| Follicle recruitment,development and ovulation are critical fators influencing the reproductive performance of human being and livestock.The activation of primordial follicles and the formation of growing follicles are of critical importance to realize female reproduction potential.Anti-müllerian hormone acts as a powerful brake on follicle transition,and the only,known to date,one autocrine and paracrine factor inhibiting follicular activation and growth.So far,roles and underlying molecular mechanisms of AMH signaling and its dynamics in follicular development have not been fully investigated in farm animals especially in pigs.Besides,better understanding of the roles and mechanisms of AMH in follicle granulosa cells would deepen our insights into controlling of folliculogenesis and evaluation of reproductive traits of higher value,which has great importance to improvement farm animal breeds.In this study,methods of immunofluorescent staining,fluorescent microscopy,RNA interference,quantitative real-time PCR,radio-immuno assay,flow cytometry,and RNA-Sequencing were employed,in order to(1)examine the expression pattern and location of AMH and AMHR2 in ovary and other tissue;(2)evaluate the effects of AMH on in vitro maturation of oocytes and their subsequent development after in vitro fertilization and parthenogenetic activation;(3)investigate effects of AMH on proliferation,apoptosis,steroidogenesis,and differentially expressed key genes of granulosa cells and theca cells cultured in vitro for determinating the mechanisms of AMH in granulosa cells and theca cells.The study contains the following five parts:Experiment ?: We aimed to explore the expression pattern of AMH and AMHR2 in pig different organs or tissues.Results of q PCR showed that AMH and AMHR2 were expressed dominantly in pig ovary and testis.Results of immunofluorescence showed that AMH was localized in the granulosa of preantral and small antral follicles,highly expressed in cumulus cell but undetectable in oocytes.Real-time quantitative(Real-time q PCR)showed the abundance of AMH m RNA was significantly decreased in granulosa cells from 1~3mm to 3~5mm diameter follicle(P<0.05)while the abundance of AMHR2 m RNA was significantly increased in granulosa cells from 3~5mm to >5mm diameter follicle(P<0.05).Intrafollicular AMH concentrations,as determined by ELISA,in antral follicles of different size classes from pigs showed that AMH concentration in 1~3mm diameter follicle was significantly higher compared with 3~5 mm and >5mm diameter follicle.And the AMH concentration in >5mm diameter follicle was almost undetected.In this section,we found that the AMH expression pattern in pig is same to other species.Experiment ? : The present study was conducted to investigate effects of AMH on IVM of pocine oocytes and the subsequent preimplantation developmental competence following in vitro fertilization and parthenogenetic activation.Different concentrations of AMH showed no significant effects among groups in rate of maturation,cumulus cell expansion index,and the rates of cleavage and blastocyst of the PAEs in routine maturation medium.When using the chemically defined medium with PG600 for IVM,we found that,in comparison to control group,10ng/m L AMH significantly promoted the maturation rate and cleavage rate after IVF(P>0.05).In comparison to control group,the GDF9 and BMP15 m RNA expression were not signifticantly different in 10 ng/m L and 100 ng/m L AMH-treated oocytes after maturation(P>0.05).CASPASE3 and BAX m RNA expression were not signifticantly different in 10 ng/m L and 100 ng/m L AMH-treated COCs after IVM.However,the expression of BAX m RNA was significantly increased in 10 ng/m L and 100 ng/m L AMH-treated COCs after IVM.This section showed that 10 ng/m L AMH could improve the nuclear and cytoplasmic maturation of porcine oocytes.Experiment ?: In comparison to control group,treatment of granulosa cells with10ng/m L AMH had no effect on FSHR and CYP19A1 m RNA expression and E2 production.FSH treatment increased FSHR and CYP19A1 m RNA expression and E2 production in granulosa cells cultured in vitro.AMH of 10 ng/m L significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 m RNA expression and E2 production.After AMHR2 si RNA,FSH treatment significantly increased FSHR and CYP19A1 m RNA expression and E2 production(P<0.05).But there was no significant difference compared to AMHR2 si RNA with FSH treatment group.This section showed that AMH significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 m RNA expression and E2 production in granulosa cells.Experiment ?: Porcoll method can use for obtain theca cells with higher purity and cell viability.LH treatment significantly increased 3?-HSD,CYP11A1,LHCGR m RNA expression and androstenedione or progesterone production(P <0.05).As compared to control group,treatment of theca cells with 10 ng/m L AMH had no effect on 3?-HSD,CYP11A1,CYP17A1,LHCGR,STAR ? CYP17A1 m RNA m RNA expression and androstenedione or progesterone production(P>0.05).AMH of10ng/m L significantly decreased the LH-stimulated effect on 3?-HSD,CYP11A1,CYP17A1,LHCGR m RNA expression and androstenedione production.To examine whether AMHR2 is required for the suppressive effects of AMH on LH-induced androstenedione production,specific si RNA for AMHR2 was used to knockdown endogenous AMHR2.The results showed that 3?-HSD m RNA expressed and androstenedione production was not significantly different between AMH and LH cotreated group and LH treated group.Transfection with si AMHR2 for 48 h obolished the suppressive effects of AMH on LH-induced 3?-HSD expression and androstenedione production.This section showed that AMH significantly decreased the LH-stimulated effect on 3?-HSD m RNA expression and androstenedione production in theca cells.Experiment ? : We also collected PFs-OGCs after being exposed to 20ng/m L AMH and cultured in vitro for 18.5 d to run RNA-Seq analysis,and finally we found that 159 genes up-regulated and 7 gene down-regulated.Q-PCR verified the trend of the genes differentially expressed between control groups and AMH treated groups,and the results were in accordance with RNA-Seq.We found that more genes were involved in biological processes by Gene Ontology analysis.The results of KEGG Pathway analysis show that AMH exerts its function mainly by participating metabolic pathway,TGF? and ECM-receptor pathways.In conclusion,we found that the AMH expression pattern is similar to other species.10 ng/m L AMH could improved the nuclear and cytoplasmic maturation of pig oocytes.AMH can compromise the FSH-stimulated effect on FSHR and CYP19A1 m RNA expression and E2 production in granulosa cells.Moreover,AMH also negatively influence the LH-stimulated effect on 3?-HSD m RNA expression and androstenedione production in theca cells.AMH exerts its function mainly by TGF?and ECM-receptor pathways. |
PDF Full Text Request