Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades
Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades
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DOI：
10.1095/biolreprod65.1.87
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发表时间：
2001-07-01
影响因子：
3.6
通讯作者：
Hoyer, PB
中科院分区：
文献类型：
--
作者：
Hu, XM;Christian, PJ;Hoyer, PB
先前的研究表明，给予4-乙烯基环己烯二环氧化物(VCD)对大鼠的卵毒性可能是通过加速闭锁(细胞凋亡)的正常速率来实现的。本研究旨在探讨与细胞凋亡相关的caspase级联在卵巢小卵泡中的作用。雌性F344大鼠给予单剂量VCD(第1天80 mg/kg，未开始卵毒性的时间)，或每天给药15天(80 mg/kg，第15天，正在进行显著的卵毒性作用的时间)。末次给药后收集卵巢。分离腔前小卵泡(直径25-100微米)，制备细胞片段，测定caspase-3、-8、-9的切割活性或蛋白表达水平。VCD处理(第1天，2.86+/-0.23天；第15天，3.25+/-0.64天，VCD/对照，n=3)可使小卵泡胞浆caspase-3活性升高(P<；0.01)。这种激活在大卵泡或有腔卵泡(不是VCD靶向)中看不到。在第15天，VCD处理组小卵泡中的Procaspase-3蛋白比对照组增加了212%(P<；0.05)，但第1天给药组大鼠的Procaspase-3蛋白没有增加。用共聚焦显微镜评价免疫荧光染色强度。Caspase-3蛋白定位于发育不同阶段的腔前卵泡卵母细胞和颗粒细胞的胞浆室，从第15天开始在原始卵泡和小初级卵泡中选择性增加(P<；0.05)。小卵泡中caspase-8活性在第15天升高，但在第1天处理组大鼠无明显变化，而caspase-9活性在第1天时在线粒体部分被VCD增强。因此，这些数据提供了证据，即VCD诱导的大鼠小卵泡加速闭锁与caspase介导的级联反应的激活有关。
Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25-100 mum in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 +/- 0.23; Day 15, 3.25 +/- 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased (P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.