CRH和皮质酮影响小鼠着床前胚胎发育的机制：体外模型研究
发布时间：2019-01-10 10:46
【摘要】：心理应激时机体激活下丘脑-垂体-肾上腺(HPA)轴,从不同水平对雌性生殖系统产生多层次的抑制作用。HPA轴的主要中枢调节因子是CRH和精氨酸加压素(AVP),两者协同作用于垂体前叶促进ACTH和其他阿黑皮素原(POMC)衍生肽(如β-内啡肽)分泌,随后ACTH作用于肾上腺皮质促进肾上腺合成并释放糖皮质激素(GCs)。CRH和其诱导产生的β-内啡肽抑制促性腺激素释放激素(GnRH)分泌,肾上腺皮质分泌的GCs则抑制促黄体激素(LH)和卵巢中雌二醇、孕酮的产生,使靶组织或器官对雌二醇产生抗性。因此,应激过程中产生的CRH和GCs对雌性生殖系统的不利影响是非常明显的。本实验室前期研究认为,小鼠合子形成初期束缚应激48 h,可使胚胎发育能力明显降低,进而导致妊娠率降低,然而有关其作用机理并不清楚。因此,本实验选取CRH和GCs,通过体外直接添加或与小鼠输卵管上皮细胞(OECs)共培养或制取条件化培养液(CM)培养小鼠原核胚,探索CRH和GCs对着床前胚胎发育的影响,并对其作用机制进行深入研究。结果表明:(1)培养液中直接添加CRH不会损伤着床前胚胎发育(P0.05),但与OECs共培养时添加CRH能明显降低原核胚发育至囊胚的比例(P0.05)。利用WB检测发现小鼠OECs表达CRHR蛋白,且CRH促进其表达(P0.05);利用激光共聚焦显微镜观察发现早期胚胎(囊胚阶段除外)都不表达CRHR蛋白,这说明CRH通过与OECs中CRHR结合,对小鼠早期胚胎体外发育产生不利作用。对OECs氧化应激指标检测发现,CRH造成细胞内GSH还原能力下降(P0.05),过氧化物TOS水平以及OSI值升高(P0.05),清除过氧化物的还原酶如SOD酶水平降低(P0.05)、但过氧化氢酶水平升高(P0.05),分泌的促胚胎发育生长因子水平降低(P0.05),说明CRH诱导OECs发生氧化应激。进一步检测发现,OECs表达Fas水平升高(P0.05),培养液中Fas L水平升高(P0.05),说明OECs启动死亡受体途径,进而检测到细胞表达Caspase-3水平升高(P0.05),细胞发生晚期凋亡的比例明显升高(P0.05)。(2)培养液中直接添加GCs不会损伤着床前胚胎发育(P0.05),但与OECs共培养时添加GCs能显著降低原核胚发育至囊胚的比例(P0.05)。利用WB检测发现小鼠OECs表达GR蛋白,且GCs下调其表达(P0.05);利用激光共聚焦显微镜观察发现不同发育阶段早期胚胎都表达GR蛋白。接着我们利用荧光定量RT-PCR检测早期胚胎,没检测到11-HSD1 mRNA表达,但GCs上调11-HSD2 mRNA表达水平(P0.05)。因此,GCs可能通过11-HSD2解除对早期胚胎发育的直接阻碍作用,但可能通过与OECs中GR结合对胚胎发育产生不利作用。对OECs氧化应激指标检测发现,GCs造成细胞内GSH还原能力下降(P0.05),过氧化物TOS水平以及OSI值升高(P0.05),清除过氧化物的还原酶如SOD酶水平降低(P0.05)、过氧化氢酶等水平升高(P0.05),分泌的促胚胎发育生长因子水平降低(P0.05),说明GCs诱导OECs发生氧化应激。进一步检测发现,OECs表达Fas水平升高(P0.05),培养液中FasL水平升高(P0.05),说明OECs启动死亡受体途径,进而检测到细胞表达Caspase-3水平升高(P0.05),细胞发生晚期凋亡的比例明显升高(P0.05)。总之,CRH或GCs通过与OECs中各自的受体结合,导致OECs发生氧化应激,并诱导OECs的凋亡。OECs凋亡进而引起囊胚的GSH还原能力下降(P0.05),Fas和Caspase-3水平升高(P0.05),说明对着床前胚胎也造成了氧化应激并发生凋亡,导致原核胚发育至4-细胞和囊胚的比例明显下降以及囊胚细胞数降低。
[Abstract]:The hypothalamic-pituitary-adrenal (HPA) axis was activated by the body at the time of psychological stress, and the female reproductive system had a multi-level inhibition effect from different levels. The major central regulatory factor of the HPA axis is the CRH and the arginine vasopressin (AVP), both of which are co-acting on the anterior pituitary of the pituitary to promote the secretion of ACTH and other opiomelas (POMC)-derived peptides (e.g., endorphin), The ACTH is then applied to the adrenal cortex to promote the synthesis of the adrenal gland and release the glucocorticoid (GCs). CRH and the induced by-endorphin inhibit the secretion of the gonadotropin-releasing hormone (GnRH), and the GCs secreted by the adrenal cortex inhibit the luteinizing hormone (LH) and the estradiol in the ovary, The production of progesterone causes the target tissue or organ to be resistant to estradiol. Therefore, the adverse effects of CRH and GCs produced in the stress process on the female reproductive system are very significant. In the early stage of this lab, the early-stage binding stress of the mouse zygote was 48 h, which could lead to a significant decrease in the development of the embryo, which in turn led to a decrease in the pregnancy rate, but the mechanism of its action was not clear. The effects of CRH and GCs on the development of preimplantation embryos were studied by co-culture with the mouse oviduct epithelial cells (OECs) or the conditioned medium (CM), and the mechanism of action of CRH and GCs was studied. The results showed that (1) The addition of CRH in the culture medium did not damage the pre-implantation embryo development (P0.05), but the addition of CRH in co-culture with OECs could significantly reduce the proportion of the development of the prokaryotes to the blastula (P0.05). The expression of CRHR in the mouse OECs was detected by WB, and the expression of CRH was promoted by CRH (P0.05). The CRHR protein was not expressed in the early embryo (except the blastula stage) by laser confocal microscope, which means that the CRH was bound