多囊卵巢综合征患者卵巢颗粒细胞凋亡的研究
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摘要
多囊卵巢综合征( PCOS)是一种以内分泌紊乱为主、多种代谢异常导致的异质性临床综合征。PCOS患者双侧卵巢有大量窦卵泡存在,但是不能周期性产生成熟卵泡,其病理机制尚不清楚。目前认为原发性、内在性卵泡生长异常可能是引起PCOS不排卵及临床内分泌改变的病理基础。研究表明,内在性的卵泡发育异常可能与卵巢颗粒细胞的凋亡调控失常有关。内质网应激(ERS)启动的凋亡途径是近年才发现的一种新的凋亡途径。通过激活未折叠蛋白反应(UPR)以保护由ERS所引起的细胞损伤,恢复细胞功能,但是如果损伤太过严重,内环境稳定不能及时恢复, ERS则引起细胞凋亡。DNA受到损害的卵巢颗粒细胞存在内质网应激标志性蛋白CHOP表达。在上述研究的基础上,本文探讨了多囊卵巢综合征患者卵巢颗粒细胞凋亡与PCOS的发病可能的相关关系。
目的:
旨在研究多囊卵巢综合征患者卵巢组织切片各级卵泡数和其颗粒细胞的凋亡情况,以及凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白的表达。
方法:
选择因多囊卵巢综合征行卵巢楔形切除的卵巢病理组织21例,同时选择年龄相匹配并于卵泡期手术的非多囊卵巢综合征正常卵巢组织15例为对照组。(1) HE染色,采用双盲读片,比较两组中各级卵泡的数目;(2) 3-末端原位标记法(TUNEL)检测各级卵泡颗粒细胞的凋亡情况,比较两组凋亡率差异;(3)免疫组化法检测凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白在各级卵泡颗粒细胞表达情况,比较两组间的差异。
结果:
1.PCOS组患者卵巢生长卵泡数增加
光镜下病理学观察:由两位病理科主治医师采取双盲法在光镜下观察其病理结构,随机挑选10个视野(10×10),记录各级卵泡的数量。两组卵巢组织切片始基卵泡、闭锁卵泡比较,差异均无统计学意义;PCOS组过渡型初级卵泡、典型初级卵泡、次级卵泡、窦卵泡较对照组增多,差异有统计学意义(P<0.05)。卵泡计数:PCOS组较对照组初级卵泡增加1.8倍,次级卵泡数和窦卵泡增加1.6倍,尤其典型初级卵泡数增加约3倍。
2.PCOS患者窦卵泡颗粒细胞凋亡率增加
TUNEL法:每张切片随机取10个视野,观察窦卵泡中TUNEL染色阳性颗粒细胞占颗粒细胞总数的百分比值,即凋亡指数AI。两组卵巢组织切片显示始基卵泡、初级卵泡、次级卵泡颗粒细胞的凋亡率比较,差异无统计学意义;窦卵泡颗粒细胞凋亡率比较,PCOS组的凋亡率高于对照组,差异有统计学意义(P<0.05)。
3.PCOS患者窦卵泡颗粒细胞凋亡调控蛋白Bcl - 2蛋白表达减少,Caspase3表达增加
免疫组化:Bcl-2、caspase3蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色,PCOS组窦卵泡颗粒细胞Bcl - 2蛋白染色强度高于对照组, Caspase3蛋白染色强度低于对照组,两组比较,差异有统计学意义(P<0.05)。
4.PCOS患者窦卵泡颗粒细胞内质网应激标志性蛋白CHOP蛋白表达增加
免疫组化:内质网应激标志性蛋白Bip蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色; CHOP蛋白主要表达于颗粒细胞的胞核,以胞核呈棕黄色颗粒为阳性。窦卵泡颗粒细胞CHOP蛋白染色强度高于对照组,两组比较,差异有统计学意义(P<0.05)。BiP蛋白在PCOS窦卵泡颗粒细胞的染色强度高于对照组,比较差异无显著性。
结论:
PCOS组患者卵巢切片早期生长卵泡及窦卵泡数增加,窦卵泡颗粒细胞凋亡增加,凋亡调控蛋白和内质网应激标志性蛋白表达异常,提示PCOS患者卵巢颗粒细胞凋亡异常,内质网应激可能参与PCOS的发病。
Polycystic ovary syndrome (PCOS) is a heterogeneity of clinical syndrome, which is caused by a wide range of metabolic abnormalities, mainly endocrine disorders. Both ovaries of PCOS patients contain a large number of antral follicle, however they are not able to produce mature follicle periodically. The pathological mechanism of PCOS has not yet been made clear. Presently the primary, intrinsic abnormalities of follicle growth are considered to be the possible pathological mechanism basis which causes PCOS non-ovulation and clinical endocrine abnormality. Studies show that the intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Recent studies point to the role of the endoplasmic reticulum (ER) in the sensing and transduction of apoptotic signals. Studies have established that expression of mutant, folding incompetent proteins causes ER stress and elicits an ER stress response, called the unfolded protein response (UPR). The initial intent of the UPR is adaptation and restoration of the normal ER function. The failure of such adaptative mechanisms leads to alarm signaling and finally to cell suicide, usually in the form of apoptosis, as a last resort to do away with dysfunctional cells.CHOP expressed in DNA-damaged ovarian granulosa cells. On the basic of these results, here we investigated test our hypothesis that differences in granulosa cell apoptosis may underlie abnormalities that affect follicular development, and to explore the pathogenesis of PCOS.
