Grant-in-Aid for Scientific Research (C)
|Allocation Type||Single-year Grants|
Obstetrics and gynecology
|Research Institution||YAMAGATA UNIVERSITY|
HIROI Masahiko Dep Ob-Gyn, School of Medicine, Yamagata University, Professor, 医学部, 教授 (60018364)
SAITO Takakazu Dep Ob-Gyn, School of Medicine, Yamagata University, Assistant, 医学部, 助手 (00272071)
NAKAHARA Kenji Dep Ob-Gyn, School of Medicine, Yamagata University, Assist. Professor, 医学部, 講師 (80250934)
SAITO Hidekazu Dep Ob-Gyn, School of Medicine, Yamagata University, Assoc. Professor, 医学部, 助教授 (90125766)
TAKAHASHI Toshifumi Dep Ob-Gyn, School of Medicine, Yamagata University, Assistant, 医学部, 助手 (20302292)
ITOH Mariko Dep Ob-Gyn, School of Medicine, Yamagata University, Assistant, 医学部, 助手 (40292429)
太田 信彦 山形大学, 医学部, 助手 (40301072)
|Project Period (FY)
1998 – 1999
Completed(Fiscal Year 1999)
|Budget Amount *help
¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1999 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥2,700,000 (Direct Cost : ¥2,700,000)
|Keywords||Ovarian follicle / Granulosa Cell / Steroidogenesis / Apoptosis / CD44 / 卵透明帯|
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.
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 steroidogenesis
When 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