This study examined the role of CRH-induced ovarian cell apoptosis in the restraint stress (RS)-induced impairment of oocyte competence. Oocyte percentages of apoptotic cumulus cells (CCs) did not differ between stressed and control mice before in vitro maturation (IVM) but became significantly higher in stressed mice after IVM without serum, growth factor, and hormone. The level of Bcl2 mRNA decreased significantly in mural granulosa cells (MGCs) and ovarian homogenates after RS. Whereas ovarian estradiol, testosterone, and IGF1 decreased, cortisol and progesterone increased significantly following RS. RS increased the level of CRH in serum, ovary, and oocyte while enhancing the expression of CRHR1 in CCs, MGCs, and thecal cells. RS down-regulated ovarian expression of glucocorticoid receptor and brain-derived neurotrophic factor. Furthermore, CRH supplementation to IVM medium impaired oocyte developmental potential while increasing apoptotic CCs, an effect that was completely overcome by addition of the CRHR1 antagonist antalarmin. Results suggest that RS impaired oocyte competence by increasing CRH but not glucocorticoids. Increased CRH initiated a latent apoptotic program in CCs and oocytes during their intraovarian development, which was executed later during IVM to impair oocyte competence. Thus, elevated CRH interacted with increased CRHR1 on thecal cells and MGCs, reducing the production of testosterone, estrogen, and IGF1 while increasing the level of progesterone. The imbalance between estrogen and progesterone and the decreased availability of growth factors triggered apoptosis of MGCs and facilitated CC expression of CRHR1, which interacted with the oocyte-derived CRH later during IVM to induce CC apoptosis and reduce oocyte competence.

Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr2+ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.