OBJECTIVE:
The purposes of this study were to score the composition of follicles in normal and PCOS ovaries, and to investigate apoptosis and the expressions of Bcl - 2 , Caspase3 , Bip and CHOP in granulosa cells of PCOS .Based on these experiments, we tried to make some exploration on the pathogenesis of PCOS.
METHODS:
We took 21 cases of ovarian wedge resection of ovarian pathology due to PCOS, and 15 cases of normal (non-PCOS) ovarian tissue from age-matched patients at early stage of the menstrual cycle as control group. Ovarian follicles of two groups were counted by morphometric analysis.Granulosa cells apoptosis in ovaries was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL).The expression of apoptosis regulatory protein caspase3, Bcl-2 and endoplasmic reticulum stress protein Bip, CHOP protein was analysed with immunohistochemical staining.
RESULTS:
1.Stockpiling of growing follicles in ovaries of women with PCOS
Two pathology physicians observed the pathological structure in the optical microscope double-blindly. 10 visual fields (10*10) were randomly selected, and recorded the number of follicles at all levels. The total number of growing follicles was significantly greater in PCOS ovaries than normal, but the number of nongrowing primordial follicles did not differ. The transitional primary follicles, typical primary follicles, secondary follicles and antral follicle are more than control group, the differences are statistically significant. (P<0.05).Differential counts showed that the number of growing follicles at each stage of development was significantly greater: PCOS had 1.8-fold more primary, 1.6-fold more secondary, and 1.6-fold more Graafian follicles than normal. The greatest effect was on the classic primary follicles where the number was almost 3-fold greater in PCOS ovaries.
2. Granulosa cells from PCOS patients have higher apoptotic rates
Ten visual fields were randomly chosen for each slice, and observed the percentage of TUNEL-positive granulosa cells of the total granulosa cell number, that is, apoptotic index (AI). The comparison of ovarian tissue biopsy revealed that the apoptosis rate of primordial follicle, primary follicle and secondary follicle granulosa cells of the two groups are not significantly different. The antral follicular granulosa cell apoptosis rate of PCOS group is higher than control group. The difference is statistically significant. (P<0.05)
3. Expression of apoptotic regulators in granulosa cells
Caspase3 and Bcl-2 protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. Significantly higher apoptotic rates were associated with stronger intensities immunostaining of the apoptotic effector caspase-3 (P<0.05) and weaker intensities immunostaining of the anti-apoptotic survival factor cellular Bcl -2 in the PCOS group (P<0.05).
4. Expression of endoplasmic reticulum stress protein in granulosa cells
Hallmark of endoplasmic reticulum stress protein CHOP protein was mainly expressed in the nuclei of granulosa cells.BIP protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. The intensities of CHOP immunostaining in PCOS ovaries were significantantly stronger compared to human normal ovaries.No significant difference of Bip expression was found between follicle of PCOS and the normal control group.
CONCLUSIONS:
PCOS patients have higher numbers of primary follicles, secondary follicles and antral follicle in ovarian compared with normal women. Significantly higher apoptotic rates were found in granulosa cells of patients with PCOS, compared with women with regular ovulatory cycles.The intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Endoplasmic reticulum stress might be involved in the pathogenesis of PCOS.
