钙调神经磷酸酶对小鼠卵泡卵母细胞发育的影响及调控机制的研究
发布时间：2019-03-11 07:28
【摘要】：Calcineurin(钙调神经磷酸酶)是一种重要的丝/苏氨酸蛋白磷酸酶,由催化亚基A和调节亚基B组成,A亚基分为α、β和γ亚型,B亚基分为1和2型。它们广泛的分布于动物的各种组织和器官中,在细胞的信号传递过程中直接受Ca2+/钙调素(Ca M)的调控,主要通过发挥去磷酸化的作用参与调节体内的许多重要过程,如细胞周期、细胞的生长及分化等。然而calcineurin在小鼠卵母细胞和颗粒细胞发育的过程中的作用及调控机制相关的报道较少。本研究通过体外培养小鼠卵母细胞、颗粒细胞、卵丘卵细胞复合体以及卵泡等,并利用calcineurin抑制剂FK506、腹腔灌胃、immunohistochemistry(IHC)、immunofluorescence(IF)、western blot(WB)、real time PCR、ELISA及流式细胞仪检测等方法,揭示calcineurin在小鼠雌雄生殖系统中的表达模式及对卵巢卵泡、颗粒细胞和卵母细胞中的作用,进而获得calcineurin在小鼠卵母细胞中的作用及调控机制。Calcineurin的定位表达结果显示,calcineurin主要定位于在雌鼠卵巢卵泡及黄体细胞、输卵管粘膜上皮和子宫肌层,并且在原始卵泡、初级卵泡、有腔卵泡以及排卵前的有腔卵泡中均有表达;在雄鼠睾丸和附睾头、附睾体和附睾尾中都有阳性着色反应,主要定位于睾丸间质细胞和附睾头、体和尾的精子和假复单层柱状上皮细胞中。而calcineurin各个亚基亚型在卵母细胞和颗粒细胞中的表达模式是:calcineurin A亚基的三个亚型(α、β和γ)在卵母细胞和颗粒细胞中都有表达,calcineurin Aβ的m RNA水平显著高于α和γ亚型,calcineurin Aβ的m RNA水平在卵母细胞发生生发泡破裂(GVBD)时发生显著的下降;calcineurin B亚基的两个亚型(1和2)在颗粒细胞中均有表达,但B1型的m RNA水平显著高于B2型,在卵母细胞则仅检测到B1型m RNA的表达,并且在GV、GVBD及MII期卵母细胞中的表达水平没有显著性差异,呈现稳定表达。进一步研究calcineurin A在卵细胞和颗粒细胞中的定位发现:calcineurin A亚基定位于GV期卵母细胞的生发泡中,当卵母细胞发生GVBD时扩散到细胞质中,并且GVBD期卵母细胞中calcineurin A亚基的蛋白水平显著低于GV和MII期卵母细胞,calcineurin A亚基在颗粒细胞中主要定位于细胞核中。利用Calcineurin的抑制剂FK506对小鼠裸卵(DOs)、卵丘卵母细胞复合体(COCs)、卵泡(FOs)及颗粒细胞、黄体细胞的培养结果显示,calcineurin抑制剂FK506可以促进DOs、COCs以及FOs中卵母细胞发生GVBD,但不影响COCs卵丘的扩展。Calcineurin抑制剂FK506可以促进卵母细胞MPF以及颗粒细胞中MAPK的磷酸化,并且在低浓度组和高浓度可以抑制颗粒细胞的增殖,但是只有高浓度组可以促进颗粒细胞的凋亡。此外,FK506可以抑制黄体细胞孕酮的分泌,利用FK506对雌鼠进行灌胃处理后,发现低浓度组的小鼠的卵巢中存在大量未排卵的大有腔卵泡。本研究结果表明calcineurin在雌鼠中可以通过调控卵巢颗粒细胞和黄体细胞的发育及激素分泌以及MPF和MAPK信号通路,从而进一步调控卵母细胞的发育,并且calcineurin Aβ可能在此过程中发挥着重要作用,为calcineurin在哺乳动物卵巢中的研究提供了参考依据。
[Abstract]:Calcineurin (calcineurin) is an important serine/ threonine protein phosphatase, consisting of a catalytic subunit A and an adjusting subunit B, and the A subunit is divided into 1 and 2 types, and the B subunit is divided into 1 and 2 types. They are widely distributed in various tissues and organs of the animal, and are directly controlled by the Ca2 +/ calmodulin (Ca M) in the signal transmission process of the cells, The growth and differentiation of the cells, and the like. However, the role of calcitrinin in the development of mouse oocytes and granulosa cells and its regulatory mechanisms are less. In this study, the mouse oocytes, the granulosa cells, the cumulus ovulus complex and the follicle were cultured in vitro, and the methods such as FK506, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, The expression pattern of calcitrinin in male and female reproductive system of mice and its effect on the ovarian follicles, granulosa cells and oocytes were revealed, and the role and regulation mechanism of calcitrinin in mouse oocytes were also obtained. The results of the localization of Calcinuria indicated that calcinin was mainly located in the ovarian and yellow cells of the female mice, the mucosal epithelium of the fallopian tube and the myometrium of the uterus, and expressed in both the primary and primary follicles, the follicle in the cavity, and the follicle in the cavity before ovulation; in the male and the epididymides, There was positive staining in the epididymis and the tail of the epididymis, which was mainly located in the testis interstitial cells and the epididymides, and the sperm of the tail and the pseudocomplex monolayer of columnar epithelial cells. The expression pattern of the sub-subunits of calcitrinin in the oocytes and granulosa cells is that the three subtypes of the calcininin A subunit are expressed in both the oocyte and the granulosa cell, and the m-RNA level of calcitrinin A-subunit is significantly higher than that of the human and the granular cells. The level of m-RNA of calcininin A was significantly lower in the generation of BBD in the oocytes; the two subtypes (1 and 2) of the calcininin B subunit were expressed in the granulosa cells, but the level of m-RNA of the B1-type was significantly higher than that of the B2 type, and only the expression of the B1-type m RNA was detected in the oocytes. There was no significant difference in the expression of GV, GVBD and MII oocytes. The localization of calcitrinin A in the oocytes and granulosa cells was further studied. The calcininin A subunit was located in the generation of the GV stage oocytes, and when the oocytes had GVBD, the calcinerein A subunit was spread to the cytoplasm, and the calcitrinin A subunit protein level was significantly lower in the GVBD oocytes than in the GV and MII oocytes. The calcininin A subunit is mainly located in the nucleus of the granulosa cells. The results showed that the calcinerein inhibitor, FK506, could promote the occurrence of GVBD in the oocytes of DOs, COCs and FOs, but did not affect the expansion of the COCs cumulus. Calcineurin inhibitor FK506 can promote the phosphorylation of MAPK in the oocytes of MPF and granulosa cells, and can inhibit the proliferation of the granulosa cells in the low concentration group and the high concentration, but only the high concentration group can promote the apoptosis of the granulosa cells. In addition, FK506 can inhibit the secretion of the luteal cell progesterone, and after the administration of the female rats by using the FK506, it is found that there is a large number of non-ovulatory large-cavity follicles in the ovary of the low-concentration group. The results of this study show that calcinin can regulate the development of the ovarian granulosa cells and the yellow cells and the secretion of the hormone and the pathway of the MPF and MAPK signals in the female, so as to further regulate the development of the oocytes, and the calcinerein A can play an important role in the process. The reference is provided for the study of calcitrinin in the ovary of a mammal.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S814
