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Studies on the regulatory mechanism of interaction between the development of ovulatry follicle and ovum in the ovary.

卵巢内排卵卵泡发育与卵子相互作用调控机制的研究。

基本信息

批准号：
10671513
负责人：
HIROI Masahiko
金额：
$ 2.43万
依托单位：
YAMAGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671513/
关键词：
Ovarian follicle Granulosa Cell Steroidogenesis Apoptosis CD44 卵透明帯

项目摘要

Regulatory mechanism of interaction between the development of the follicle and ovum in the ovary is not still completely understood yet. It is so important scientifically and also clinically to know its mechanism. Following studies were undertaken in our Department during recent two years.(1) Apoptotic bodies in the granulosa cell :The incidence of apoptotic bodies in membrana granulosa of patients with endometriosis was significantly higher than that of the control (male factor infertility) group and increased as the revised AFS classification advanced. The incidence of apoptotic bodies in membrana granulosa was significantly higher in patients with chocolate cysts than in those without chocolate cysts. The patients with endometriosis had smaller numbers of developed follicles (≧15 mm), harvested oocytes, and mature oocytes than the male factor infertility patients. The existence of chocolate cysts corresponded with a reduced number of both harvested oocytes and mature oocytes.(2) El … More imination of atretic follcles :Numerous ovarian follicles from 32-35 day virgin mice were examined by transmission electron microscopy and light microscopic immunohistochemistry. No macrophages were seen, but various stages of apoptotic granulosa cells were encountered. Presumably a granulosa cell or its debris in an advanced stage of apoptosis was destined to be phagocytosed by adjacent normal-looking granulosa cells. Other granulosa cells of normal appearance were seen in the region of the zona pellucida in contact with and apparently phagocytosing atrophic oocytes. Such granulosa cells were characterized by the presence of gap junctions with other cells and frequently contained annular gap junctions in the cytoplasm.(3) CD44 and the ovum :CD44 is a polymorphic and polyfunctional transmembrane glycoprotein widely expressed in many types of cells. The expression of this protein on human membrana granulosa was studied by two techniques. In the flow cytometric analysis of 32 patients, the incidence of CD44 expression in cumulus cells (62.6【minus-plus】1.3%) was significantly higher than that in mural granulosa cells (38.5【minus-plus】3.2%). In the comparison of CD44 expression by flow cytometry according to the maturation of each cumulus-oocyte complex, the incidence of CD44 expression of cumulus cells was significantly higher in the mature group than in the immature group. In a flow cytometric analysis, patients with endometriosis showed a significantly lower incidence of CD44 expression in cumulus cells compared to the infertility of unknown origin group, and compared to both the male infertility group and the unknown origin group in mural granulosa cells. These findings suggest that the standard from of CD44 is expressed in human membrana granulosa with polarity and may play and important role in oocyte maturation.(4) Effects of aging on the CaィイD12+ィエD1 release :A transient increase in cytosolic free CaィイD12+ィエD1 concentration ([CaィイD12+ィエD1]. I) was induced by photolysis of fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rete of increase in [CaィイD12+ィエD1] I significantly decreased in the aged oocytes. Reduced ER CaィイD12+ィエD1 release in the aged oocyte may not be attributable to aging-related desensitization of the InsPィイD23ィエD2-sensitive CaィイD12+ィエD1 channels in the ER because concentrations of caged InsPィイD23ィエD2 for half maximal [CaィイD12+ィエD1] i increase were identical for fresh and aged oocytes. The peak [CaィイD12+ィエD1] i response following administration of 5 μm trapsigargin, a specific ER CaィイD12+ィエD1 ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER CaィイD12+ィエD1 stores. We conclude from these results that reduction of CaィイD12+ィエD1 release from the InspィイD23ィエD2-sensitive CaィイD12+ィエD1 stores in the aged oocyte arises from depletion of the ER CaィイD12+ィエD1 stores with aging. These aging-related changes in CaィイD12+ィエD1 release and reuptake may account for alterations in CaィイD12+ィエD1 oscillations in aged fertilized oocytes.(5) Granulosa cell and steroidogenesisWhen porcine granulosa cells were cultured on dishes, progesterone was secreted in the culture medium continuously. However, when cells were overlaid with a type I collagen (TIC) gel progesterone production was suppressed to 0.34-fold (day 3) and 0.16-fold (day 4) compared with that of the cells without TIC gel (p<0.01). Treatment with human FSH(hFSH) enhanced progesterone production in a dose-dependent manner in cells without TIC gel and, to a lesser extent, in cells with TIC gel. The effects of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) was also examined. This is the rate-limiting enzyme for conversion of cholesterol to progesterone. P450scc gene expression in cells overlaid with TIC gel was suppressed to 0.62-fold (day 3) and to 0.36-fold (day 4) compared with without TIC gel. When pregnenolone, which is the direct precursor of progesterone, was added to the culture medium, progesterone production increased of TIC gel addition. Moreover, hFSH induces P450scc gene expression in cells with and without TIC gel overlay. Less