目的:
旨在研究多囊卵巢综合征患者卵巢组织切片各级卵泡数和其颗粒细胞的凋亡情况,以及凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白的表达。
方法:
选择因多囊卵巢综合征行卵巢楔形切除的卵巢病理组织21例,同时选择年龄相匹配并于卵泡期手术的非多囊卵巢综合征正常卵巢组织15例为对照组。(1) HE染色,采用双盲读片,比较两组中各级卵泡的数目;(2) 3-末端原位标记法(TUNEL)检测各级卵泡颗粒细胞的凋亡情况,比较两组凋亡率差异;(3)免疫组化法检测凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白在各级卵泡颗粒细胞表达情况,比较两组间的差异。
结果:
1.PCOS组患者卵巢生长卵泡数增加
光镜下病理学观察:由两位病理科主治医师采取双盲法在光镜下观察其病理结构,随机挑选10个视野(10×10),记录各级卵泡的数量。两组卵巢组织切片始基卵泡、闭锁卵泡比较,差异均无统计学意义;PCOS组过渡型初级卵泡、典型初级卵泡、次级卵泡、窦卵泡较对照组增多,差异有统计学意义(P<0.05)。卵泡计数:PCOS组较对照组初级卵泡增加1.8倍,次级卵泡数和窦卵泡增加1.6倍,尤其典型初级卵泡数增加约3倍。
2.PCOS患者窦卵泡颗粒细胞凋亡率增加
TUNEL法:每张切片随机取10个视野,观察窦卵泡中TUNEL染色阳性颗粒细胞占颗粒细胞总数的百分比值,即凋亡指数AI。两组卵巢组织切片显示始基卵泡、初级卵泡、次级卵泡颗粒细胞的凋亡率比较,差异无统计学意义;窦卵泡颗粒细胞凋亡率比较,PCOS组的凋亡率高于对照组,差异有统计学意义(P<0.05)。
3.PCOS患者窦卵泡颗粒细胞凋亡调控蛋白Bcl - 2蛋白表达减少,Caspase3表达增加
免疫组化:Bcl-2、caspase3蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色,PCOS组窦卵泡颗粒细胞Bcl - 2蛋白染色强度高于对照组, Caspase3蛋白染色强度低于对照组,两组比较,差异有统计学意义(P<0.05)。
4.PCOS患者窦卵泡颗粒细胞内质网应激标志性蛋白CHOP蛋白表达增加
免疫组化:内质网应激标志性蛋白Bip蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色; CHOP蛋白主要表达于颗粒细胞的胞核,以胞核呈棕黄色颗粒为阳性。窦卵泡颗粒细胞CHOP蛋白染色强度高于对照组,两组比较,差异有统计学意义(P<0.05)。BiP蛋白在PCOS窦卵泡颗粒细胞的染色强度高于对照组,比较差异无显著性。
结论:
PCOS组患者卵巢切片早期生长卵泡及窦卵泡数增加,窦卵泡颗粒细胞凋亡增加,凋亡调控蛋白和内质网应激标志性蛋白表达异常,提示PCOS患者卵巢颗粒细胞凋亡异常,内质网应激可能参与PCOS的发病。
Polycystic ovary syndrome (PCOS) is a heterogeneity of clinical syndrome, which is caused by a wide range of metabolic abnormalities, mainly endocrine disorders. Both ovaries of PCOS patients contain a large number of antral follicle, however they are not able to produce mature follicle periodically. The pathological mechanism of PCOS has not yet been made clear. Presently the primary, intrinsic abnormalities of follicle growth are considered to be the possible pathological mechanism basis which causes PCOS non-ovulation and clinical endocrine abnormality. Studies show that the intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Recent studies point to the role of the endoplasmic reticulum (ER) in the sensing and transduction of apoptotic signals. Studies have established that expression of mutant, folding incompetent proteins causes ER stress and elicits an ER stress response, called the unfolded protein response (UPR). The initial intent of the UPR is adaptation and restoration of the normal ER function. The failure of such adaptative mechanisms leads to alarm signaling and finally to cell suicide, usually in the form of apoptosis, as a last resort to do away with dysfunctional cells.CHOP expressed in DNA-damaged ovarian granulosa cells. On the basic of these results, here we investigated test our hypothesis that differences in granulosa cell apoptosis may underlie abnormalities that affect follicular development, and to explore the pathogenesis of PCOS.
OBJECTIVE:
The purposes of this study were to score the composition of follicles in normal and PCOS ovaries, and to investigate apoptosis and the expressions of Bcl - 2 , Caspase3 , Bip and CHOP in granulosa cells of PCOS .Based on these experiments, we tried to make some exploration on the pathogenesis of PCOS.
METHODS:
We took 21 cases of ovarian wedge resection of ovarian pathology due to PCOS, and 15 cases of normal (non-PCOS) ovarian tissue from age-matched patients at early stage of the menstrual cycle as control group. Ovarian follicles of two groups were counted by morphometric analysis.Granulosa cells apoptosis in ovaries was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL).The expression of apoptosis regulatory protein caspase3, Bcl-2 and endoplasmic reticulum stress protein Bip, CHOP protein was analysed with immunohistochemical staining.
RESULTS:
1.Stockpiling of growing follicles in ovaries of women with PCOS
Two pathology physicians observed the pathological structure in the optical microscope double-blindly. 10 visual fields (10*10) were randomly selected, and recorded the number of follicles at all levels. The total number of growing follicles was significantly greater in PCOS ovaries than normal, but the number of nongrowing primordial follicles did not differ. The transitional primary follicles, typical primary follicles, secondary follicles and antral follicle are more than control group, the differences are statistically significant. (P<0.05).Differential counts showed that the number of growing follicles at each stage of development was significantly greater: PCOS had 1.8-fold more primary, 1.6-fold more secondary, and 1.6-fold more Graafian follicles than normal. The greatest effect was on the classic primary follicles where the number was almost 3-fold greater in PCOS ovaries.
