慢性应激对卵母细胞发育潜能和卵巢局部调控因子BDNF的作用研究
发布时间：2018-12-14 03:57
【摘要】：目的:建立慢性不可预见性温和应激小鼠模型;观察慢性不可预见性温和应激对小鼠卵巢反应性、卵母细胞发育潜能的影响,探讨与卵母细胞发育潜能相关的局部调控因子BDNF是否参与了慢性不可预见性温和应激对小鼠卵巢反应性和卵母细胞发育潜能的影响。 方法: (1)利用孤养、饥饿、冷水游泳等连续温和应激相继施与小鼠30天,建立慢性不可预见性温和应激雌性小鼠模型,旷场试验检测行为学、ELISA方法检测血清学皮质酮、免疫组化方法检测下丘脑CRH阳性神经元变化鉴定慢性应激动物模型。(2)观察PMSG+HCG促排卵后的获卵数、卵母细胞孤雌激活后体外培养至囊胚阶段观察慢性应激对卵巢反应性和卵母细胞发育潜能的影响。(3)透视电子显微镜观察卵母细胞和颗粒细胞超微结构、卵巢组织免疫组化方法检测卵巢BDNF水平探讨慢性应激对小鼠卵巢卵巢反应性和卵母细胞发育潜能可能的机制。 结果: (1)通过连续30天的慢性不可预见性温和应激后,行为学检测显示小鼠中央格停留时间延长、水平运动和直立运动明显减少;血清学检测显示慢性应激后小鼠皮质酮水平明显升高;免疫组化方法显示慢性应激后下丘脑室旁核CRH阳性神经元增多。(2)慢性应激后小鼠促排卵获卵数明显减少;卵母细胞孤雌激活后体外培养,囊胚形成率显著降低。(3)慢性应激后透视电子显微镜下观察小鼠卵巢超微结构显示卵母细胞与颗粒细胞间缝隙连接破坏、颗粒细胞凋亡增多;卵巢组织免疫组化结果显示卵巢窦卵泡、成熟卵泡内BDNF表达降低。 结论:慢性不可预见性温和应激后小鼠行为学呈抑郁样改变、HPA轴活性升高,说明小鼠处于应激状态;慢性应激后小鼠获卵数减少、囊胚形成率降低,提示慢性应激降低了卵巢反应性和卵母细胞发育潜能;慢性应激小鼠卵泡内颗粒细胞凋亡增加、卵母细胞与颗粒细胞间的缝隙连接破坏;慢性应激可致窦卵泡和成熟卵泡内BDNF表达水平下降。以上结果提示慢性应激可能通过抑制卵巢内自分泌和旁分泌因子BDNF的表达,进而抑制小鼠颗粒细胞生长和增殖,并抑制卵母细胞和颗粒细胞之间的缝隙连接交流,从而对卵泡的生长,卵母细胞的发育潜能产生影响。
[Abstract]:Objective: to establish a mouse model of chronic unpredictable mild stress. To observe the effect of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. To investigate whether the local regulatory factor BDNF associated with oocyte development potential is involved in the effects of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. Methods: (1) the female mice model of chronic unpredictable mild stress was established by continuous mild stress, such as solitary rearing, starvation and cold water swimming for 30 days. Behavior was detected by open field test and serum corticosterone was detected by ELISA method. The changes of CRH positive neurons in hypothalamus were detected by immunohistochemical method to identify the chronic stress animal model. (2) the number of eggs obtained after ovulation induced by PMSG HCG was observed. The effects of chronic stress on ovarian reactivity and oocyte development potential were observed in vitro after parthenogenetic activation. (3) the ultrastructure of oocytes and granulosa cells were observed by fluoroscopy. Detection of ovarian BDNF level by immunohistochemical method in ovarian tissues to explore the possible mechanism of chronic stress on ovarian responsiveness and oocyte development potential in mice. Results: (1) after 30 days of chronic unpredictable mild stress, the behavior test showed that the retention time of central lattice was prolonged, the horizontal movement and upright movement were decreased obviously. The serum level of corticosterone was significantly increased after chronic stress, the number of CRH positive neurons in paraventricular nucleus of hypothalamus was increased after chronic stress. (2) the number of ovulation induction was significantly decreased after chronic stress. The blastocyst formation rate was significantly decreased after parthenogenetic activation of oocytes in vitro. (3) the ultrastructure of mouse ovary after chronic stress showed that the gap junctions between oocytes and granulosa cells were damaged. Granulosa cell apoptosis increased; The expression of BDNF in ovarian antral follicles was lower than that in mature follicles. Conclusion: after chronic unpredictable mild stress, the behavior of mice showed depressive changes and the activity of HPA axis increased, indicating that the mice were in stress state. The number of oocytes was decreased and the blastocyst formation rate was decreased after chronic stress, indicating that chronic stress decreased ovarian reactivity and oocyte development potential. The apoptosis of granulosa cells in follicles increased and the gap junction between oocytes and granulosa cells was destroyed in chronic stress, and the expression of BDNF in antral follicles and mature follicles decreased after chronic stress. These results suggest that chronic stress may inhibit the growth and proliferation of mouse granulosa cells by inhibiting the expression of autocrine and paracrine factor BDNF, and inhibit the gap junction communication between oocytes and granulosa cells. Thus, the growth of follicles and the developmental potential of oocytes are affected.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321
