A conditional tetracycline-regulated increase in gamma amino butyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat
A conditional tetracycline-regulated increase in gamma amino butyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat
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DOI：
10.1210/en.142.5.2102
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发表时间：
2001-05-01
期刊：
影响因子：
4.8
通讯作者：
Ojeda, SR
中科院分区：
文献类型：
--
作者：
Bilger, M;Heger, S;Ojeda, SR
γ-氨基丁酸（GABA）是哺乳动物下丘脑中控制LH释放激素（LHRH）分泌的主要抑制性神经递质。已知GABA作用靶点的神经内分泌脑的离散区域（如正中隆起）内GABA稳态的改变是否会破坏LHRH释放系统维持生殖周期的能力尚不清楚，但可以通过实验进行详细检查。目前的实验是为了研究这个问题。通过用可调节的逆转录病毒载体感染永生化的BAS-8.1星形胶质细胞进行遗传修饰，以在四环素（泰特）控制的基因表达系统的控制下表达编码GABA合成酶谷氨酸脱羧酶-67（GAD-67）的基因。在该系统中，目标基因的表达被泰特抑制，并在不存在抗生素的情况下被激活。携带该调节盒并在不存在泰特（“GAD开启”）的情况下培养的BAS-8.1细胞表达丰富水平的GAD-67信使RNA和GAD酶活性，并且当用谷氨酸激发时释放GABA。所有这些反应在暴露于泰特的24小时内被抑制（“GAD关闭”）。将“GAD on”细胞移植到幼年晚期雌性大鼠的正中隆起，靠近LHRH神经末梢，不影响阴道开口的年龄，但极大地破坏了随后的发情周期。这些动物表现出长时间的持续发情，偶尔在发情前期和发情间期中断，表明发生不规则排卵事件。在饮用水中加入四环素类似物强力霉素（DOXY）可抑制GAD-67的合成，并使动情周期恢复到与移植有天然BAS-8.1细胞的对照脂肪不可区分的模式。携带“GAD on”细胞的动物显示血清LH和雌二醇水平略有增加，血清雄烯二酮显著升高，所有这些都通过关闭移植细胞中的GAD-67合成而消除。卵巢的形态测定分析显示，与移植天然BAS 8.1细胞的动物相比，移植GABA产生细胞的两组动物具有增加的大窦卵泡（> 500 μ m）的发生率，但是在这一类别中，“GAD on”大鼠中类固醇生成更活跃的卵泡（即大于600 μ m）的发生率高于“GAD off”大鼠。这些结果表明，一个区域离散的，时间控制的增加GABA的LHRH神经末梢在正中隆起的下丘脑足以破坏大鼠的动情周期，并提高了可能性，类似的局部变化GABA的稳态可能有助于下丘脑闭经/月经过少的病理人类。
Gamma amino butyric acid (GABA) is the main inhibitory neuro-transmitter controlling LH-releasing hormone (LHRH) secretion in the mammalian hypothalamus. Whether alterations in GABA homeostasis within discrete regions of the neuroendocrine brain known to be targets of GABA action, such as the median eminence, can disrupt the ability of the LHRH releasing system to maintain reproductive cyclicity is not known but amenable to experimental scrutiny. The present experiments were undertaken to examine this issue. Immortalized BAS-8.1 astroglial cells were genetically modified by infection with a regulatable retroviral vector to express the gene encoding the GABA synthesizing enzyme glutamic acid decarboxylase-67 (GAD-67) under the control of a tetracycline (tet) controlled gene expression system. In this system, expression of the gene of interest is repressed by tet and activated in the absence of the antibiotic. BAS-8.1 cells carrying this regulatory cassette, and cultured in the absence of tet ("GAD on"), expressed abundant levels of GAD-67 messenger RNA and GAD enzymatic activity, and released GABA when challenged with glutamate. All of these responses were inhibited within 24 h of exposure to tet ("GAD off"). Grafting "GAD on" cells into the median eminence of late juvenile female rats, near LHRH nerve terminals, did not affect the age at vaginal opening, but greatly disrupted subsequent estrous cyclicity. These animals exhibiting long periods of persistent estrus, interrupted by occasional days in proestrus and diestrus, suggesting the occurrence of irregular ovulatory episodes. Administration of the tetracycline analog doxycycline (DOXY) in the drinking water inhibited GAD-67synthesis and restored estrous cyclicity to a pattern indistinguishable from that of control fats grafted with native BAS-8.1 cells. Animals carrying "GAD on" cells showed a small increase in serum LH and estradiol levels, and a marked elevation in serum androstenedione, all of which were obliterated by turning GAD-67 synthesis off in the grafted cells. Morphometric analysis of the ovaries revealed that both groups grafted with GABA-producing cells had an increased incidence of large antral follicles (> 500 mum) compared with animals grafted with native BAS8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 mum) was greater in "GAD on" than in "GAD off' rats. These results indicate that a regionally discrete, temporally controlled increase in GABA availability to LHRH nerve terminals in the median eminence of the hypothalamus suffices to disrupt estrous cyclicity in the rat, and raise the possibility that similar local alterations in GABA homeostasis may contribute to the pathology of hypothalamic amenorrhea/oligomenorrhea in humans.