2. Granulosa cells from PCOS patients have higher apoptotic rates
Ten visual fields were randomly chosen for each slice, and observed the percentage of TUNEL-positive granulosa cells of the total granulosa cell number, that is, apoptotic index (AI). The comparison of ovarian tissue biopsy revealed that the apoptosis rate of primordial follicle, primary follicle and secondary follicle granulosa cells of the two groups are not significantly different. The antral follicular granulosa cell apoptosis rate of PCOS group is higher than control group. The difference is statistically significant. (P<0.05)
3. Expression of apoptotic regulators in granulosa cells
Caspase3 and Bcl-2 protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. Significantly higher apoptotic rates were associated with stronger intensities immunostaining of the apoptotic effector caspase-3 (P<0.05) and weaker intensities immunostaining of the anti-apoptotic survival factor cellular Bcl -2 in the PCOS group (P<0.05).
4. Expression of endoplasmic reticulum stress protein in granulosa cells
Hallmark of endoplasmic reticulum stress protein CHOP protein was mainly expressed in the nuclei of granulosa cells.BIP protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. The intensities of CHOP immunostaining in PCOS ovaries were significantantly stronger compared to human normal ovaries.No significant difference of Bip expression was found between follicle of PCOS and the normal control group.
CONCLUSIONS:
PCOS patients have higher numbers of primary follicles, secondary follicles and antral follicle in ovarian compared with normal women. Significantly higher apoptotic rates were found in granulosa cells of patients with PCOS, compared with women with regular ovulatory cycles.The intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Endoplasmic reticulum stress might be involved in the pathogenesis of PCOS.
引文
1. Azziz R, Woods KS, Reyna R,et al. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab.2004 ,89(6):2745-2749.
2. Webber LJ, Stubbs S, Stark J,et al. Formation and early development of follicles in the polycystic ovary. Lancet,2003,362(9389):1017-1021.
3. Frank S, Stark J and Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Human Reproduction Update, 2008,14(4):367-378.
4. Cataldo NA , Dumesic DA , Goldsmith PC , et al . Immunolocalization of Fas and Fas ligand in ovaries of women with polycystic ovary syndrome :relationship to apoptosis. Hum Reprod , 2000,15(9) : 1889 - 1897.
5. Honnma H, Endo T,Henmi H,et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis,2006,11(9):1525-1533.
6. Salvetti NR, Panzani CG, Gimeno EJ, et al.An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats.Reprod Biol Endocrinol. 2009 1,7:68.
7. Das M, Djahanbakhch O, Hacihanefioglu B, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome[J]. J Clin Endocrinol Metab, 2008, 93(3):881-887.
8. Voeltz G K, Rolls MM, Rapoport TA. Structural organization of the endoplasmic reticulum. EMBO Rep, 2002,3(10): 944-950。
9. Hampton RY. ER-associated degradation in protein quality control and cellular regulation. Curr Opin Cell Biol, 2002,14(4): 476-482.
10. Tanimura A, Yujiri T, Tanaka Y,et al. The anti-apoptotic role of the unfolded protein response in Bcr-Abl-positive leukemia cells. Leuk Res. 2009,33(7):924-928.
11. Sundar Rajan S, Srinivasan V, Balasubramanyam M, et al. Endoplasmic reticulum (ER) stress & diabetes. Indian J Med Res. 2007,125(3):411-424.
12. Yoshimura FK, Luo X. Induction of endoplasmic reticulum stress in thymic lymphocytes by theenvelope precursor polyprotein of a murine leukemia virus during the preleukemic period. J Virol. 2007 ,81(8):4374-4377.
13. Szegezdi E, Logue SE, Gorman AM,et al.. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006,7(9):880-885.
14. Oktay K, Briggs D, Gosden RG.Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab .1997,82 (11):3748-3751.
15. Kim JM , Yoon YD, Tsang BK. Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia. Endocrinology Philadelphia, 1999, 140(5): 2307-2317.
16. Hughesdon PE. Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called“hyperthecosis”. Obstet Gynecol Surv,1982,37(2);59-37.
17. Maciel GA, Baracat EC, Benda JA,et al. Stockpiling of transitional and classic primary follicles in ovaries of woman polycystic ovary syndrome. J Clin Endocrinol Metab. 2004,89(11),5321-5327.
18. Webber LJ, Stubbs SA, Stark J,et al.. Prolonged survival in culture of preantral follicles from polycystic ovaries. J Clin Endocrinol Metab. 2007,92(5):1975-1978.
19. Stubbs SA, Stark J, Dilworth SM,et al. Abnormal Preantral Folliculogenesis in Polycystic Ovaries is Associated with Increased Granulosa Cell Division. J Clin Endocrinol Metab,2007, 92(11):4418-4426.
20. Hillier SG. Gonadotropin control of ovarian folliclar growth and development. Mol Cell Endocrinol, 2001,179(1-2):39-46.