to the CRHR in OECs. It has a negative effect on the in vitro development of the early embryo of the mouse. The results showed that the level of GSH in the cells decreased (P0.05), the level of the TOS and the level of the OSI were higher (P0.05), and the level of the reductase, such as the SOD, was decreased (P0.05), but the level of the catalase was increased (P0.05). The level of the secreted pro-embryonic development growth factor (P <0.05) showed that CRH induced oxidative stress in OECs. The results showed that the expression of Fas was higher in OECs (P0.05), and the level of Fas L in the culture medium was increased (P0.05). (2) The direct addition of GCs in the culture medium did not damage the pre-implantation embryo development (P0.05), but the addition of GCs in co-culture with OECs could significantly reduce the proportion of the development of the prokaryotes to the blastocysts (P0.05). The expression of GR in mouse OECs was detected by WB, and the expression of GR was down-regulated by GCs (P0.05). Then we used the fluorescence quantitative RT-PCR to detect the early embryo, and the expression of 11-CD1 mRNA was not detected, but the GCs up-regulate the expression level of 11-CD2 mRNA (P0.05). As a result, GCs may remove the direct barrier to early embryonic development by 11-HAC2, but may have an adverse effect on the development of embryos by binding to GR in OECs. The results showed that GCs decreased the GSH-reduction ability in the cells (P0.05), the level of the peroxide TOS and the increase of the OSI value (P0.05), and the levels of the reductase such as the SOD (P0.05), catalase and the like were decreased (P0.05). The level of the secreted pro-embryonic development growth factor decreased (P0.05), indicating that the GCs induced oxidative stress in OECs. The results showed that the expression of Fas was higher in OECs (P0.05), and the level of FasL in the culture medium was increased (P0.05). In conclusion, CRH or GCs, by binding to the respective receptors in OECs, leads to oxidative stress in OECs and induces apoptosis in OECs. The apoptosis of OECs leads to a decrease in the level of GSH reduction of the blastula (P0.05), and the level of Fas and Caspase-3 (P. 05), which indicates that the pre-implantation embryos also cause oxidative stress and apoptosis, resulting in a significant decrease in the percentage of the development of the prokaryotes to the 4-cell and the blastula, and the decrease in the number of blastocysts.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q132
本文编号：2406219
[Abstract]:The hypothalamic-pituitary-adrenal (HPA) axis was activated by the body at the time of psychological stress, and the female reproductive system had a multi-level inhibition effect from different levels. The major central regulatory factor of the HPA axis is the CRH and the arginine vasopressin (AVP), both of which are co-acting on the anterior pituitary of the pituitary to promote the secretion of ACTH and other opiomelas (POMC)-derived peptides (e.g., endorphin), The ACTH is then applied to the adrenal cortex to promote the synthesis of the adrenal gland and release the glucocorticoid (GCs). CRH and the induced by-endorphin inhibit the secretion of the gonadotropin-releasing hormone (GnRH), and the GCs secreted by the adrenal cortex inhibit the luteinizing hormone (LH) and the estradiol in the ovary, The production of progesterone causes the target tissue or organ to be resistant to estradiol. Therefore, the adverse effects of CRH and GCs produced in the stress process on the female reproductive system are very significant. In the early stage of this lab, the early-stage binding stress of the mouse zygote was 48 h, which could lead to a significant decrease in the development of the