Corticosterone Regulates The Development Of Mouse Ovarian Follicles And The Expression Of LHR In Ovarian Granulosa Cells

In modern intensive livestock and poultry production,stress is an important cause of female animal follicle development disorders,leading to low fertility.Studies have shown that stress-induced follicular dysplasia may be related to glucocorticoids produced by stress.The main component of glucocorticoids is cortisol(cortisol,cortisol in non-blocking dentate mammals)or corticosterone(corticosterone,corticosterone in some dentates),which is referred to herein as CORT.According to reports,the body encounters stress and causes a sharp increase in CORT content,which affects the secretion of luteinizing hormone(LH).As a result,the ovarian follicles lose the support of LH,which can support the development of follicles.On the other hand,when the body is under stress,the reduced binding of LH to the LH receptor(LHR)on the surface of granulosa cells(GCs)leads to the inhibition of its biological function and reduced number of ovulations.Studies had shown that the biological effects of CORT can be achieved by binding to glucocorticoid reporter(GR)and can activate downstream target genes’ expression.So,the issue is that whether the ovulation disorder caused by CORT related to the regulation of LHR expression in GCs?In order to prove this idea,this article chiefly carried out the following research contents:(1)After continuous injection of CORT,mice were tested for body weight,ovarian weight,ovarian index,and follicular development at different developmental stages during the same period of estrus;(2)Through the hormone ovulation method to clarify the effect of increased amount of CORT in vivo on ovulation;(3)By detecting the expression of estradiol and progesterone and their synthetase genes at different periods of follicular development to further clarify the relationship between CORT and female reproductive hormone secretion;(4)To explore the effect of CORT on the expression of LHR through the in vivo injection of CORT experiment in vivo and the culture of ovarian GCs in vitro;(5)By inhibiting the function of GR to explore the pathway of CORT on LHR inhibition;(6)By detecting the changes in the expression levels of LHR-related transcription factors CREB and AP1 to further clarify the mechanism of CORT regulating LHR expression in ovarian GCs cultured.The specific test results are as follows:1.Inhibitory of ovulation by continuous injection of CORT in miceContinuous injection of CORT into mice was used to construct a stress model in vivo.The results showed that after injecting CORT into superovulated mice,the number of oocytes decreased,but the ovulation rate of mice did not change significantly.This indicates that if stress is given,exogenous LH/hCG would not inhibit the ovulation process in mice and the decrease in oocytes number may be due to the inhibitory effect of CORT on the development of mature follicles.2.Inhibitory of ovarian gain and ovarian follicular development by Continuous injection of CORT in miceContinuous injection of CORT reduced the body weight,ovarian weight and ovarian index of mice.At the same time,HE staining results showed that CORT had the most obvious inhibitory effect on the development of secondary follicles and mature follicles.This result indicates that continuous injection of CORT can cause growth and ovarian development in mice to be blocked.3.Effect of CORT injection on the transcription level of steroid hormones and steroid synthase genes in miceEstradiol and progesterone are important hormones that regulate follicular development,ovulation and pregnancy in animals.We tested the levels of progesterone and estradiol in mouse plasma and the level of steroid synthase gene transcription in ovarian GCs.The results showed that after CORT injection,the transcription level of the steroid synthase gene in ovarian GCs showed relatively consistent changes with plasma estradiol and progesterone levels.This indicates that CORT may down-regulate peripheral estradiol and progesterone levels by inhibiting follicular development and suppressing the expression of steroid synthase-related genes in GCs.4.Inhibitory of LHR expression in MGCs by Continuous injection of CORTTo investigate whether the surge of CORT caused by stress will inhibit the expression of LHR in mouse ovarian granulosa cells.We detected the expression of LHR in mice from the CORT injection group and granulocytes cultured in vitro,and found that LHR transcription and translation were inhibited by CORT.This indicates that CORT may be a key regulator of LHR expression under stress.5.The inhibitory effect of CORT on the expression of LHR depends on GRCORT needs to bind to its receptor to play a role,so we explored the relationship between CORT and GR.We had confirmed in vivo and in vitro experiments that CORT upregulates GR expression.Furthermore,the addition of RU486(GR inhibitor)to GCs in vitro can block the down-regulation of LHR expression caused by CORT,suggesting that GR pathway may be involved in the inhibition of LHR expression by granule cells by CORT.6.Inhibitory CREB and AP1 expression by GR activationAfter activation,GR can enter the nucleus and interacts with the promoter of the target gene,thereby enhancing or inhibiting genes transcription.We therefore selected two classical transcription factors CREB and AP1 of LHR to check whether their expression is regulated by GR.Through Western blot and qRT-PCR,we found that CORT inhibited the expression of CREB and the expression of two subunits of AP1 including c-Fos and c-Jun.When GR inhibitor RU486 blocked GR activity,the expression levels of CREB,p-CREB,c-Fos and c-Jun expression levels were restored.This result indicates that CORT can suppress the expression of LHR by inhibiting the expression of the transcription factors CREB and AP1.In summary,this article used CORT intraperitoneal injection of mice to simulate the stress response under physiological conditions,and established a stress model with simple operation and stable effect.At the same time,we found that CORT can inhibit ovulation in female follicles and can reduce the level of synthesis related steroid hormone.In addition,the results also showed that CORT can inhibit the expression of CREB and AP1 as LHR transcripts by activating GR,which ultimately led to downregulation of LHR expression.This may be one of the reasons why stress can cause a decrease in ovulation in mice.The above research results reveal the potential molecular mechanism of stress affecting follicular development and ovulation,and provide new clues to explain the reasons for the decline in litter performance of animals under stress.