21. Amsterdam A, Keren-Tal I, Aharoni D.Cross-talk between cAMP and p53-generated signals in induction of differentiation and apoptosis in steroidogenic granulosa cells.Steroids. 1996,61(4):252-256.
22. Kugu K, Ratts VS, Piquette GN,et al. Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ, 1998(1),5:67-76.
23. Depalo R, Nappi L, Loverro G,et al. Evidence of apoptosis in human primordial and primaryfollicles. Hum Reprod, 2003,18(2):2678-2682.
24. Albamonte MS, Willis MA, Albamonte MI,et al. The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis. Hum Reprod. 2008,23(8):1895-1901.
25. Matikainen T, Perez GI, Zheng TS,et al. Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary. Endocrinology. 2001,142(6):2468-2480
26. Glamoclija V, Vilovi? K, Saraga-Babi? M,et al. Apoptosis and active caspase-3 expression in human granulosa cells. Fertil Steril. 2005,83(2):426-431.
27. Isobe N, Yoshimura Y. Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle. J Reprod Dev. 2007 ,53(5):1119-1124.
28. Isobe N, Yoshimura Y. Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows. Theriogenology. 2000,54(7):1159-1169.
29. Baravalle C, Salvetti NR, Mira GA, et al. The role of ACTH in the pathogenesis of polycystic ovarian syndrome in rats: hormonal profiles and ovarian morphology. Physiol Res. 2007,56(1):67-78.
30. Rutkowski DT. A trip to the ER: coping with stress. Trends Cell Biol, 2004,14(1): 20-28.
31. Honnma H, Endo T,Henmi H,et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis,2006,11(9):1525-1533.
32. Ron D, Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev, 1992,6(3): 439-453.
33. Gotoh T, Oyadomari S, Mori K, et al.Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J Biol Chem. 2002,277(14):12343-12350.
34. Katayama T, Imaizumi K, Honda A, Yoneda T, Kudo T, Takeda M, Mori K, Rozmahel R, Fraser P, George-Hyslop PS, Tohyama M. Disturbed activation of endoplasmic reticulum stresstransducers by familial Alzheimer’s disease-linked presenilin-1 mutations. J Biol Chem, 2001,276(46): 43446-43454.
35. Chua CE, Tang BL. Alpha-synuclein and Parkinson’s disease: the first roadblock. J Cell Mol Med, 2006,10(4): 837-846.
36. Eizirik D L, Cardozo A K, Cnop M. The role for endoplasmic reticulum stress in diabietes mellitus. Endocr Rev, 2008,29(1): 42-61.
37. Michael C. Allemand1, Brian A. et al.Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance. PLoS One. 2009,4(1):e4274.
38. Andrea Dunaif, Jinru Xia, Carol-Beth Book,et al.Excessive Insulin Receptor Serine Phosphorylation in Cultured Fibroblasts and in Skeletal Muscle A Potential Mechanism for Insulin Resistance in the Polycystic Ovary Syndrome.J Clin Invest. 1995,96(2):801-810.
39. Kaneto H, Nakatani Y, Kawamori D,. Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance. Int J Biochem Cell Biol. 2006,38(5-6):782-93.
40. Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes . Science,2004, 306 (5695): 4574 - 4561.
41. Muoio DM, Newgard CB. Biomedicine. Insulin resistance takes a trip through the ER. Science, 2004, 306 (5695) : 425 - 426.
42. Oyadomari S, Koizumi A, Takeda K, et al. Targeted disrup tion of the Chop gene delays endop lasmic reticulum stress2mediated diabetes. J Clin Invest, 2002, 109(4): 525 - 532.
43. Nishitoh H,Matsuzawa A, Tobiume K, et al. ASK1 is essential to endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev, 2002,16(11): 1345 - 1355.
1. Matsuda-Minehata F, Inoue N, Goto Y, et al.Regulation of Ovarian Granulosa Cell Death by Pro- and Anti-apopotic Molecules[J]. J Reprod Dev, 2006, 52(6):695-705.
2. Hussein MR. Apoptosis in the ovary : molecular mechanisms[J]. Hum Reprod Update, 2005, 11 ( 2 ) : 161-177.
3. Inoue N, Maeda A, Matsuda-Minehata F, et al. Expression and localization of Fas ligand and Fas during atresia in porcine ovarian follicles[J]. J Reprod Dev,2006, 52(6):723-730.
4. Vital-Reyes V, Rodríguez-Burford C, Chhieng DC, et al.Ovarian expression of markers associated with proliferation or apoptosis in women with diminished ovarian reserve[J]. Fertil Steril,2006 ,86(1):176-185.
5. Buchsbaum DJ, Zhou T, Lobuglio AF, et a.l TRAIL receptor-targeted therapy[J]. Future Oncol,2006 ,2(4): 493-508.
6. J??skel?inen M, Kyr?nlahti A, Anttonen M, et al. TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary[J]. Differentiation, 2009, 77(4):369-376.
7. Das M, Djahanbakhch O, Hacihanefioglu B, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome[J]. J Clin Endocrinol Metab, 2008, 93(3):881-887.
