Study On The Effect And Mechanism Of Psychological Stress On The Estrous Cycle Of Female Rats

Objective:Due to the long-term high-intensity training and the impact of psychological stress before and after the competition,female athletes often have exercise-induced menstrual disorders.A large number of clinical data show that physical and psychological stress can inhibit or damage female reproductive endocrine function at multiple levels in the HPO axis,resulting in symptoms such as sub-health,clinical gynecological diseases and premature senility.In this paper,the effects of psychological stress on the estrous cycle of female rats and the related mechanism of HPO axis were studied by establishing psychological stress models in different time periods,in order to improve it.Methods:Eighty female rats with two consecutive estrous cycles were selected into the experiment.Eighty female SD rats were randomly divided into 8 groups:control group（0.5,1,2,3 months）and psychological stress group（0.5,1,2,3 months）with 10 rats in each group.The experimental group was placed in the retainer to immobilize stress once a day（9:00 to 15:00）.The female rats were weighed 6 hours a day and weighed every week.The estrous cycle of female rats was observed by vaginal exfoliated cell smear every day.After restraint stress,3%pentobarbital sodium was injected into abdominal cavity according to 40mg/kg during estrus.Blood was taken from abdominal aorta.Blood leukocyte were detected by automatic blood cell analyzer,blood biochemical indexes were detected by automatic biochemical analyzer,and serum inflammatory factors,oxidative stress response,sex hormones,stress hormones,neurotransmitters and hormones related to ovarian reserve function were detected by ELISA.The hypothalamus,pituitary,ovary and uterus were taken and weighed to calculate the organ coefficient.The hypothalamic GnRH hormone was detected by radioimmunoassay.The pathological changes of ovary and uterus were observed by HE staining and the thickness of endometrium was calculated.The levels of tissue inflammatory factors and oxidative stress were detected by ELISA.Hypothalamic GnRH,Kiss-1 and GPR54 mRNA;pituitary GnRHR,FSH and LH mRNA;ovary FSHR,LHR and ERαmRNA;were measured by PCR.The relative expression level of GnRHR and ERαmRNA in uterus.Proteomics and transcriptome were used to analyze the effects of psychological stress on the expression of proteins and mRNA in ovary,and the up-and down-regulated mRNA and proteins in four periods of psychological stress were screened and verified by real-time fluorescence quantitative PCR,WB and ELISA.Results:1.Effects of psychological stress on body weight and estrous cycle.The body weight of rats slowed down from 1 week of restraint（P<0.01）,and the disorder of estrous cycle appeared after 2 weeks of restraint,and the disorder rate gradually increased with the prolongation of restraint stress time,mainly characterized by prolonged interestrus.2.Changes in blood biochemical indicators caused by psychological stress.Compared with the control group,0.5 and 1 month of restraint resulted in neutrophil decline and monocyte increase（all P<0.05）;ALT,AST,and ALP activities increased significantly（all P<0.01）,suggesting abnormal liver function;LDL significantly decreased when restrained for 0.5 months（P<0.05）,CHO and HDL contents significantly decreased when restrained for 2 months（all P<0.01）;serum IL-1βcaused by psychological stress,TNF-α,IL-6 and MDA levels increased significantly（all P<0.01）.3.Psychological stress causes changes in hormone levels.Compared with the control group,the GnRH hormone levels in the hypothalamus decreased significantly（P<0.01）;the blood neurotransmitters ACH and DA levels decreased significantly（all P<0.01）,and 5-HT and Gly levels increased significantly（all P<0.01）;the levels of CORT,NE,ACTH and FSH in blood increased significantly,while the levels of LH,T,P,E₂ and PRL decreased significantly（all P<0.01）.4.Effects of psychological stress on ovary and uterus.Pathological examination showed that psychological stress could lead to loose medulla structure,obvious hyperemia,decrease of follicles at all levels,infiltration of inflammatory cells in uterine tissue,and obvious infiltration of inflammatory cells in uterine tissue,and the levels of AMH and INHB and the thickness of endometrium decreased significantly（all P<001）.The levels of IL-1β,TNF-αand MDA in ovarian and uterine tissues increased significantly,while the levels of SOD and GSH decreased significantly.5.Effects of psychological stress on important genes in hypothalamus-pituitary-ovary axis.The levels of GnRH,GPR54 and Kiss-1 mRNA in hypothalamus increased at first and then decreased;the levels of GnRHR and FSH mRNA in pituitary increased at first and then decreased,while LH mRNA decreased at first and then increased;the levels of FSHR and LHR mRNA in ovary were significantly higher than those in the control group,ERαmRNA increased at first and then decreased,and the levels of GnRHR and ERαmRNA in uterus decreased at first and then increased.6.Psychological stress leads to changes in ovarian transcriptome and proteome.1700differentially expressed proteins were screened,which mainly played a role in the process of small molecular catabolism,mitochondrial matrix and coenzyme binding,and participated in fatty acid metabolism,branched chain amino acid catabolism,steroid metabolism and neutrophil degranulation.1216 differential mRNA mainly play a role in the process of development,positive regulation of biological process and phosphorus metabolism,and participate in the pathways of metabolism,ovarian steroid synthesis and steroid hormone biosynthesis.Joint analysis showed that the mRNA and protein of Fabp6,Acat1,Igh-6 and Nt5e were differentially expressed in different stress groups.Conclusion:1.Psychological stress can reduce the body weight and prolong the estrous cycle,especially the interestrus of female rats.2.Psychological stress can lead to changes in the indexes of HPA axis and hormones in HPO axis,which may disrupt the estrous cycle by interfering with the endocrine regulation of HPA axis and HPO axis.3.Psychological stress can lead to changes in the organ coefficients of ovary and uterus in female rats,and damage in varying degrees.Ovary and uterus are the target organs.4.Psychological stress changes the mRNA expression of GnRH,Kiss-1,GPR54,GnRHR,FSH,LH,FSHR,LHR and ERαin hypothalamus-pituitary-ovary axis of pubertal female rats.It is suggested that psychological stress may interfere with the endocrine regulation of hypothalamus-pituitary-ovary axis by affecting the expression level of these genes,thus having adverse effects on gonadal development and reproductive function.It can also affect the reproductive function of uterus by affecting the expression of ERαand GnRHR.5.Psychological stress leads to the change of mRNA and protein expression profile of ovary.Fabp6,Acat1,Igh-6 and Nt5e may play a role in ovarian injury induced by psychological stress.