The Molecular Mechanism Of Ovarian Apoptosis Induced By Restraint Stress

Restraint is an experimental procedure developed for biomedical studies of stress, primarily in connection with''psychogenic''ulcer induction.''Psychogenic''or''psychosomatic''refers to the implication that no invasive physical procedure or tissue trauma is involved, but rather the stress response is initiated in the brain in response to the psychological distress of being unable to move freely. The hypothalamic–pituitary–adrenal (HPA) axis, when activated by stress, exerts an inhibitory effect on the female reproductive system.In our study, a restraint system was established to allow mice intake of food and water freely in case of additional effects of hunger and thirsty, and at the same time restrain their movement to ensure that mice could move back and forth to some extent, but could not turn around while in the micro-cage. The period of restraint stress lasted 24 hours, ranging from 24-48h(0 is the time for PMSG injection). Effects of restraint stress conducted following gonadotropin stimulation on apoptosis, estradiol secretion, expression of CRHR1 mRNA, IGF-1 mRNA in intact ovary ,unintact ovary, MGC and COCs was investigated. The results indicated that: (ⅰ) Compared with control group, stress decreased the expression of Bcl2/1 mRNA in intact ovary and MGC significantly ,although there is no difference in the expression of Bax or Caspase3. The apoptosis of cumulus received from GV stage COCs is not different between stress group and control group, the same as the COCs matured in maturing culture medium for 14 h, while COCs matured in incomplete culture medium ( lack of hormones and serum in the maturing culture medium), the apoptosis of cumulus received from stress group is higher than that received from control group( Hoechst 33342 staining), which is strengthed by the increase in the expression of caspase3 and bax mRNA in the stress group, suggesting that restraint stress can induce the initiation of apoptosis.(ⅱ)stress decreased ovarian and plasma estradiol secretion compared with control group,which accounted for the initiation of apoptosis.(ⅲ)stress decreased the expression of IGF-1 mRNA in MGC , which might not only partly account for the decrease in estradiol in plasma and ovary, but also contributed to the apoptosis related gene-Bcl2/1 mRNA decline in stress group. Conversely, expression of CRHR1 mRNA in ovary increased,which may inhibit the production of testone , subsequently inhibit the production of estradiol indirectly, thus the increasement of CRHR1 mRNA in ovary may be also responsible for the decrease in estradiol in plasma and ovary. (ⅳ) stress increased expression of CRHR1 mRNA in the cumulus cells compared with control group, which may be responsible for the Apoptosis increased in stress group when matured in incomplete culture medium.In conclusion, we took use of this self-established syestem to prove that restraint stress applied to the female during follicular maturation induced by gonadotropin stimulation affected the expression of IGF-1 and CRHR1 mRNA, which is the mechanism by which stress affected the estradiol level and induced the initiation of apoptosis, this will help us to figure out the mechanisms of action of stress on female reproductive system.