Characterization of a rat in vitro ovarian culture system to study the ovarian toxicant 4-vinylcyclohexene diepoxide
Characterization of a rat in vitro ovarian culture system to study the ovarian toxicant 4-vinylcyclohexene diepoxide
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DOI：
10.1006/taap.2002.9502
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发表时间：
2002-10-15
影响因子：
3.8
通讯作者：
Hoyer, PB
中科院分区：
文献类型：
--
作者：
Devine, PJ;Sipes, IG;Hoyer, PB
每天重复给大鼠服用职业化学物质4-乙烯基环己烯二环氧化物(VCD)会选择性地耗尽最小的腔前卵泡(原始卵泡和初级卵泡)。这些目标人群很难研究，因为他们只占卵巢组织总量的很小一部分。此外，它们随机分布在整个卵巢中。因此，建立了一个以原始卵泡和初级卵泡为主的新生大鼠卵巢培养系统，并对其对VCD的敏感性进行了评估。新生大鼠在出生后4-19天每天注射VCD(80 mg/kg/day)，首次证实了其对VCD剂量的体内敏感性。PND 19时，原始卵泡和小初级卵泡明显枯竭。然后利用漂浮器官培养系统建立卵巢培养系统，体外处理PND 4Fischer 344大鼠的卵巢。在培养的卵巢中，初始卵泡群由原始卵泡(81%)和小初级卵泡(19%)组成，而在培养15天后发育出较大的腔前卵泡(67%为原始卵泡，31%为小初级卵泡，2%为大初级卵泡)。培养的大鼠卵巢对VCD(大于或等于30微米)孵育的卵泡耗竭非常敏感，并且卵泡的丢失具有时间依赖性(8-15天)。用免疫组织化学方法获得体内证实的VCD暴露的卵巢细胞凋亡的证据。与对照组相比，VCD(30um，15d)孵育的卵巢中凋亡相关活性caspase-3和TUNEL的表达显著增加。这些结果表明，培养的新生大鼠卵巢腔前小卵泡对VCD暴露敏感。VCD在体外和体内的作用相似，这表明该系统在未来与VCD或其他卵毒化学物质引起的卵巢卵泡丢失相关的机制研究中是有用的。(C)2002年埃尔塞维尔科学公司(美国)。
Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) causes selective depletion of the smallest preantral ovarian follicles (primordial and primary). These targeted populations are difficult to study because they comprise very little of the overall mass of ovarian tissue. Additionally, they are randomly distributed throughout the ovary. Therefore, a neonatal rat ovarian culture system containing predominantly primordial and primary follicles was developed and its susceptibility to VCD was assessed. The in vivo sensitivity of neonatal rats to VCD dosing was first confirmed by daily injection of VCD (80 mg/kg/day ip) on postnatal days (PND) 4-19. On PND 19, depletion of primordial and small primary follicles was evident. Ovarian cultures were then established utilizing a floating organ culture system to treat ovaries from PND 4 Fischer 344 rats in vitro. Initial follicle populations in cultured ovaries consisted of primordial (81%) and small primary (19%) follicles, whereas larger-sized preantral follicles had developed after 15 days in culture (67% primordial, 31% small primary, and 2% large primary). Cultured rat ovaries were sensitive to follicle depletion by incubation with VCD (greater than or equal to30 muM), and follicle loss occurred in a time-dependent manner (8-15 days). Evidence for apoptosis in VCD-exposed ovaries, as demonstrated in vivo, was obtained using immunohistochemistry. There was significantly more staining for apoptosis-associated active caspase-3 and TUNEL in ovaries incubated with VCD (30 muM, 15 days) compared with matched controls. These results demonstrate that small preantral follicles of cultured neonatal rat ovaries are sensitive to VCD exposure. The similarities between VCD's effects in vitro and in vivo demonstrate the usefulness of this system for future mechanistic studies related to ovarian follicle loss induced by VCD or other ovotoxic chemicals. (C) 2002 Elsevier Science (USA).