A conditional tetracycline-regulated increase in gamma amino butyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat

Gamma amino butyric acid (GABA) is the main inhibitory neuro-transmitter co ntrolling LH-releasing hormone (LHRH) secretion in the mammalian hypothalam us. 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. Immortalize d BAS-8.1 astroglial cells were genetically modified by infection with a re gulatable retroviral vector to express the gene encoding the GABA synthesiz ing enzyme glutamic acid decarboxylase-67 (GAD-67) under the control of a t etracycline (tet) controlled gene expression system. In this system, expres sion of the gene of interest is repressed by tet and activated in the absen ce 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 challe nged with glutamate. All of these responses were inhibited within 24 h of e xposure to tet ("GAD off"). Grafting "GAD on" cells into the median eminenc e of late juvenile female rats, near LHRH nerve terminals, did not affect t he age at vaginal opening, but greatly disrupted subsequent estrous cyclici ty. 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 do xycycline (DOXY) in the drinking water inhibited GAD-67synthesis and restor ed estrous cyclicity to a pattern indistinguishable from that of control fa ts grafted with native BAS-8.1 cells. Animals carrying "GAD on" cells showe d 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 re vealed that both groups grafted with GABA-producing cells had an increased incidence of large antral follicles (> 500 mum) compared with animals graft ed with native BAS8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 mum) was gre ater in "GAD on" than in "GAD off' rats. These results indicate that a regi onally discrete, temporally controlled increase in GABA availability to LHR H nerve terminals in the median eminence of the hypothalamus suffices to di srupt estrous cyclicity in the rat, and raise the possibility that similar local alterations in GABA homeostasis may contribute to the pathology of hy pothalamic amenorrhea/oligomenorrhea in humans.