Mechanism of melatonin inhibiting chronic stress-induced follicular dysplasia in mice by inhibiting the PTEN/AKT/FOXO3a pathway
Objective To explore the mechanism of melatonin inhibiting chronic stress-induced follicular dysplasia by inhibiting the activation of PTEN/AKT/FOXO3a pathway in ovaries of mice.Method Sixty 6-week-old female C57BL/6N mice were randomly divided into normal control group,chronic stress group,and melatonin group,20 mouse in each group.The body mass and ovarian mass of the mice were recorded using a laboratory balance.Enzyme-linked immunosorbent assay(ELISA)kits were used to measure the serum hormone levels in mice.Histological analysis was performed to determine the number of follicles at different stages in ovaries of mice.ELISA and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression of anti-Müllerian hormone(AMH)in serum of mice.Western blotting analysis was conducted to examine the expression of the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)/Forkhead box transcription factor 3a(FOXO3a)signaling pathway.PCR was used to measure the transcription levels of PI3K/AKT/FOXO3a in ovaries of mice.Statistical methods were used for LSD t-test,x2 tests,one-way analysis of variance(ANOVA)or repeated measurement data analysis of variance.Result Chronic stress group were higher than those with normal control group in number of primary follicles(174±30 vs 107±17,t=-148.098),p-phosphatase and tensin homolog deleted on chromosome ten(p-PTEN)proteins(2.03±0.19 vs 1.26±0.16,t=-32.207),p-AKT proteins(1.99±0.18 vs 1.07±0.05,t=-70.021),p-FOXO3a proteins(2.18±0.20 vs 1.18±0.11,t=-64.621),cortistatin(CORT)(137±12 vs 83±7,t=-124.235),follicle-stimulating hormone(FSH)[(13.1±1.9)IU/L vs(8.2±1.6)IU/L,t=-25.388](all P＜0.001).Chronic stress group were lower than those with normal control group in body mass[(22.5±2.5)g vs(28.4±4.7)g,t=12.906],ovarian mass[(9±3)mg vs(23±5)mg,t=45.302],estradiol levels[(36±8)μg/L vs(75±10)μg/L,t=88.937],androgen levels[(0.13±0.01)μg/L vs(0.20±0.02)μg/L,t=23.123],luteinizing hormone(LH)levels[(3.2±0.3)IU/L vs(4.6±0.5)IU/L,t=31.170]and the number of primary follicles(87±9 vs 143±27,t=1 11.829),secondary follicles(92±11 vs 150±21,t=130.837)and sinus follicles(43±5 vs 96±8,t=144.851)(all P＜0.001).Melatonin group were lower than those with chronic stress group in p-PTEN/PTEN(1.15±0.14 vs 2.03±0.19,t=4.983),p-AKT/AKT(1.21±0.13 vs 1.99±0.18,t=-12.108),p-FOXO3a/FOXO3a(1.35±0.17 vs 2.18±0.20,t=-11.274),CORT[(106±10)μg/L vs(137±12)μg/L,t=-50.392)and FSH levels[(10.2±1.5)IU/L vs(13.1±1.9)IU/L,t=-10.317)](all P＜0.001).Melatonin group were higher than those with chronic stress group in body mass of mice[(26.5±4.1)g vs(22.5±2.5)g,t=5.182],ovarian mass[(17±5)mg vs(9±3)mg,t=19.588],estradiol levels[(66±6)μg/L vs(36±8)μg/L,t=20.485],androgen[(0.21±0.02)μg/L vs(0.13±0.01)μg/L,t=6.458],LH levels[(4.3±0.4)IU/L vs(3.2±0.3)IU/L,t=6.924]and the number of primary follicles[(125±15)vs(87±9),t=38.784],secondary follicles[137±16 vs 92±11,t=29.063]and sinus follicles[76±6 vs 43±5,t=49.447](all P＜0.001).Conclusion Melatonin inhibits the phosphorylation of PTEN/AKT/FOXO3a pathway members,increases the serum AMH levels in mice,and ultimately alleviates the loss of primordial follicles and follicular dysplasia in the ovaries of mice induced by chronic stress.
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