8. Stubbs SA, Stark J, Dilworth SM, et al. Abnormal Preantral Folliculogenesis in Polycystic Ovaries is Associated with Increased Granulosa Cell Division[J]. J Clin Endocrinol Metab, 2007, 92(11):4418-4426.
9. Honnma H, Endo T, Henmi H, et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles withindehydroepiandrosterone-induced polycystic ovaries in rats[J]. Apoptosis, 2006, 11(9):1525-1533.
10. Schimmer AD, Dalili S, Batey RA , et al. Targeting XIAP for treatment of malignancy[J]. Cell Death Differ, 2006, 13(2):179-188.
11. Cheng Y, Maeda A, Goto Y, Matsuda F, et al. Changes in expression and localization of X-linked inhibitor of apoptosis protein (XIAP) in follicular granulosa cells during atresia in porcine ovaries[J]. J Reprod Dev, 2008, 54(6):454-459.
12. Webber LJ,Stubbs S, Stark J, et al. Prolonged survival in culture of preantral follicles form polycystic ovaries[J]. J Clin Endocrinol Metab, 2007, 92(5):1975-1978.
13. Inoue N,Manabe N,Matsut T,et a1. Roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries[J]. J Reprod Dev,2003, 49 (4):313—321.
14. Johnson AL, Ratajczak C, Haugen MJ,et al. Tumor necrosis factor-related apoptosis inducing ligand expression and activity in hen granulosa cells.Reproduction. 2007,133(3):609-616.
15. Azziz R,Carmina E,Dewailly D, et a1. Criteria for defining polycystic ovary syndrome as a predominan tly hyperandmgenie syndrome:all an drogen excess society guideline[J].J Clin Endocrinol Metab, 2006, 91(11):4237-4245.
16. Ska?ba P. Progress in gynecological endocrinology[J]. Ginekol Pol, 2008, 79(12):877-881.
17. Okutsu Y, Itoh MT, Takahashi N, et a1. Exogenous androstenedione induces formation of follicular cysts and premature luteinization of granulosa cells in the ovary[J]. Fertil Steril, 2008 Dec 17. [Epub ahead of print]
18. Kewalramani G, Puthanveetil P, Wang F, et al. AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death[J]. Cardiovasc Res, 2009, 28. [Epub ahead of print]
19. Onalan G, Selam B, Baran Y, et al. Serum and follicular fluid levels of soluble Fas, soluble Fas ligand and apoptosis of luteinized granulosa cells in PCOS patients undergoing IVF. Hum Reprod, 2005, 20(9):2391-2395.
20. Tosca L, Uzbekova S, Chabrolle C, et al. Effects of Metformin on Bovine Granulosa Cells Steroidogenesis: Possible Involvement of Adenosine 50 Monophosphate-Activated Protein Kinase (AMPK) [J]. Biol Reprod, 2007, 76(3):368-378.
21. Eng GS, Sheridan RA, Wyman A.AMP kinase activation increases glucose uptake, decreases apoptosis, and improves pregnancy outcome in embryos exposed to high IGF-I concentrations[J]. Diabetes, 2007, 56(9):2228-2234.
2. Webber LJ, Stubbs S, Stark J,et al. Formation and early development of follicles in the polycystic ovary. Lancet,2003,362(9389):1017-1021.
3. Frank S, Stark J and Hardy K. Follicle dynamics and anovulation in polycystic ovary syndrome. Human Reproduction Update, 2008,14(4):367-378.
4. Cataldo NA , Dumesic DA , Goldsmith PC , et al . Immunolocalization of Fas and Fas ligand in ovaries of women with polycystic ovary syndrome :relationship to apoptosis. Hum Reprod , 2000,15(9) : 1889 - 1897.
5. Honnma H, Endo T,Henmi H,et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis,2006,11(9):1525-1533.
6. Salvetti NR, Panzani CG, Gimeno EJ, et al.An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats.Reprod Biol Endocrinol. 2009 1,7:68.
7. Das M, Djahanbakhch O, Hacihanefioglu B, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome[J]. J Clin Endocrinol Metab, 2008, 93(3):881-887.
8. Voeltz G K, Rolls MM, Rapoport TA. Structural organization of the endoplasmic reticulum. EMBO Rep, 2002,3(10): 944-950。
9. Hampton RY. ER-associated degradation in protein quality control and cellular regulation. Curr Opin Cell Biol, 2002,14(4): 476-482.
10. Tanimura A, Yujiri T, Tanaka Y,et al. The anti-apoptotic role of the unfolded protein response in Bcr-Abl-positive leukemia cells. Leuk Res. 2009,33(7):924-928.
11. Sundar Rajan S, Srinivasan V, Balasubramanyam M, et al. Endoplasmic reticulum (ER) stress & diabetes. Indian J Med Res. 2007,125(3):411-424.