，
本文编号：2438077
[Abstract]:Calcineurin (calcineurin) is an important serine/ threonine protein phosphatase, consisting of a catalytic subunit A and an adjusting subunit B, and the A subunit is divided into 1 and 2 types, and the B subunit is divided into 1 and 2 types. They are widely distributed in various tissues and organs of the animal, and are directly controlled by the Ca2 +/ calmodulin (Ca M) in the signal transmission process of the cells, The growth and differentiation of the cells, and the like. However, the role of calcitrinin in the development of mouse oocytes and granulosa cells and its regulatory mechanisms are less. In this study, the mouse oocytes, the granulosa cells, the cumulus ovulus complex and the follicle were cultured in vitro, and the methods such as FK506, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, IHC, The expression pattern of calcitrinin in male and female reproductive system of mice and its effect on the ovarian follicles, granulosa cells and oocytes were revealed, and the role and regulation mechanism of calcitrinin in mouse oocytes were also obtained. The results of the localization of Calcinuria indicated that calcinin was mainly located in the ovarian and yellow cells of the female mice, the mucosal epithelium of the fallopian tube and the myometrium of the uterus, and expressed in both the primary and primary follicles, the follicle in the cavity, and the follicle in the cavity before ovulation; in the male and the epididymides, There was positive staining in the epididymis and the tail of the epididymis, which was mainly located in the testis interstitial cells and the epididymides, and the sperm of the tail and the pseudocomplex monolayer of columnar epithelial cells. The expression pattern of the sub-subunits of calcitrinin in the oocytes and granulosa cells is that the three subtypes of the calcininin A subunit are expressed in both the oocyte and the granulosa cell, and the m-RNA level of calcitrinin A-subunit is significantly higher than that of the human and the granular cells. The level of m-RNA of calcininin A was significantly lower in the generation of BBD in the oocytes; the two subtypes (1 and 2) of the calcininin B subunit were expressed in the granulosa cells, but the level of m-RNA of the B1-type was significantly higher than that of the B2 type, and only the expression of the B1-type m RNA was detected in the oocytes. There was no significant difference in the expression of GV, GVBD and MII oocytes. The localization of calcitrinin A in the oocytes and granulosa cells was further studied. The calcininin A subunit was located in the generation of the GV stage oocytes, and when the oocytes had GVBD, the calcinerein A subunit was spread to the cytoplasm, and the calcitrinin A subunit protein level was significantly lower in the GVBD oocytes than in the GV and MII oocytes. The calcininin A subunit is mainly located in the nucleus of the granulosa cells. The results showed that the calcinerein inhibitor, FK506, could promote the occurrence of GVBD in the oocytes of DOs, COCs and FOs, but did not affect the expansion of the COCs cumulus. Calcineurin inhibitor FK506 can promote the phosphorylation of MAPK in the oocytes of MPF and granulosa cells, and can inhibit the proliferation of the granulosa cells in the low concentration group and the high concentration, but only the high concentration group can promote the apoptosis of the granulosa cells. In addition, FK506 can inhibit the secretion of the luteal cell progesterone, and after the administration of the female rats by using the FK506, it is found that there is a large number of non-ovulatory large-cavity follicles in the ovary of the low-concentration group. The results of this study show that calcinin can regulate the development of the ovarian granulosa cells and the yellow cells and the secretion of the hormone and the pathway of the MPF and MAPK signals in the female, so as to further regulate the development of the oocytes, and the calcinerein A can play an important role in the process. The reference is provided for the study of calcitrinin in the ovary of a mammal.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S814
，
本文编号：2438077
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