卵巢中卵泡和卵子发育相互作用的调控机制至今仍不完全清楚。了解其发生机制具有重要的科学意义和临床意义。近两年来，我系开展了以下研究。(1)颗粒细胞中的凋亡小体：子宫内膜异位症患者颗粒膜中凋亡小体的发生率显著高于对照组（男性因素不育），并随着修订的AFS分类的进展而增加。有巧克力囊肿的患者颗粒膜中凋亡小体的发生率明显高于无巧克力囊肿的患者。与男性因素不孕患者相比，子宫内膜异位症患者的发育卵泡（<15 mm）、收获的卵母细胞和成熟卵母细胞数量较少。巧克力囊肿的存在与收获的卵母细胞和成熟卵母细胞的数量减少相对应。(2)El ...更多信息闭锁卵泡的免疫化：用透射电镜和光镜免疫组织化学方法观察了32-35天未孕小鼠的大量卵泡。没有看到巨噬细胞，但遇到了不同阶段的凋亡颗粒细胞。据推测，处于凋亡晚期的颗粒细胞或其碎片注定会被相邻的正常颗粒细胞吞噬。在透明质膜的区域内，还可见到其它外观正常的颗粒细胞，它们与萎缩的卵母细胞接触并明显吞噬萎缩的卵母细胞。这种颗粒细胞的特点是与其他细胞的缝隙连接的存在，并经常在细胞质中含有环形缝隙连接。(3)CD 44和卵子：CD 44是一种多形态和多功能的跨膜糖蛋白，广泛表达于多种类型的细胞中。通过两种技术研究了该蛋白在人颗粒膜上的表达。在32例患者的流式细胞术分析中，卵丘细胞中CD 44表达的发生率（62.6[-+]1.3%）显著高于壁颗粒细胞（38.5[-+]3.2%）。根据各卵丘-卵母细胞复合体的成熟程度，用流式细胞术比较卵丘细胞CD 44表达，成熟组卵丘细胞CD 44表达的发生率显著高于未成熟组。在流式细胞术分析中，子宫内膜异位症患者的卵丘细胞中CD 44表达的发生率显著低于不明原因不育组，并且与男性不育组和不明原因组的壁颗粒细胞相比。这些结果表明，人卵母细胞颗粒膜上表达的标准型CD 44具有极性，可能在卵母细胞成熟过程中起重要作用。(4)衰老对Ca ~（2+）D_1释放的影响：胞浆游离Ca ~（2+）D_1浓度（[Ca ~（2+）D_1]）短暂增加。[Ca ~（2+）D_（12）+Ca ~（2+）D_（1+）]_I的最大增加率在老化卵母细胞中显著降低。老化卵母细胞中ER Ca ~（2+）D12+ Ca ~（2+）D1释放的减少可能不是由于ER中InsP_（23）D2-敏感性Ca ~（2+）D12+ Ca ~（2+）D1通道的老化相关脱敏，因为新鲜和老化卵母细胞中笼状InsP_（23）D23_（2+）D12+ Ca ~（2+）D1最大增加一半的浓度是相同的。5 μm trapsigargin（一种特异性ER Ca ~（2+）D_1 ATP酶抑制剂）处理后，衰老卵母细胞的[Ca ~（2+）D_1]_i峰值反应显著降低，提示ER Ca ~（2+）D_1储备减少。我们从这些结果中得出结论，衰老卵母细胞中Insp β D23 β D2-敏感性Ca β D12+ β D1储存的Ca β D12 + β D1释放的减少是由于ER Ca β D12+ β D1储存随着衰老而耗尽。这些年龄相关的变化，在Ca ~（2+）D_12 + Ca ~（2+）D_1释放和再摄取可能解释的改变，在Ca ~（2+）D_12 + Ca ~（2+）D_1振荡在老化的受精卵母细胞。(5)颗粒细胞与甾体激素的生成猪颗粒细胞在培养皿上培养时，孕酮在培养液中持续分泌。然而，当细胞覆盖有I型胶原（TIC）凝胶时，与没有TIC凝胶的细胞相比，孕酮产生被抑制至0.34倍（第3天）和0.16倍（第4天）（p<0.01）。在没有TIC凝胶的细胞中，用人FSH（hFSH）处理以剂量依赖性方式增强孕酮的产生，在较小程度上，在有TIC凝胶的细胞中。TIC凝胶覆盖对细胞色素P450胆固醇侧链裂解酶（P450 SCC）的影响也进行了检查。这是胆固醇转化为孕酮的限速酶。与不使用TIC凝胶相比，用TIC凝胶覆盖的细胞中P450 SCC基因表达被抑制至0.62倍（第3天）和0.36倍（第4天）。当将孕酮的直接前体--双烯醇酮加入到培养基中时，TIC凝胶的加入增加了孕酮的产生。此外，hFSH诱导P450 SCC基因表达的细胞与TIC凝胶覆盖和不。少