12. Yoshimura FK, Luo X. Induction of endoplasmic reticulum stress in thymic lymphocytes by theenvelope precursor polyprotein of a murine leukemia virus during the preleukemic period. J Virol. 2007 ,81(8):4374-4377.
13. Szegezdi E, Logue SE, Gorman AM,et al.. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006,7(9):880-885.
14. Oktay K, Briggs D, Gosden RG.Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab .1997,82 (11):3748-3751.
15. Kim JM , Yoon YD, Tsang BK. Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia. Endocrinology Philadelphia, 1999, 140(5): 2307-2317.
16. Hughesdon PE. Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called“hyperthecosis”. Obstet Gynecol Surv,1982,37(2);59-37.
17. Maciel GA, Baracat EC, Benda JA,et al. Stockpiling of transitional and classic primary follicles in ovaries of woman polycystic ovary syndrome. J Clin Endocrinol Metab. 2004,89(11),5321-5327.
18. Webber LJ, Stubbs SA, Stark J,et al.. Prolonged survival in culture of preantral follicles from polycystic ovaries. J Clin Endocrinol Metab. 2007,92(5):1975-1978.
19. Stubbs SA, Stark J, Dilworth SM,et al. Abnormal Preantral Folliculogenesis in Polycystic Ovaries is Associated with Increased Granulosa Cell Division. J Clin Endocrinol Metab,2007, 92(11):4418-4426.
20. Hillier SG. Gonadotropin control of ovarian folliclar growth and development. Mol Cell Endocrinol, 2001,179(1-2):39-46.
21. Amsterdam A, Keren-Tal I, Aharoni D.Cross-talk between cAMP and p53-generated signals in induction of differentiation and apoptosis in steroidogenic granulosa cells.Steroids. 1996,61(4):252-256.
22. Kugu K, Ratts VS, Piquette GN,et al. Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ, 1998(1),5:67-76.
23. Depalo R, Nappi L, Loverro G,et al. Evidence of apoptosis in human primordial and primaryfollicles. Hum Reprod, 2003,18(2):2678-2682.
24. Albamonte MS, Willis MA, Albamonte MI,et al. The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis. Hum Reprod. 2008,23(8):1895-1901.
25. Matikainen T, Perez GI, Zheng TS,et al. Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary. Endocrinology. 2001,142(6):2468-2480
26. Glamoclija V, Vilovi? K, Saraga-Babi? M,et al. Apoptosis and active caspase-3 expression in human granulosa cells. Fertil Steril. 2005,83(2):426-431.
27. Isobe N, Yoshimura Y. Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle. J Reprod Dev. 2007 ,53(5):1119-1124.
28. Isobe N, Yoshimura Y. Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows. Theriogenology. 2000,54(7):1159-1169.
29. Baravalle C, Salvetti NR, Mira GA, et al. The role of ACTH in the pathogenesis of polycystic ovarian syndrome in rats: hormonal profiles and ovarian morphology. Physiol Res. 2007,56(1):67-78.
30. Rutkowski DT. A trip to the ER: coping with stress. Trends Cell Biol, 2004,14(1): 20-28.
31. Honnma H, Endo T,Henmi H,et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis,2006,11(9):1525-1533.
32. Ron D, Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev, 1992,6(3): 439-453.
33. Gotoh T, Oyadomari S, Mori K, et al.Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J Biol Chem. 2002,277(14):12343-12350.
34. Katayama T, Imaizumi K, Honda A, Yoneda T, Kudo T, Takeda M, Mori K, Rozmahel R, Fraser P, George-Hyslop PS, Tohyama M. Disturbed activation of endoplasmic reticulum stresstransducers by familial Alzheimer’s disease-linked presenilin-1 mutations. J Biol Chem, 2001,276(46): 43446-43454.
35. Chua CE, Tang BL. Alpha-synuclein and Parkinson’s disease: the first roadblock. J Cell Mol Med, 2006,10(4): 837-846.
36. Eizirik D L, Cardozo A K, Cnop M. The role for endoplasmic reticulum stress in diabietes mellitus. Endocr Rev, 2008,29(1): 42-61.
37. Michael C. Allemand1, Brian A. et al.Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance. PLoS One. 2009,4(1):e4274.
38. Andrea Dunaif, Jinru Xia, Carol-Beth Book,et al.Excessive Insulin Receptor Serine Phosphorylation in Cultured Fibroblasts and in Skeletal Muscle A Potential Mechanism for Insulin Resistance in the Polycystic Ovary Syndrome.J Clin Invest. 1995,96(2):801-810.
39. Kaneto H, Nakatani Y, Kawamori D,. Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance. Int J Biochem Cell Biol. 2006,38(5-6):782-93.
40. Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes . Science,2004, 306 (5695): 4574 - 4561.
41. Muoio DM, Newgard CB. Biomedicine. Insulin resistance takes a trip through the ER. Science, 2004, 306 (5695) : 425 - 426.
