Search

Much has been learned in recent years about the central mechanisms controlling the initiation of mammalian puberty. It is now clear that this process requires the interactive participation of several genes. Using a combination of high throughput, molecular, and bioinformatics strategies, in combination with a system biology approach, we singled out from the hypothalamus of nonhuman primates and rats a group of related genes whose expression increases at the time of female puberty. Although these genes [henceforth termed tumor-related genes (TRGs)] have diverse cellular functions, they share the common feature of having been earlier identified as involved in tumor suppression/tumor formation. A prominent member of this group is KiSS1, a gene recently shown to be essential for the occurrence of puberty. Cis-regulatory analysis revealed the presence of a hierarchically arranged gene set containing five major hubs (CDP/CUTL1, MAF, p53, YY1, and USF2) controlling the network at the transcriptional level. In turn, these hubs are heavily connected to non-TRGs involved in the transcriptional regulation of the pubertal process. TRGs may be expressed in the mammalian hypothalamus as components of a regulatory gene network that facilitates and integrates cellular and cell-cell communication programs required for the acquisition of female reproductive competence.

γ-Aminobutyric acid (GABA) inhibits the embryonic migration of GnRH neurons and regulates hypothalamic GnRH release. A subset of GnRH neurons expresses GABA along their migratory route in the nasal compartment before entering the brain, suggesting that GABA produced by GnRH neurons may help regulate the migratory process. To examine this hypothesis and the possibility that persistence of GABA production by GnRH neurons may affect subsequent reproductive function, we generated transgenic mice in which the expression of glutamic acid decarboxylase-67 (GAD-67), a key enzyme in GABA synthesis, is targeted to GnRH neurons under the control of the GnRH gene promoter. On embryonic d 15, when GnRH neurons are still migrating, the transgenic animals had more GnRH neurons in aberrant locations in the cerebral cortex and fewer neurons reaching the hypothalamic-preoptic region, whereas migration into the brain was not affected. Hypothalamic GnRH content in mutant mice was low during the first week of postnatal life, increasing to normal values during infantile development (second week after birth) in the presence of increased pulsatile GnRH release. Consistent with these changes, serum LH and FSH levels were also elevated. Gonadotropin release returned to normal values by the time steroid negative feedback became established (fourth week of life). Ovariectomy at this time demonstrated an enhanced gonadotropin response in transgenic animals. Although the onset of puberty, as assessed by the age at vaginal opening and first ovulation, was not affected in the mutant mice, estrous cyclicity and adult reproductive capacity were disrupted. Mutant mice had reduced litter sizes, increased time intervals between deliveries of litters, and a shorter reproductive life span. Thus, GABA produced within GnRH neurons does not delay GnRH neuronal migration, but instead serves as a developmental cue that increases the positional diversity of these neurons within the basal forebrain. In addition, the results suggest that the timely termination of GABA production within the GnRH neuronal network is a prerequisite for normal reproductive function. The possibility arises that similar abnormalities in GABA homeostasis may contribute to syndromes of hypothalamic amenorrhea/oligomenorrhea in humans.

Gamma amino butyric acid (GABA) is the main inhibitory neurotransmitter 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 rats 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 micrometer) compared with animals grafted with native BAS-8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 micrometer) 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.

Mammalian reproductive cyclicity requires the periodic discharge of GnRH from hypothalamic neurons into the portal vessels connecting the neuroendocrine brain to the pituitary gland. GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The transcriptional control of this process is not well understood, but it appears to involve several genes. One of them, termed enhanced at puberty 1 (EAP1), has been postulated to function in the female hypothalamus as an upstream regulator of neuroendocrine reproductive function. RNA interference-mediated inhibition of EAP1 expression, targeted to the preoptic region, delays puberty and disrupts estrous cyclicity in rodents, suggesting that EAP1 is required for the normalcy of these events. Here, we show that knocking down EAP1 expression in a region of the medial basal hypothalamus that includes the arcuate nucleus, via lentiviral-mediated delivery of RNA interference, results in cessation of menstrual cyclicity in female rhesus monkeys undergoing regular menstrual cycles. Neither lentiviruses encoding an unrelated small interfering RNA nor the placement of viral particles carrying EAP1 small interfering RNA outside the medial basal hypothalamus-arcuate nucleus region affected menstrual cycles, indicating that region-specific expression of EAP1 in the hypothalamus is required for menstrual cyclicity in higher primates. The cellular mechanism by which EAP1 exerts this function is unknown, but the recent finding that EAP1 is an integral component of a powerful transcriptional-repressive complex suggests that EAP1 may control reproductive cyclicity by inhibiting downstream repressor genes involved in the neuroendocrine control of reproductive function.

The initiation of mammalian puberty requires an increase in pulsatile release of GnRH from the hypothalamus. This increase is brought about by coordinated changes in transsynaptic and glial-neuronal communication. As the neuronal and glial excitatory inputs to the GnRH neuronal network increase, the transsynaptic inhibitory tone decreases, leading to the pubertal activation of GnRH secretion. The excitatory neuronal systems most prevalently involved in this process use glutamate and the peptide kisspeptin for neurotransmission/neuromodulation, whereas the most important inhibitory inputs are provided by γ-aminobutyric acid (GABA)ergic and opiatergic neurons. Glial cells, on the other hand, facilitate GnRH secretion via growth factor-dependent cell-cell signaling. Coordination of this regulatory neuronal-glial network may require a hierarchical arrangement. One level of coordination appears to be provided by a host of unrelated genes encoding proteins required for cell-cell communication. A second, but overlapping, level might be provided by a second tier of genes engaged in specific cell functions required for productive cell-cell interaction. A third and higher level of control involves the transcriptional regulation of these subordinate genes by a handful of upper echelon genes that, operating within the different neuronal and glial subsets required for the initiation of the pubertal process, sustain the functional integration of the network. The existence of functionally connected genes controlling the pubertal process is consistent with the concept that puberty is under genetic control and that the genetic underpinnings of both normal and deranged puberty are polygenic rather than specified by a single gene. The availability of improved high-throughput techniques and computational methods for global analysis of mRNAs and proteins will allow us to not only initiate the systematic identification of the different components of this neuroendocrine network but also to define their functional interactions.