CRH和皮質(zhì)酮影響小鼠著床前胚胎發(fā)育的機制：體外模型研究
發(fā)布時間：2019-01-10 10:46
【摘要】：心理應激時機體激活下丘腦-垂體-腎上腺(HPA)軸,從不同水平對雌性生殖系統(tǒng)產(chǎn)生多層次的抑制作用。HPA軸的主要中樞調(diào)節(jié)因子是CRH和精氨酸加壓素(AVP),兩者協(xié)同作用于垂體前葉促進ACTH和其他阿黑皮素原(POMC)衍生肽(如β-內(nèi)啡肽)分泌,隨后ACTH作用于腎上腺皮質(zhì)促進腎上腺合成并釋放糖皮質(zhì)激素(GCs)。CRH和其誘導產(chǎn)生的β-內(nèi)啡肽抑制促性腺激素釋放激素(GnRH)分泌,腎上腺皮質(zhì)分泌的GCs則抑制促黃體激素(LH)和卵巢中雌二醇、孕酮的產(chǎn)生,使靶組織或器官對雌二醇產(chǎn)生抗性。因此,應激過程中產(chǎn)生的CRH和GCs對雌性生殖系統(tǒng)的不利影響是非常明顯的。本實驗室前期研究認為,小鼠合子形成初期束縛應激48 h,可使胚胎發(fā)育能力明顯降低,進而導致妊娠率降低,然而有關其作用機理并不清楚。因此,本實驗選取CRH和GCs,通過體外直接添加或與小鼠輸卵管上皮細胞(OECs)共培養(yǎng)或制取條件化培養(yǎng)液(CM)培養(yǎng)小鼠原核胚,探索CRH和GCs對著床前胚胎發(fā)育的影響,并對其作用機制進行深入研究。結果表明:(1)培養(yǎng)液中直接添加CRH不會損傷著床前胚胎發(fā)育(P0.05),但與OECs共培養(yǎng)時添加CRH能明顯降低原核胚發(fā)育至囊胚的比例(P0.05)。利用WB檢測發(fā)現(xiàn)小鼠OECs表達CRHR蛋白,且CRH促進其表達(P0.05);利用激光共聚焦顯微鏡觀察發(fā)現(xiàn)早期胚胎(囊胚階段除外)都不表達CRHR蛋白,這說明CRH通過與OECs中CRHR結合,對小鼠早期胚胎體外發(fā)育產(chǎn)生不利作用。對OECs氧化應激指標檢測發(fā)現(xiàn),CRH造成細胞內(nèi)GSH還原能力下降(P0.05),過氧化物TOS水平以及OSI值升高(P0.05),清除過氧化物的還原酶如SOD酶水平降低(P0.05)、但過氧化氫酶水平升高(P0.05),分泌的促胚胎發(fā)育生長因子水平降低(P0.05),說明CRH誘導OECs發(fā)生氧化應激。進一步檢測發(fā)現(xiàn),OECs表達Fas水平升高(P0.05),培養(yǎng)液中Fas L水平升高(P0.05),說明OECs啟動死亡受體途徑,進而檢測到細胞表達Caspase-3水平升高(P0.05),細胞發(fā)生晚期凋亡的比例明顯升高(P0.05)。(2)培養(yǎng)液中直接添加GCs不會損傷著床前胚胎發(fā)育(P0.05),但與OECs共培養(yǎng)時添加GCs能顯著降低原核胚發(fā)育至囊胚的比例(P0.05)。利用WB檢測發(fā)現(xiàn)小鼠OECs表達GR蛋白,且GCs下調(diào)其表達(P0.05);利用激光共聚焦顯微鏡觀察發(fā)現(xiàn)不同發(fā)育階段早期胚胎都表達GR蛋白。接著我們利用熒光定量RT-PCR檢測早期胚胎,沒檢測到11-HSD1 mRNA表達,但GCs上調(diào)11-HSD2 mRNA表達水平(P0.05)。因此,GCs可能通過11-HSD2解除對早期胚胎發(fā)育的直接阻礙作用,但可能通過與OECs中GR結合對胚胎發(fā)育產(chǎn)生不利作用。對OECs氧化應激指標檢測發(fā)現(xiàn),GCs造成細胞內(nèi)GSH還原能力下降(P0.05),過氧化物TOS水平以及OSI值升高(P0.05),清除過氧化物的還原酶如SOD酶水平降低(P0.05)、過氧化氫酶等水平升高(P0.05),分泌的促胚胎發(fā)育生長因子水平降低(P0.05),說明GCs誘導OECs發(fā)生氧化應激。進一步檢測發(fā)現(xiàn),OECs表達Fas水平升高(P0.05),培養(yǎng)液中FasL水平升高(P0.05),說明OECs啟動死亡受體途徑,進而檢測到細胞表達Caspase-3水平升高(P0.05),細胞發(fā)生晚期凋亡的比例明顯升高(P0.05)�？傊�,CRH或GCs通過與OECs中各自的受體結合,導致OECs發(fā)生氧化應激,并誘導OECs的凋亡。OECs凋亡進而引起囊胚的GSH還原能力下降(P0.05),Fas和Caspase-3水平升高(P0.05),說明對著床前胚胎也造成了氧化應激并發(fā)生凋亡,導致原核胚發(fā)育至4-細胞和囊胚的比例明顯下降以及囊胚細胞數(shù)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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q132
本文編號：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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q132
【參考文獻】
相關期刊論文 前2條
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
，本文編號：2406219
本文鏈接：http://sikaile.net/shoufeilunwen/jckxbs/2406219.html
最近更新
教材專著