42. Oyadomari S, Koizumi A, Takeda K, et al. Targeted disrup tion of the Chop gene delays endop lasmic reticulum stress2mediated diabetes. J Clin Invest, 2002, 109(4): 525 - 532.
43. Nishitoh H,Matsuzawa A, Tobiume K, et al. ASK1 is essential to endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev, 2002,16(11): 1345 - 1355.
1. Matsuda-Minehata F, Inoue N, Goto Y, et al.Regulation of Ovarian Granulosa Cell Death by Pro- and Anti-apopotic Molecules[J]. J Reprod Dev, 2006, 52(6):695-705.
2. Hussein MR. Apoptosis in the ovary : molecular mechanisms[J]. Hum Reprod Update, 2005, 11 ( 2 ) : 161-177.
3. Inoue N, Maeda A, Matsuda-Minehata F, et al. Expression and localization of Fas ligand and Fas during atresia in porcine ovarian follicles[J]. J Reprod Dev,2006, 52(6):723-730.
4. Vital-Reyes V, Rodríguez-Burford C, Chhieng DC, et al.Ovarian expression of markers associated with proliferation or apoptosis in women with diminished ovarian reserve[J]. Fertil Steril,2006 ,86(1):176-185.
5. Buchsbaum DJ, Zhou T, Lobuglio AF, et a.l TRAIL receptor-targeted therapy[J]. Future Oncol,2006 ,2(4): 493-508.
6. J??skel?inen M, Kyr?nlahti A, Anttonen M, et al. TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary[J]. Differentiation, 2009, 77(4):369-376.
7. Das M, Djahanbakhch O, Hacihanefioglu B, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome[J]. J Clin Endocrinol Metab, 2008, 93(3):881-887.
8. Stubbs SA, Stark J, Dilworth SM, et al. Abnormal Preantral Folliculogenesis in Polycystic Ovaries is Associated with Increased Granulosa Cell Division[J]. J Clin Endocrinol Metab, 2007, 92(11):4418-4426.
9. Honnma H, Endo T, Henmi H, et al. Altered expression of Fas/Fas ligand/caspase8 and membrane type1-matrix metalloproteinase in atretic follicles withindehydroepiandrosterone-induced polycystic ovaries in rats[J]. Apoptosis, 2006, 11(9):1525-1533.
10. Schimmer AD, Dalili S, Batey RA , et al. Targeting XIAP for treatment of malignancy[J]. Cell Death Differ, 2006, 13(2):179-188.
11. Cheng Y, Maeda A, Goto Y, Matsuda F, et al. Changes in expression and localization of X-linked inhibitor of apoptosis protein (XIAP) in follicular granulosa cells during atresia in porcine ovaries[J]. J Reprod Dev, 2008, 54(6):454-459.
12. Webber LJ,Stubbs S, Stark J, et al. Prolonged survival in culture of preantral follicles form polycystic ovaries[J]. J Clin Endocrinol Metab, 2007, 92(5):1975-1978.
13. Inoue N,Manabe N,Matsut T,et a1. Roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries[J]. J Reprod Dev,2003, 49 (4):313—321.
14. Johnson AL, Ratajczak C, Haugen MJ,et al. Tumor necrosis factor-related apoptosis inducing ligand expression and activity in hen granulosa cells.Reproduction. 2007,133(3):609-616.
15. Azziz R,Carmina E,Dewailly D, et a1. Criteria for defining polycystic ovary syndrome as a predominan tly hyperandmgenie syndrome:all an drogen excess society guideline[J].J Clin Endocrinol Metab, 2006, 91(11):4237-4245.
16. Ska?ba P. Progress in gynecological endocrinology[J]. Ginekol Pol, 2008, 79(12):877-881.
17. Okutsu Y, Itoh MT, Takahashi N, et a1. Exogenous androstenedione induces formation of follicular cysts and premature luteinization of granulosa cells in the ovary[J]. Fertil Steril, 2008 Dec 17. [Epub ahead of print]
18. Kewalramani G, Puthanveetil P, Wang F, et al. AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death[J]. Cardiovasc Res, 2009, 28. [Epub ahead of print]
19. Onalan G, Selam B, Baran Y, et al. Serum and follicular fluid levels of soluble Fas, soluble Fas ligand and apoptosis of luteinized granulosa cells in PCOS patients undergoing IVF. Hum Reprod, 2005, 20(9):2391-2395.
20. Tosca L, Uzbekova S, Chabrolle C, et al. Effects of Metformin on Bovine Granulosa Cells Steroidogenesis: Possible Involvement of Adenosine 50 Monophosphate-Activated Protein Kinase (AMPK) [J]. Biol Reprod, 2007, 76(3):368-378.
21. Eng GS, Sheridan RA, Wyman A.AMP kinase activation increases glucose uptake, decreases apoptosis, and improves pregnancy outcome in embryos exposed to high IGF-I concentrations[J]. Diabetes, 2007, 56(9):2228-2234.