【摘要】：Calcineurin(鈣調(diào)神經(jīng)磷酸酶)是一種重要的絲/蘇氨酸蛋白磷酸酶,由催化亞基A和調(diào)節(jié)亞基B組成,A亞基分為α、β和γ亞型,B亞基分為1和2型。它們廣泛的分布于動(dòng)物的各種組織和器官中,在細(xì)胞的信號(hào)傳遞過(guò)程中直接受Ca2+/鈣調(diào)素(Ca M)的調(diào)控,主要通過(guò)發(fā)揮去磷酸化的作用參與調(diào)節(jié)體內(nèi)的許多重要過(guò)程,如細(xì)胞周期、細(xì)胞的生長(zhǎng)及分化等。然而calcineurin在小鼠卵母細(xì)胞和顆粒細(xì)胞發(fā)育的過(guò)程中的作用及調(diào)控機(jī)制相關(guān)的報(bào)道較少。本研究通過(guò)體外培養(yǎng)小鼠卵母細(xì)胞、顆粒細(xì)胞、卵丘卵細(xì)胞復(fù)合體以及卵泡等,并利用calcineurin抑制劑FK506、腹腔灌胃、immunohistochemistry(IHC)、immunofluorescence(IF)、western blot(WB)、real time PCR、ELISA及流式細(xì)胞儀檢測(cè)等方法,揭示calcineurin在小鼠雌雄生殖系統(tǒng)中的表達(dá)模式及對(duì)卵巢卵泡、顆粒細(xì)胞和卵母細(xì)胞中的作用,進(jìn)而獲得calcineurin在小鼠卵母細(xì)胞中的作用及調(diào)控機(jī)制。Calcineurin的定位表達(dá)結(jié)果顯示,calcineurin主要定位于在雌鼠卵巢卵泡及黃體細(xì)胞、輸卵管粘膜上皮和子宮肌層,并且在原始卵泡、初級(jí)卵泡、有腔卵泡以及排卵前的有腔卵泡中均有表達(dá);在雄鼠睪丸和附睪頭、附睪體和附睪尾中都有陽(yáng)性著色反應(yīng),主要定位于睪丸間質(zhì)細(xì)胞和附睪頭、體和尾的精子和假?gòu)?fù)單層柱狀上皮細(xì)胞中。而calcineurin各個(gè)亞基亞型在卵母細(xì)胞和顆粒細(xì)胞中的表達(dá)模式是:calcineurin A亞基的三個(gè)亞型(α、β和γ)在卵母細(xì)胞和顆粒細(xì)胞中都有表達(dá),calcineurin Aβ的m RNA水平顯著高于α和γ亞型,calcineurin Aβ的m RNA水平在卵母細(xì)胞發(fā)生生發(fā)泡破裂(GVBD)時(shí)發(fā)生顯著的下降;calcineurin B亞基的兩個(gè)亞型(1和2)在顆粒細(xì)胞中均有表達(dá),但B1型的m RNA水平顯著高于B2型,在卵母細(xì)胞則僅檢測(cè)到B1型m RNA的表達(dá),并且在GV、GVBD及MII期卵母細(xì)胞中的表達(dá)水平?jīng)]有顯著性差異,呈現(xiàn)穩(wěn)定表達(dá)。進(jìn)一步研究calcineurin A在卵細(xì)胞和顆粒細(xì)胞中的定位發(fā)現(xiàn):calcineurin A亞基定位于GV期卵母細(xì)胞的生發(fā)泡中,當(dāng)卵母細(xì)胞發(fā)生GVBD時(shí)擴(kuò)散到細(xì)胞質(zhì)中,并且GVBD期卵母細(xì)胞中calcineurin A亞基的蛋白水平顯著低于GV和MII期卵母細(xì)胞,calcineurin A亞基在顆粒細(xì)胞中主要定位于細(xì)胞核中。利用Calcineurin的抑制劑FK506對(duì)小鼠裸卵(DOs)、卵丘卵母細(xì)胞復(fù)合體(COCs)、卵泡(FOs)及顆粒細(xì)胞、黃體細(xì)胞的培養(yǎng)結(jié)果顯示,calcineurin抑制劑FK506可以促進(jìn)DOs、COCs以及FOs中卵母細(xì)胞發(fā)生GVBD,但不影響COCs卵丘的擴(kuò)展。Calcineurin抑制劑FK506可以促進(jìn)卵母細(xì)胞MPF以及顆粒細(xì)胞中MAPK的磷酸化,并且在低濃度組和高濃度可以抑制顆粒細(xì)胞的增殖,但是只有高濃度組可以促進(jìn)顆粒細(xì)胞的凋亡。此外,FK506可以抑制黃體細(xì)胞孕酮的分泌,利用FK506對(duì)雌鼠進(jìn)行灌胃處理后,發(fā)現(xiàn)低濃度組的小鼠的卵巢中存在大量未排卵的大有腔卵泡。本研究結(jié)果表明calcineurin在雌鼠中可以通過(guò)調(diào)控卵巢顆粒細(xì)胞和黃體細(xì)胞的發(fā)育及激素分泌以及MPF和MAPK信號(hào)通路,從而進(jìn)一步調(diào)控卵母細(xì)胞的發(fā)育,并且calcineurin Aβ可能在此過(guò)程中發(fā)揮著重要作用,為calcineurin在哺乳動(dòng)物卵巢中的研究提供了參考依據(jù)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S814
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本文編號(hào)：2438077