Transgenerational Genetic Effects And Programming Mechanism Of Estrogen-synthesis Inhibition In Ovary Of Offspring Rats Induced By Prenatal Dexamethasone Exposure

Objective Dexamethasone has been routinely administered to pregnant women who have reached the preterm birth period of 24 weeks but less than 34 weeks.Epidemiological investigation found that the more dexamethasone treatments,the more obvious weight loss of newborns,which was closely related to the occurrence of various chronic diseases in adulthood.Animal experiments showed that prenatal dexamethasone exposure（PDE）could lead to abnormal reproductive function and reduced ovarian reserve in female offspring.Our previous research also found that PDE increased ovarian apoptosis in female fetal rats and caused abnormal follicular development postnatally,which could even exist in F3 generation.This study intends to establish a PDE rat model to observe the ovarian estrogen synthesis function,follicular development changes and their transgenerational inheritance effects in the female offspring,and further confirm the intrauterine programming mechanism through the intervention experiments of animals,in vitro organs and cells.Methods From GD9 to GD20,pregnant rats were injected subcutaneously with 0.2mg/kg dexamethasone once a day,and the control rats were mock-treated with the same volume of carrier（saline solution）.At GD20,some pregnant rats were randomly selected and sacrificed by anesthesia with isoflurane.Pregnant rats with a pup size of 10 to 14（male/female ratio of approximately 1:1）were considered eligible（n=15）,and female pups were collected for blood and ovaries.Pups were randomly selected from each litter for subsequent analysis.Fetal blood samples were collected and serum was separated.For morphological purposes,the fetal ovaries were randomly selected for 24 h in 4%paraformaldehyde,then dehydrated in ethanol and embedded in paraffin.The remaining ovaries were immediately frozen in liquid nitrogen and stored at-80°C for further analysis.Radioimmunoassay was used to detect serum estradiol（E₂）levels,and hematoxylin-eosin staining was used to observe ovarian morphology.Detection of glucocorticoid receptor（GR）,Runt related transcription factor 2（RUNX2）and cytochrome P450 aromatase（P450arom）gene/protein levels in ovarian tissue,and the expression of mi RNA320a-3p in ovarian tissues and oocytes.By using rat fetal ovary tissue and human granulosa-like tumor cell line（KGN）cultured in vitro,we treated with 500 n M dexamethasone for 72 h,and further intervened the key molecules in the regulatory pathway（GR/mi RNA320a-3p/RUNX2）and then detected the corresponding indicators as animal experiments.Results Animal experiments:（1）Compared with the control group,PDE can reduce the serum E₂ level and inhibit the m RNA expression of P450arom in F1 generation of female adult offspring rats（P<0.01）;Immunohistochemical staining showed that P450arom was mainly expressed in ovarian granulosa cells and presented as yellow granules.P450arom positive cells in F1 generation of PDE offspring were significantly reduced,while quantitative analysis found that P450arom protein expression was also significantly reduced（P<0.01）.At the same time,the number of secondary ovarian follicles was decreased,and the number of antral follicles was increased（P<0.05,P<0.01）in PDE group.It was further found that the F2generation of PDE female offspring still showed significantly decreased serum E₂ level and P450arom m RNA&protein expression,markedly decreased ovarian sinus follicles and increased atresia follicles（P<0.05,P<0.01）.In addition,serum E₂ level,P450arom m RNA&protein expression were significantly decreased in F3 generation of PDE group,as well as the decreased original ovarian follicles and increased atresia follicles（P<0.05,P<0.01）.（2）Compared with the control group,PDE inhibited the expression of mi RNA320a-3p in F1 female offspring（P<0.05）,and up-regulate RUNX2 m RNA expression（P<0.05）.Immunohistochemical staining showed that RUNX2 was mainly expressed in ovarian granulosa cells,presenting yellow particles,the number of RUNX2 positive cells in F1generation of PDE female offspring was significantly increased.At the same time,quantitative analysis of immunohistochemistry revealed that protein expression level of RUNX2 in ovary of PDE group was significantly increased（P<0.05）.Further chromatin immunoprecipitation revealed that the combination of RUNX2 and P450arom promoter region in the ovaries of F1generation of PDE group was increased（P<0.01）.We further found that F2 generation of the PDE female offspring still showed inhibited mi RNA320a-3p expression,significantly increased RUNX2 m RNA and protein expression,and increased binding of RUNX2 to P450arom promoter region（P<0.05,P<0.01）.In addition,the F3 generation of the PDE female offspring continued to present inhibited mi RNA320a-3p expression,and increased RUNX2 m RNA and protein expression,as well as the increased increased binding of RUNX2 to the P450arom promoter region.（P<0.05,P<0.01）.