Andrew Wolfe

Timing and completion of puberty in female mice depend on estrogen receptor α-signaling in kisspeptin neurons

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms—a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor α (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus.

Neuroendocrine Regulation of the Luteinizing Hormone-Releasing Hormone Pulse Generator in the Rat

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.

On Feature Spreading and the Representation of Place of Articulation

Since Clements (1985) introduced feature geometry, four major innovations have been proposed: Unified Feature Theory, Vowel-Place Theory, Strict Locality, and Partial Spreading. We set out the problems that each innovation encounters and propose a new model of feature geometry and feature spreading that is not subject to these problems. Of the four innovations, the new model-Revised Articulator Theory (RAT)-keeps Partial Spreading, but rejects the rest. RAT also introduces a new type of unary feature-one for each articulator-to indicate that the articulator is the designated articulator of the segment.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.

Estrogenic Regulation of the GnRH Neuron

Reproductive function is regulated by the secretion of luteinizing hormone (LH) and follicle-stimulating hormone from the pituitary and the steroid hormones from the gonads. The dynamic changes in the levels of the reproductive hormones regulate secondary sex characteristics, gametogenesis, cellular function, and behavior. Hypothalamic GnRH neurons, with cell bodies located in the basal hypothalamus, represent the final common pathway for neuronally derived signals to the pituitary. As such, they serve as integrators of a dizzying array of signals including sensory inputs mediating information about circadian, seasonal, behavioral, pheromonal, and emotional cues. Additionally, information about peripheral physiological function may also be included in the integrative signal to the GnRH neuron. These signals may communicate information about metabolic status, disease, or infection. Gonadal steroid hormones arguably exert the most important effects on GnRH neuronal function. In both males and females, the gonadal steroid hormones exert negative feedback regulation on axis activity at both the level of the pituitary and the hypothalamus. These negative feedback loops regulate homeostasis of steroid hormone levels. In females, a cyclic reversal of estrogen feedback produces a positive feedback loop at both the hypothalamic and pituitary levels. Central positive feedback results in a dramatic increase in GnRH secretion (Moenter et al., 1992; Xia et al., 1992; Clarke, 1993; Sisk et al., 2001). This is coupled with an increase in pituitary sensitivity to GnRH (Savoy-Moore et al., 1980; Turzillo et al., 1995), which produces the massive surge in secretion of LH that triggers ovulation. While feedback regulation of the axis in males is in part mediated by estrogen receptors (ER), there is not a clear consensus as to the relative role of ER versus AR signaling in males (Lindzey et al., 1998; Wersinger et al., 1999). Therefore, this review will focus on estrogenic signaling in the female.

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between -1250 and -587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.

Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo

Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (−3446/+23 bp, −2078/+23 bp, and −1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5′-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both −2078 and −1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissuesin vivo. We demonstrate that DNA sequences contained within the proximal −1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to −2078 bp mediate the repression of ovarian GnRH.

Insulin augments GnRH-stimulated LHβ gene expression by Egr-1

Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHβ gene expression by GnRH and insulin was examined in LβT2 cells. Endogenous LHβ mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences −140 to +1 in the mouse LHβ gene. Insulin together with GnRH stimulates activity of an LHβ-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences −51 to −42 in the LHβ gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHβ DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line--an immortalized GnRH-secreting neuronal cell line--insulin maximally increases Egr-1 mRNA after 30min of treatment and Egr-1 protein and GnRH mRNA after 60min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between -67 and -76bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.

The Gonadotropin-Releasing Hormone Cell-Specific Element Is Required for Normal Puberty and Estrous Cyclicity

Appropriate tissue-specific gene expression of gonadotropin-releasing hormone (GnRH) is critical for pubertal development and maintenance of reproductive competence. In these studies, a common element in the mouse GnRH (mGnRH) promoter, between −2806 and −2078 bp, is shown to mediate differential regulation of hypothalamic and ovarian mGnRH expression. To further characterize this region, we generated a knock-out mouse (GREKO−/−) with a deletion of the mGnRH promoter fragment between −2806 and −2078 bp. GnRH mRNA expression in the brain of GREKO−/− was less than the expression in wild-type mice; however, immunohistochemical analysis revealed no difference between the numbers of GnRH neurons among groups. GnRH mRNA expression in the ovary was fivefold higher in GREKO−/−. The immunohistochemical staining for GnRH in the ovary increased in surface epithelial and granulosa cells and also in the corpora lutea of GREKO−/− mice. The reproductive phenotype revealed that the mean day of vaginal opening was delayed, and additionally, there was a significant decrease in the length of proestrus and diestrus-metestrus phases of the estrous cycle, resulting in a shortened estrous cycle in GREKO−/− mice. This work supports the hypothesis that the region of the GnRH promoter contained between −2806 and −2078 bp acts as a cell-specific enhancer in the GnRH neuron and as a repressor in the ovary. Deletion of this region in vivo implicates the GnRH promoter in mediating pubertal development and periodic reproductive cycling, and forms the foundation to define the nuclear proteins important for puberty and estrous cycling in mammals.