Effects Of Acupuncture On HPO Axis And FSH-cAMP Signaling Pathway In A Rat Model Of Diminished Ovarian Reserve

BackgroundDiminished ovarian reserve(DOR)refers to a decrease in the number of oocytes in the ovary and/or decline in the quality,accompanied by a reduction in anti-Mullerian hormone(AMH)levels,decreased antal follicle count(AFC),and increased follicle stimulating hormone(FSH)levels.With the development of society,women are facing increasing pressure in life and work,and the DOR incidence is increasing year by year.Infertility caused by the DOR seriously affects women's reproductive health and life quality,and brings heavy mental and economic burden to their families.FSH is one of the main hormones of the hypothalamic-pituitary-ovarian(HPO)axis,which can promote the proliferation and differentiation of granulosa cells,affect the development,maturation and ovulation of follicles,and affect the synthesis of estrogen.The binding of FSH to its specific FSH receptor(FSHR)is the basis of its biological effects.FSH binding to FSHR further activates cascade amplification of downstream signaling molecules in the cyclic adenosine monophosphate Cyclic adenosine monophosphate(cAMP)and protein kinase A(PKA)signaling pathway,and affects granulosa cell proliferation and differentiation and ovarian sex hormone synthesis.P450 aromatase(P450arom)is a speed limiting enzyme in granulosa cells.It catalyzes the transformation of androgen into estrogen and participated in follicular recruitment.Clinical trials showed that acupuncture could reduce serum FSH levels,increase serum estradiol(E2)levels,and improve the ovarian reserve by adjusting abnormal sex hormone levels.Animal experiments also showed that acupuncture could adjust the disordered state of the HPO axis and restore the excessively secreted Gonadotropin-releasing hormone(GnRH)levels to normal level.The FSH-cAMP signaling pathway was important for follicular development and ovarian function,but there were few acupuncture researches of DOR animal model on this signaling pathway.This experiment explored the mechanism of acupuncture to improve ovarian reserve function from two aspects:overall regulation of HPO axis and local changes of FSH-cAMP signaling pathway in ovary.ObjectiveTo explore the effects of acupuncture on the hypothalamus-pituitary-ovary axis and FSH-cAMP signaling pathway in a rat model of diminished ovarian reserve.MethodExperiment 1:Establishment and validation of DOR animal modelsTwenty specific pathogen free(SPF)female Sprague Dawley(SD)rats,including 10 of 3 months old as control group and another 10 of 9 month old retired breeding rats as DOR model group.The daily vaginal smear of rats was observed to determine the estrous cycle,and the rats in the diestrus were selected.ELISA detection of serum sex hormone(FSH?LH?E2?AMH?inhibitory B(INH-B)levels,).The antral follicle count was determined by frozen sections and HE staining.Experiment 2:Effects of acupuncture on HPO axis and FSH-cAMP signaling pathway in DOR ratsForty female SD rats,including 10 of 3 months old as control group and 30 of 9 months old retired breeding rats randomly divided into three groups:DOR model group(DOR),restraintriction group(DOR+res),acupuncture group(DOR+acu group)according to serum FSH level in the orbital blood.The control group and the DOR group did not receive any intervention,DOR+res group did not received handling and restriction as DOR+acu group did except acupuncture treatment.DOR+acu group received acupuncture at three acupoints:GV20,BL32 and CV4.The regimen of the treatment was once every other day,each time for 20 minutes,a total of 10 times acupuncture treatment.The status of the estrous cycle were monitored by daily vaginal smears,and rats at diestrus were selected when acupuncture treatment was finished.The concentration of GnRH content in the hypothalamus,FSH and LH content in pituitary tissue,cAMP content in ovarian tissue,and serum hormone levels were detected by ELISA.The relative content of FSHR protein in ovarian tissue was detected by Western Blot.The relative change in contents of FSHR mRNA and P450 mRNA in rat ovarian tissue were detected by real-time PCR.The frozen section-HE staining method was used to determine the morphological changes of the left ovary and Antal follicle count.ResultsExperiment 1:Establishment and validation of DOR animal model1.Estrous cycleThe irregular rate of estrous cycle in DOR group was significantly higher than that in control group(P<0.05).2.Serum hormone levelsCompared with control group,the serum FSH level of rats in the DOR group was significantly increased,the E2 and AMH level was significantly decreased(P<0.05).There was no significant difference in serum LH and INH-B(P>0.05)3.Antral follicle count(AFC)Compared with control group,AFC in rats of DOR group was significantly decreased(P<0.05).Experiment 2:Effects of acupuncture on HPO axis and FSH-cAMP signaling pathway in DOR rats1.The effect of acupuncture on the HPO axis in DOR rats(1)GnRH content in hypothalamus tissue:Compared with control group,GnRH levels in the hypothalamus tissue of rats in the DOR group were significantly increased(P<0.01).Compared with the DOR group,the GnRH levels in DOR+res group and DOR+acu group were significantly reduced(P<0.01).Compared with DOR+res group,the GnRH level in DOR+acu group was significantly reduced,and difference was statistically significant(P<0.05).