofFSH-HP on the3rd day of their menstrual cycle to superstimulate ovulation.Intramuscular HCG (10,000IU) was administered when3or more follicles were C16mm in diameter, and after34-36h follicles were aspirated by vaginal oocyte retrievalunder the guidance of ultrasound. Methods：The level of AMH and SCF protein secretion in serum were examined by ELISAin235cases of infertile woman, the level of AMH and SCF protein in follicular andrelative mRNA in granulose cells were detected by ELISA and real-time RT-PCR in78cases who received IVF-ET treatment.The relationship between the expression ofAMH and SCF was analyzed by Pearson correlation.Results:1.There is not a significant correlation between AMH and SCF in235cases ofinfertile woman;2. The expression of AMH and SCF shows a significant negative correlation infollicular fluid from55IVF-ET patients;3The expression of AMH and SCF mRNA shows a significant negativecorrelation in granulose cells from31IVF-ET patients.Conclusion：1.The expressions of AMH and SCF shows a significant negative correlation inhuman granulosa cells.2.The expression of AMH and SCF shows a significant negative correlation infollicular fluid.3. The level of AMH shows a significant positive correlation with ovary highresponse.4. There is not a significant correlation between AMH and SCF in serum.Chapter2The effect of AMH on SCF expression in granulosa cellsObjective:This study was conducted in15patients who underwent IVF-ET. The inclusioncriteria were female age under35years with infertility due to male or tubal factors.Patients with endometriosis and polycystic ovariansyndrome were excluded. Patientsreceived an intramuscular injection of1.25mg of long-acting Diphereline (Ipsen) during the middle of their luteal phase to downregulate the pituitary and anintramuscular injection of150–450IU/d of FSH-HP on the3rd day of their menstrualcycle to superstimulate ovulation. Intramuscular HCG (10,000IU) was administeredwhen3or more follicles were C16mm in diameter, and after34-36h follicles wereaspirated by vaginal oocyte retrieval under the guidance of ultrasound.Methods:1.The cells were then plated, at a concentration of10,000cells per well, in35mm culture dishes at37℃in a95%air,5%CO2humidified environment for24hours. After seeding, the cells were rinsed with PBS and then subjected to treatmentswith rhAMH（10ng/ml,15ng/ml,20ng/ml）;2. Pick up the special siRNA-AMH sequence and chemically synthesize in vivo,and transfect the ovarian granulosa tumor cell line COV434withHiperFectRTransfection Reagent, then test the expression of SCF mRNA and protein.Results:1. The effect of rhAMH on SCF expression showed a dose-dependent response.2. The expression of AMH mRNA and protein obviously decreased insiRNA-AMH groups, and the expression of SCF in mRNA and protein obviouslyincreased（both P＜0.05）.Conclusions:1. AMH down-regulates SCF mRNA and protein expression in granulasa cells.2. The expression of SCF mRNA and Protein increased significantly in COV434with pGCsi-shRNA-AMH. Chapter3rhAMH down-regulates SCF mRNA and proteinexpression via the cAMP/PKA pathwayObjective:We enrolled a total of51patients with ages27-43years from The Jones Institutefor Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia, USAfrom April to November2013. Couples with tubal, unexplained or male infertilitywere included. Patients with endometriosis and polycystic ovarian syndrome wereexcluded from this study.Methods:The cells were then plated, at a concentration of10,000cells per well, in35mmculture dishes at37℃in a95%air,5%CO2humidified environment for24hours.After seeding, the cells were rinsed with PBS and then subjected to treatments withrhAMH and/or cAMP in the presence or absence of an inhibitor of PKA (H89,1mM)for48h.Results:There was a significant negative correlation between AMH and SCF in FF, andin the mRNA expression of AMH and SCF in hGC. Conversely, there was nocorrelation between AMH and SCF in serum. In primary cultures of hGCs, SCFmRNA and protein expression were evaluated after pretreatment with AMH andcAMP (alone or in combination) in the presence or absence of a PKA inhibitor (H89).SCF was down-regulated in AMH groups and was increased in the cAMP groups.The effect of rhAMH on SCF expression showed a dose-dependent response. H89abolished the effect of rhAMH on SCF expression.Conclusions:This is the first report on a modulatory role for AMH as an ovarian/follicularautocrine/paracrine factor controlling SCF expression. We demonstrated that AMH regulates SCF via the cAMP/PKA pathway.