| Background: Ageing is a key factor of the female infertility.With the increase of age,female ovarian function declines,which leads to the diminished ovarian reserve(DOR),dysfunction of primary follicles activation and another symptoms,blocking the first recruitment and development of follicles,resulting in the female infertility.In vitro activation(IVA)technology is to stimulate the resting primary follicles in ovarian tissues in vitro by physical and chemical methods,so that they have the potential to continue to develop be mature follicles.In the development follicle IVA technology on ovarian tissues,PTEN-PI3 K signaling pathway is one of the most critical signaling pathways to activate primordial follicles.In recent years,IVA technology has been proved to be able to treat patients with ovarian insufficiency(POI)(age < 40 years old),by activating the primary follicles in the ovarian tissues of patients with POI to continue to develop.However,there is no research on whether IVA technology can also activate and even promote the development of the primary follicles in those ovarian tissues of DOR patients due to the older age.Objective: Primordial follicles of elderly mice or women ovarian tissues were cultured in vitro and by activating the PTEN-PI3 K signaling pathway,to investigate the effects on the development of follicles in elderly mice or women.Methods: Collectd fresh ovarian tissues of ICR mice(40 weeks old)and thawed ovarian tissues of elderly women(40-49 years old),and divided them into activation group(IVA)and control group(CON)for subsequent culturing in vitro.In the activated group,the PI3 K stimulator 740Y-P(150 μg/ml)and the PTEN inhibitor bp V(HOpic)(30 μM)were added to the ovarian tissues’ culture medium for 24 hours,and then the PI3 K stimulator 740Y-P(150 μg/ml)was added alone for another 24 hours.While in the control group,the same volumn of dd H2 O was added to the culture medium of the ovarian tissues.The ovarian tissues were collected on the test day(D0),the second day(D2),and the sixth day(D6)for embedding,slicing or extracting total proteins.Observing the morphology of primordial follicles,primary follicles and secondary follicles by HE staining,counting and analyzing the proportions of the follicles at each stage.The immunohistochemistry was performed to observe the amount of the dormancy factor FOXO3 a protein,a downstream of PTEN-PI3 K,moving out from the oocyte nucleus,and analyze the corresponding follicles proportion of those FOXO3 a in the activated group and the control group,as well as detecting the protein expression of GDF9 in oocytes.Used WB to test the protein expression of the p-PTEN/PTEN and p-Akt/Akt in the PTEN-PI3 K signaling pathway.The ovarian tissues of elder patients were transplanted to the latissimus dorsi of nude mice to observe the growth of the activated ovarian tissue and the development of follicles.Result:IVA of ovarian tissue in aged mice: 1.On the second day after IVA,the proportion of primordial follicles in the activated group was lower than that in the control group,with a significant difference(P<0.001);the proportion of primary follicles in the activated group was higher than that in the control group,with a significant difference(P<0.01).On the 6th day after IVA,the proportion of primordial follicles in the activated group was also lower than that in the control group,with a significant difference(P<0.01);the proportion of primary follicles in the activated group was also higher than that in the control group,with a significant difference(P<0.05).For the secondary follicles,there was no significant difference between the two groups on the second day and the sixth day(P>0.05,P>0.05),but the proportion of activated group was higher than that of the control group.2.The follicles proportion of the dormancy factor FOXO3 a protein moving out from the oocyte nucleus in the activated group was higher than that in the control group,with a significant difference(P<0.05).3.The p-Akt protein expression level of the activated group on the 2nd and 6th day were higher than that of the control group,with significant difference(P<0.05,P<0.05);The p-PTEN protein expression level of the activated group on day 2 and day 6 were also higher than that of the control group,with significant differences(P<0.05,P<0.01).IVA of ovarian tissue in elder women: 1.On the second day after IVA,the proportion of primordial follicles in the activated group was lower than that in the control group,with a significant difference(P<0.001);the proportion of primary follicles in the activated group was higher than that in the control group,with a significant difference(P<0.001).On the 6th day after IVA,the proportion of primordial follicles in the activated group was also lower than that in the control group,with a significant difference(P<0.01);the proportion of primary follicles in the activated group was also higher than that in the control group,with a significant difference(P<0.05).For the secondary follicles,there was no significant difference between the two groups on the second day and the sixth day(P>0.05,P>0.05),but the proportion of activated group was higher than that of the control group,especially on the 6th day.2.The expression of the specific expression factor GDF9 protein can be detected in the oocytes of primordial follicles and primary follicles in the activated group,and the granulosa cells of follicles developed from flat fusiform to cubic shape.3.There is no significant difference in the increase of p-Akt protein expression level of the activated group on day 2 and day 6 compared with the control group(P>0.05,P>0.05),but the expression level of p-Akt in the activated group has an increasing trend;and the protein expression level of p-PTEN was no significantly increased in protein expression level of the activated group on the 2nd and 6th day compared with the control group(P>0.05,P>0.05),while the expression level of p-PTEN of the activated group also had an increasing trend.The reason for the insignificant difference may be that the ovarian reserve of the elderly is decreased,which leads to the decrease of the expression of follicle protein in the total protein of the tissue,so that the increasing trend of the expression of p-Akt and p-PTEN protein in the activated group is not obvious.4.Two weeks after allogeneic transplantation,new blood vessels and intact follicles can be observed in both the control group and the activated group.Two months later,the ovarian tissue in the control group shrank significantly.The weight of the ovarian tissue in the activated group was significantly greater than that in the control group,with significant difference(P<0.01).And the growth of antral follicles was found in the ovarian tissue of the activated group.Conclusions: 1.In vitro addition of PI3 K stimulator 740Y-P and PTEN inhibitor bp V(hopic)can activate primordial follicles in ovarian tissues of fresh in elder mice(40weeks)and thawed in elder women(40-49 years).2.In vitro activation of primordial follicles in the aged ovarian may be mediated by PTEN-PI3 K signaling pathway. |
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