（3）Compared with the control group,the number of oocytes in the ovary of the PDE fetal rats did not change significantly,but the maximum cross-sectional area of the ovary decreased（P<0.05）,and the m RNA expression of P450arom in the ovary of the PDE group was suppressed（P<0.01）.The results of immunohistochemistry and quantitative analysis also showed that P450arom protein expression was reduced（P<0.05）,and serum E₂ level was significantly reduced（P<0.05）.We further examined the expression of GR,mi RNA320a-3p and RUNX2 in fetal rat ovary.Compared with the control group,GR m RNA expression in fetal ovary of PDE group was significantly increased（P<0.05）,while mi RNA320a-3p expression was down-regulated（P<0.05）and RUNX2 m RNA expression was increased（P<0.01）.The results of immunohistochemistry and quantitative analysis also showed increased expression of RUNX2 protein（P<0.05）,accompanied by increased binding of RUNX2 to the P450arom promoter region（P<0.05）.In vitro culture of fetal rat ovary:To directly mimic the effects of PDE on fetal ovaries,we dissected the ovaries from GD20 fetal rat for in vitro organ culture and treated with dexamethasone.In vitro culture for 3 days had no significant effect on the morphology and apoptosis of the fetal ovary.After treating with 500 n M dexamethasone for 72 hours,GR m RNA expression in fetal ovary increased significantly,mi RNA320a-3p expression was suppressed but RUNX2 m RNA expression was increased,and P450arom m RNA expression was significantly down-regulated（P<0.05,P<0.01）.After treating with mi RNA320a-3p mimics,the mi RNA320a-3p expression in fetal ovary was significantly increased,and it could reverse the inhibition of m RNA expression of RUNX2and P450arom induced by dexamethasone（P<0.05,P<0.01）.Immunohistochemistry and quantitative analysis also showed that mi RNA320a-3p mimics could reverse the effect of dexamethasone on the expression of RUNX2 and P450arom proteins in the fetal ovary（P<0.05,P<0.01）.Further GR intervention results showed that,compared with the control group,after treating with RU486,mi RNA320a-3p expression was up-regulated in fetal ovarian and could reverse the inhibition of m RNA expression of RUNX2 and P450arom induced by dexamethasone（P<0.05,P<0.01）.Immunohistochemistry and quantitative analysis also showed that RU486 could reverse the effect of dexamethasone on the expression of RUNX2and P450arom proteins in the fetal ovary（P<0.05,P<0.01）.In vitro culture of human cell line:Compared with the control group,treatment with500 n M dexamethasone for 72 h caused increased GR nuclear translocation in ovarian granulosa cells（P<0.05）,down-regulated mi RNA320a-3p expression（P<0.01）,increased RUNX2expression as well as inhibited P450arom expression（P<0.05）.Overexpression of mi RNA320a-3p was induced by plasmid transfection of mi RNA320a-3p into KGN human ovarian granulosa cell line,the expression of mi RNA320a-3p was significantly increased（P<0.05）and reversed the effect of dexamethasone on RUNX2 and P450arom m RNA expression in KGN cell line（P<0.05）.Immunohistochemistry and quantitative analysis also showed that mi RNA320a-3p mimics could reverse the effect of dexamethasone on RUNX2 and P450arom protein expression（P<0.05,P<0.01）.The result showed that the group with co-transfected mi RNA320a-3p mimics with luciferase vectors bearing RUNX2 m RNA 3′-UTR target sequences showed the lowest luciferase activity.The luciferase activity was not significantly different between co-transfected control and mi RNA320a-3p mimics with luciferase vectors bearing the mutated RUNX2 m RNA 3′-UTR group,it was suggested that mi RNA320a-3p is indeed binding to 3′UTR of RUNX2 m RNA（P<0.05,P<0.01）.Further GR intervention experiment showed that,after intervention with RU486,the expression of mi RNA320a-3p was up-regulated in KGN cell lines,and it could reverse the influence of RUNX2 m RNA expression and P450arom m RNA expression caused by dexamethasone（P<0.05,P<0.01）.It also showed that RU486 could reverse the effect of dexamethasone on the protein expression of RUNX2 and P450arom（P<0.05,P<0.01）.Further,the binding experiment of RUNX2 and P450arom showed that dexamethasone caused increased binding of RUNX2 to P450arom by increasing RUNX2（P<0.01）.After the administration of RUNX2si RNA,the expression of P450arom increased（P<0.05,P<0.01）.Oocytes experiment:Compared with the control group,the expression of mi RNA320a-3p in oocytes of both PDE F1 and F2 generation were consistent inhibited（P<0.01）,and consistent with the results of ovarian expression in vivo and in vitro,as well as in KGN cell line.It was suggested that the inhibition of mi RNA320a-3p expression induced by PDE in maternal germ cells is stable.Furthermore,we examined the methylation status of Cp G sites in the mi RNA320a-3p promoter region of fetal rat ovary.The results showed that compared with the control group,the methylation modification of several Cp G site in the promoter region of mi RNA320a-3p was changed in the PDE group,but showed no significance.Conclusion:PDE could activate GR,which targeted up-regulation of RUNX2 expression by inhibiting the mi RNA320a-3p expression,and further inhibited estrogen synthesis function by down-regulating the P450arom in fetal ovaries.PDE-induced ovarian mi RNA320a-3p inhibition could affect F2 generation through the oocytes of F1 generation.Due to its genetic stability,F2 generation oocytes indirectly exposed to dexamethasone also showed inhibition of mi RNA320a-3p expression and were inherited to F3 generation.The cascade effect of mi RNA320a-3p/RUNX2/P450arom finally resulted in the transgenerational inheritance effect that inhibited the ovarian estrogen synthesis function of PDE offspring.