(2)FSH and LH content in pituitary tissue:There were no significant difference in FSH and LH content in pituitary tissue between among the four groups of rats(P>0.05).(3)Acupuncture improves the ovarian reserve function of DOR rats:? The irregular rate of estrous cycle:Compared with control group,the irregular rate of estrous cycle in the DOR group was significantly higher(P<0.01).Compared with DOR group,the irregular rate in the DOR+res group had an increasing trend,and the difference was not statistically significant(P>0.05).Compared with DOR group,the irregular rate in DOR+acu group had a decreasing trend,and the difference was not statistically significant(P>0.05).Compared with DOR+res group,the irregular rate in DOR+auc group was significant decreased(P<0.05).? Morphological changes of the ovary:In the control group,the cortex of ovary section was more continuous,the granular cells in the follicles were closely arranged,the medulla was dense,and the blood vessels were intact.The cortical sections of the ovaries of DOR group were discontinuous,the arrangement of granular cells in the follicles was disordered,the medulla was sparse,and the vascular morphology was incomplete.In DOR+res group,cortical continuity in ovarian sections was poor,granular cell layer was poor,medulla was loose,and blood vessels were less distributed.In DOR+acu group,the ovary section cortex was more continuous,the ovarian granulosa cell layer increased,the medulla structure was less loose,and the blood vessels were less distributed? Antal follicle count(AFC):Compared with control group,the AFC in DOR group decreased significantly(P<0.05).But there was no significant difference in the number of sinus follicles between DOR,DOR+res and DOR+acu groups? Serum hormone levels:Compared with control group,the levels of serum FSH and LH in DOR group were significantly increased and the levels of AMH and E 2 were significantly reduced(P<0.05),but there was no significant difference in INH-B level(P>0.05).Compared with DOR group,there was no significant difference in serum levels of 5 hormones in DOR+res group(P>0.05).Compared with DOR group,serum FSH levels decreased significantly and E2 and AMH levels were significant increased in DOR+acu group(P<0.05),and the other 2 hormone levels were not significantly different(P>0.05)Compared with DOR+res group,there was no significant difference in serum levels of 5 hormones in DOR+acu group(P>0.05)2.Effects of acupuncture on FSH-cAMP signaling pathway in D OR rats(1)Relative level of expression of FSHR in ovarian tissue:Compared with the control group,the relative level of expression of FSHR protein in the ovarian tissue of the DOR group was significantly reduced(P<0.01).Compared with DOR group,there was no significant difference in relative level of expression of FSHR protein in the ovarian tissue of DOR+res group(P>0.05).Compared with the DOR group and DOR+res group,the relative level of expression of FSHR protein in the rat ovarian tissue of the DOR+acu group was significantly increased(P<0.01).(2)Relative expression levels of FSHR mRNA and P450 mRNA in ovarian tissue:? Relative expression levels of FSHR mRNA in ovary tissue:Compared with control group,the relative level of expression of FSHR mRNA of DOR group was significantly decreased(P<0.01).Compared with DOR group,there was no significant difference in the relative expression FSHR mRNA in DOR+res group(P>0.05).Compared with DOR group,the relative level of expression of FSHR mRNA DOR+acu group was significantly increased(P<0.05).Compared with DOR+res group,there was no significant difference in the relative expression of ovarian tissue FSHR mRNA in DOR+acu group(P>0.05)? Relative expression levels of P450 mRNA in ovary tissue:Compared with control group,the relative level of expression of P450 mRNA of DOR group was significantly decreased(P<0.01).Compared with DOR group,the relative expression P450 mRNA in DOR+res group and DOR+acu group were significantly increased(P<0.01).Compared with DOR+res group,there was no significant difference in the relative expression of ovarian tissue P450 mRNA in DOR+acu group(P>0.05).(3)cAMP content in ovarian tissue:Compared with control group,the cAMP content in the DOR group was significantly reduced(P<0.01).Compared with DOR group,there was no significant difference in the cAMP content of ovarian tissue in DOR+res group(P>0.05).Compared with DOR group and DOR+res group,the cAMP of ovarian tissue in DOR+acu group was increased significantly(P<0.01).Conclusion1.Compared with three-month-old female SD rats,the ovarian reserve function of nine-month-old female retired breeding rats was significantly diminished,thus can be used as a DOR animal model.2.Acupuncture can reduce the GnRH content of hypothalamic tissue in DOR rats.3.Acupuncture could improve ovarian reserve function in DOR rats to some extent by adjusting the disturbed emotional cycle and abnormal sex hormone levels.4.Signal transduction pathway FSH-cAMP of ovarian granulosa cells may be involved in the regulation of ovarian reserve function in DOR rats by acupuncture treatment and more experimental evidence is needed.