Synergistic Regulatary Effect And Mechanism Of INH And AMH On Follicular Development In Mouse

In female animals,anti-Mullerian Hormone?AMH?is mainly secreted by preantral follicles and small antral follicles,which can inhibit the recruitment of primordial follicles and the transition of primary follicles to antral follicles.Inhibin?INH?is mainly secreted by large antral follicles,which inhibits the development and maturation of antral follicles.AMH and INH belong to the transforming growth factor?TGF-??superfamily members,and both are able to inhibit the follicular development.However,whether there are interactions or synergestic actions between AMH and INH are still unclear.Therefore,this study analyzed the regulatory effect and mechanism of AMH and INHA?Inhibin A?on steroid hormone secretion in granulosa cells of mice.Moreover,the effects of AMH and INH combined immunization on steroid hormone secretion,ovarian weight and litter size of mice were investigated using gene immunization.The aim is to clarify the interaction between AMH and INH in follicular development,which not only contributes to the enrichment of endocrine theory,but also lays the foundation for the development of new techniques improving animal fecundity.The main research contents and results are as follows:1.The regulation of AMH and INHA treatment on the synthesis and secretion of steroids hormone in granulosa cellsMouse granulosa cells were treated with different concentrations?1 ng/mL,10ng/mL and 100 ng/mL?of AMH or INHA at the basal level or FSH induced level for 48 h,and the estradiol?E₂?and progesterone?P₄?levels in supernatant was detected by ELISA.It was found that the addition of AMH alone had no significant effect on the production of basal levels of E₂ and P₄;whereas low dose?1 ng/mL?and moderate dose?10 ng/mL?INHA significantly inhibited the production of progesterone at the basic level?P<0.05?;notably,both AMH and INHA significantly inhibited the synthesis of E₂ and P₄ at FSH induced level?P<0.01?.Real-time PCR was used to detect the expression of romatase-related genes in granulosa cells.It was found that AMH and INHA did not change the expression of CYP19A1,FSHR,P450scc,3?HSD and StAR mRNA at the basal level,but significantly inhibited the expression of CYP19A1,FSHR,P450scc,3?HSD and StAR mRNA at FSH induced levels?P<0.01?.2.The synegistic effect and mechanism of AMH and INHA combined treatment on the synthesis and secretion of steroids hormone in granulosa cellsAfter AMH and INHA were added into granulosa cells for 48 h,the combination of AMH and INHA significantly inhibited the secretion of E₂ in FSH-induced level compared with AMH or INHA alone treatment?P<0.01?.In addition,the inhibitory effect of AMH and INHA combined group on FSH-induced P₄ secretion was extremely significantly stronger than that of the group supplement with AMH alone?P<0.01?and significantly stronger than that of INHA alone treatment group?P<0.05?,suggesting that AMH and INHA can synergistically negatively regulate FSH induced estradiol and progesterone secretion.Furthermore,both AMH and INHA significantly inhibited FSH-induced intracellular cAMP levels?P<0.05?and the phosphorylation of CREB phosphorylation,and FSH also can induce the expression of phosphorylated Smad 2/3?P<0.01?,while INHA inhibits the FSH-mediated pSmad2/3 signaling pathway?P<0.05?.Therefore,AMH and INH inhibit the regulation of steroid hormones mainly through inhibiting FSH-mediated cAMP-PKA-CREB signaling pathway,INH also available through the Smad2/3 signaling pathway.3.Effect of co-immunization with AMH and INHA on the fertility of mice120 Kunming female mice of 5-weeks were randomly divided into 4 groups,intramuscular injection of pVAX-tPA-SAMH-asd plasmids?group A?,pVAX-tPA-SINH-asd plasmids?group I?,pVAX-tPA-SAMH-asd plasmids and pVAX-tPA-SINH-asd plasmids?group AI?and normal saline?group C?were performed,and the same dose was given as booster after 2 weeks.The results showed that the all plasmid immunization group could induce the specific anti-AMH and anti-INH immune response in mice,and had high antibody positive rate and antibody titer.Among them,the plasma estradiol level and ovarian weight of INH antibody-positive mice and AMH and INH double antibody-positive mice were significantly higher than control group?P<0.05?;the AMH antibody-positive mice had significantly higher litter size in comparision to antibody-negative mice?14.88±2.16 VS.13.43±2.28,P<0.05?,the number of litters of AMH and INH double antibody positive mice was extremely significant higher than antibody negative mice?15.61±1.79 VS.13.43±2.28,P<0.01?.In addition,the study also found that the immunization of the AMH and INH vaccine did not affect the body weight gain and organ index of the mice.In conclusion,AMH and INHA synergistic inhibit the synthesis and secretion of steroid hormones in granulosa cells,and the combination of AMH and INHA with gene immunization can significantly improve the fertility of mice.