Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.
Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.
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DOI：
10.1210/endo.137.4.8625923
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发表时间：
1996-04
期刊：
影响因子：
4.8
通讯作者：
S. Chun;K. Eisenhauer;S. Minami;H. Billig;E. Perlas;A. Hsueh
中科院分区：
文献类型：
--
作者：
S. Chun;K. Eisenhauer;S. Minami;H. Billig;E. Perlas;A. Hsueh
在培养的排卵前卵泡中研究了激素对细胞凋亡的调节。由于早期有腔卵泡在体内生理条件下最容易发生闭锁变性，因此本研究旨在通过体外培养早期有腔卵泡来研究激素对细胞凋亡的调节作用。在大鼠24日龄时植入己烯雌酚以刺激早期有腔卵泡的发育，并在27日龄时收集卵巢。解剖早期窦状卵泡并在有或无激素处理的情况下培养（每小瓶4个）24小时。培养后，从卵泡中提取DNA，并使用3 '-末端标记和凝胶电泳来确定凋亡DNA片段化的程度。凋亡DNA片段的原位分析表明，颗粒细胞在这些卵泡中的主要细胞类型进行凋亡。在不存在激素的情况下培养的卵泡显示凋亡DNA片段化水平增加12倍，FSH以剂量依赖性方式（60%最大抑制和30 ng/ml的表观ED 50）处理可防止凋亡DNA片段化。同样地，用（Bu）2cAMP处理也抑制卵泡凋亡。然而，用LH或人CG治疗，最低限度地抑制了凋亡DNA片段化（最大抑制35%）。胰岛素样生长因子-I（IGF-I）也抑制了45%的细胞凋亡。此外，FSH对细胞凋亡的抑制作用部分逆转与IGF结合蛋白-3共孵育，表明内源性IGF-I的潜在介导作用。然而，重组牛生长激素对卵泡凋亡没有影响，尽管它能够刺激IGF-I信使RNA（mRNA）水平。与表皮生长因子（EGF）和碱性成纤维细胞生长因子孵育的卵泡最大抑制卵泡凋亡，分别只有32%和42%。配体结合分析表明，EGF对早期有腔卵泡细胞凋亡的最小有效性，与早期报道的排卵前卵泡中的有效作用相比，可能是由于成熟卵泡中EGF受体浓度增加了3.5倍。高剂量（150或500 ng/ml）的白细胞介素-1 β也抑制了48%的细胞凋亡，而用NO发生器，硝普钠或环GMP类似物治疗抑制细胞凋亡与FSH一样有效。此外，用激活素处理导致剂量相关的卵泡凋亡抑制，达到最大40%的抑制。与此相反，激活素与其结合蛋白，卵泡抑素的共同治疗，取消了这种效果。总的来说，这些数据表明卵泡凋亡的激素调节存在阶段依赖性差异。虽然FSH、LH/人CG、GH、IGF-I、EGF、碱性成纤维细胞生长因子和白细胞介素-1 β都是排卵前卵泡的有效存活因子，但FSH是早期有腔卵泡的主要存活因子，在此期间，大多数卵泡在生理条件下发生闭锁。
Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.