embryo, which in turn led to a decrease in the pregnancy rate, but the mechanism of its action was not clear. The effects of CRH and GCs on the development of preimplantation embryos were studied by co-culture with the mouse oviduct epithelial cells (OECs) or the conditioned medium (CM), and the mechanism of action of CRH and GCs was studied. The results showed that (1) The addition of CRH in the culture medium did not damage the pre-implantation embryo development (P0.05), but the addition of CRH in co-culture with OECs could significantly reduce the proportion of the development of the prokaryotes to the blastula (P0.05). The expression of CRHR in the mouse OECs was detected by WB, and the expression of CRH was promoted by CRH (P0.05). The CRHR protein was not expressed in the early embryo (except the blastula stage) by laser confocal microscope, which means that the CRH was bound to the CRHR in OECs. It has a negative effect on the in vitro development of the early embryo of the mouse. The results showed that the level of GSH in the cells decreased (P0.05), the level of the TOS and the level of the OSI were higher (P0.05), and the level of the reductase, such as the SOD, was decreased (P0.05), but the level of the catalase was increased (P0.05). The level of the secreted pro-embryonic development growth factor (P <0.05) showed that CRH induced oxidative stress in OECs. The results showed that the expression of Fas was higher in OECs (P0.05), and the level of Fas L in the culture medium was increased (P0.05). (2) The direct addition of GCs in the culture medium did not damage the pre-implantation embryo development (P0.05), but the addition of GCs in co-culture with OECs could significantly reduce the proportion of the development of the prokaryotes to the blastocysts (P0.05). The expression of GR in mouse OECs was detected by WB, and the expression of GR was down-regulated by GCs (P0.05). Then we used the fluorescence quantitative RT-PCR to detect the early embryo, and the expression of 11-CD1 mRNA was not detected, but the GCs up-regulate the expression level of 11-CD2 mRNA (P0.05). As a result, GCs may remove the direct barrier to early embryonic development by 11-HAC2, but may have an adverse effect on the development of embryos by binding to GR in OECs. The results showed that GCs decreased the GSH-reduction ability in the cells (P0.05), the level of the peroxide TOS and the increase of the OSI value (P0.05), and the levels of the reductase such as the SOD (P0.05), catalase and the like were decreased (P0.05). The level of the secreted pro-embryonic development growth factor decreased (P0.05), indicating that the GCs induced oxidative stress in OECs. The results showed that the expression of Fas was higher in OECs (P0.05), and the level of FasL in the culture medium was increased (P0.05). In conclusion, CRH or GCs, by binding to the respective receptors in OECs, leads to oxidative stress in OECs and induces apoptosis in OECs. The apoptosis of OECs leads to a decrease in the level of GSH reduction of the blastula (P0.05), and the level of Fas and Caspase-3 (P. 05), which indicates that the pre-implantation embryos also cause oxidative stress and apoptosis, resulting in a significant decrease in the percentage of the development of the prokaryotes to the 4-cell and the blastula, and the decrease in the number of blastocysts.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q132
【参考文献】
相关期刊论文 前2条
1 ;Deficit of mitochondria-derived ATP during oxidative stress impairs mouse MII oocyte spindles[J];Cell Research;2006年10期
2 ;Expression of Fas ligand and Caspase-3 contributes to formation of immune escape in gastric cancer[J];World Journal of Gastroenterology;2003年07期
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