本文编号：2377876
[Abstract]:Objective: to establish a mouse model of chronic unpredictable mild stress. To observe the effect of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. To investigate whether the local regulatory factor BDNF associated with oocyte development potential is involved in the effects of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. Methods: (1) the female mice model of chronic unpredictable mild stress was established by continuous mild stress, such as solitary rearing, starvation and cold water swimming for 30 days. Behavior was detected by open field test and serum corticosterone was detected by ELISA method. The changes of CRH positive neurons in hypothalamus were detected by immunohistochemical method to identify the chronic stress animal model. (2) the number of eggs obtained after ovulation induced by PMSG HCG was observed. The effects of chronic stress on ovarian reactivity and oocyte development potential were observed in vitro after parthenogenetic activation. (3) the ultrastructure of oocytes and granulosa cells were observed by fluoroscopy. Detection of ovarian BDNF level by immunohistochemical method in ovarian tissues to explore the possible mechanism of chronic stress on ovarian responsiveness and oocyte development potential in mice. Results: (1) after 30 days of chronic unpredictable mild stress, the behavior test showed that the retention time of central lattice was prolonged, the horizontal movement and upright movement were decreased obviously. The serum level of corticosterone was significantly increased after chronic stress, the number of CRH positive neurons in paraventricular nucleus of hypothalamus was increased after chronic stress. (2) the number of ovulation induction was significantly decreased after chronic stress. The blastocyst formation rate was significantly decreased after parthenogenetic activation of oocytes in vitro. (3) the ultrastructure of mouse ovary after chronic stress showed that the gap junctions between oocytes and granulosa cells were damaged. Granulosa cell apoptosis increased; The expression of BDNF in ovarian antral follicles was lower than that in mature follicles. Conclusion: after chronic unpredictable mild stress, the behavior of mice showed depressive changes and the activity of HPA axis increased, indicating that the mice were in stress state. The number of oocytes was decreased and the blastocyst formation rate was decreased after chronic stress, indicating that chronic stress decreased ovarian reactivity and oocyte development potential. The apoptosis of granulosa cells in follicles increased and the gap junction between oocytes and granulosa cells was destroyed in chronic stress, and the expression of BDNF in antral follicles and mature follicles decreased after chronic stress. These results suggest that chronic stress may inhibit the growth and proliferation of mouse granulosa cells by inhibiting the expression of autocrine and paracrine factor BDNF, and inhibit the gap junction communication between oocytes and granulosa cells. Thus, the growth of follicles and the developmental potential of oocytes are affected.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321
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