Regulation And Mechanism Of AMH On Sertoli Cells And Follicular Development In Mice

The current research is focused on the role and molecular mechanism of AntiMüllerian Hormone(AMH)on Sertoli cells in the male and follicular development in the female of mice.This thesis comprises of literature review and experimental researches.Experimental researches include two aspects;the first part covers the functional role of AMH on the Sertoli cells regulations and progression at one week postnatal male mice.While the second demonstrates transcriptome profiling of preantral/small antral mouse ovary follicles treated with AMH.I: Effects of AMH on Sertoli cells in male mice and its molecular mechanism:Sertoli cells produce AMH,a glycoprotein belonging to the transforming growth factor-beta family.AMH mediates the regression of Müllerian ducts in the developing male fetus.However,the role of AMH in the regulation of primary Sertoli cells remains unclear.The present study was designed to investigate the effect of AMH on the viability and proliferation of Sertoli cells,with an additional focus on stem cell factor(SCF).Sertoli cells were treated with increasing concentrations of rh-AMH(0,10,50,100,and 800 ng/m L)for two days.The results revealed that AMH increased apoptosis,which were confirmed by a significant increase in Caspase-3 and Bax and a decrease in Bcl-2 protein and m RNA expression(P<0.01),at high concentration(800 ng/m L),and promoted proliferation,which was verified by an increase in PCNA m RNA(P<0.05),at low concentration(10 ng/m L).Furthermore,rh-AMH activated the non-canonical ERK signaling pathway,the phosphorylation of which can be inhibited by ERK-inhibitor(U0126)only at low concentrations.In addition,a minimum of 500 ng/m L of rh-AMH activated SMAD phosphorylation in SCs in vitro.Similarly,low concentrations of rhAMH(10-50 ng/m L)significantly increased(P<0.05)SCF m RNA and SCF protein levels.These findings indicate that AMH differentially regulates the fate of Sertoli cells in vitro by promoting proliferation at low concentrations and apoptosis at high concentrations.In addition,AMH increased the expression of SCF,an important regulator of Sertoli cell development.Therefore,AMH may play a role in Sertoli cell development.II: Effects of AMH on preantral/ small antral mouse ovary follicles by means of transcriptome profilingThe follicles grow from a pool of primordial follicles which retain the major functions in the entire reproductive life of a female.AMH has an inhibitory effect on ovarian follicle development.The key regulatory target genes in primordial follicle development is of paradigm importance in reproductive biology of female for which a systems biology method was used to find regulatory genes performing critical role in primordial follicle development.A complete in-depth bioinformatics analysis was performed to investigate the changes in transcriptome of preantral and small antral follicles in mice treated for 12 h and 24 h with AMH in two concentrations of 50 and 200 ng/m L,and thereby identified the candidate genes in time and concentration manner designated as set 1 and 2,respectively.Firstly,we found DEGs,121 and 162 genes in time and 56 and 63 genes in concentration dependent manner in response to AMH.Then a total of 19 and 13 significantly related GO annotations in set 1 and 2 of genes were found,respectively.The enriched GO biological processes in set 1(AZGP1,PODXL2,DSG1 A,PCDHB18,CDH18)were closely related to cell adhesion,biological adhesion,and innate immune response.At the same time,4 pathways were enriched,and 8 genes participated in the same pathway called neuroactive ligand-receptor interaction.In addition,CCR7 and CXCR2 were involved in two pathways: Chemokine signaling pathway and Cytokine-cytokine receptor interaction,while in gene set 2,KCNK15,SLC34A1,KCTD16,SLC30A8,1300017J02 RIK showed the same trend of taking part in three BP terms at the same time: metal ion transport,ion transport and cation transport.We also figured out the co-expression network comprising 720 and 460 pairs of genes in set 1 and set 2 respectively.A total of 222 nodes and 720 edges in set 1,whereas 109 nodes and 460 edges were involved in set 2 respectively.The network analysis of these DEGs provided with new candidate genes and pathways associated with inhibitory action of AMH on the primordial follicle development.We also found the related 56 and 33 TFs and 16 and 14 mi RNAs which targets gene in the co-expression net-work in set 1 and set 2 respectively.In addition,we further performed genetic analysis comprising phylogenetic,epigenetic and mutation analysis as well as the functional domain identification of the most important genes and receptors.To further emphasize the function of AMH,the key identified genes protein-protein docking was carried out to find intracellular and extracellular protein-protein interaction.This study elucidates one of the novel mechanism of AMH is involvement in inhibition of ovarian follicular development which could prolong the productive life of a female.