I. Introduction II. Effects of Estrogen on GnRH Neurons A. Sexual differentiation B. Biosynthesis of GnRH C. Biosynthesis of galanin D. Electrical activity of GnRH neurons E. GnRH secretion F. GnRH degradation G. Ultrastructural changes involving GnRH neurons H. Summary of estrogen’s influence on GnRH cells III. Brain Sites of Estrogen Influence on GnRH Neurons A. Sites of estrogen’s stimulatory sites B. Sites of estrogen’s inhibitory action C. Summary IV. Pathways for Estrogen Regulation of GnRH Neurons A. Direct regulation of GnRH neurons B. Estrogen-receptive neurons as intermediaries C. Glial cells as intermediaries D. Summary V. Model of Estrogen Action on GnRH Neurons VI. Concluding Remarks

Multimodal Influence of Estrogen upon Gonadotropin-Releasing Hormone Neurons

To characterize the spectrum of pulsatile gonadotropin secretion during the course of the normal menstrual cycle, we studied normal women during 51 ovulatory cycles. Plasma gonadotropin concentrations were measured at 10-min intervals for 20-24 h during the early, mid-, and late follicular phases and the early, mid-, and late luteal phases. LH data series were analyzed using 2 different computer-assisted algorithms for pulse detection. The LH interpulse interval decreased during the follicular phase (FP) from 94 +/- 4 (+/- SEM) min in the early FP (EFP) to 71 +/- 4 min by the late FP (LFP; P less than 0.001). The estimation of LH pulse frequency in the EFP was significantly affected by slowing of episodic LH secretion during sleep. In the luteal phase (LP), the LH interpulse interval progressively increased from 103 +/- 8 min in the early LP (ELP) to 216 +/- 39 min by the late LP (LLP; P less than 0.001). Sleep-associated slowing of episodic LH secretion also occurred in the ELP. The mean LH pulse amplitude in the EFP (6.5 +/- 0.4 mIU/ml) decreased significantly by the midfollicular phase (MFP; 5.1 +/- 0.8 mIU/ml; P less than 0.05) and increased once again by the LFP (7.2 +/- 1.2 mIU/ml). LH pulse amplitude was highest in the ELP (14.9 +/- 1.7 mIU/ml), decreased by the midluteal phase (MLP) to 12.2 +/- 2.0 mIU/ml, and declined further by the LLP to 7.6 +/- 1.1 mIU/ml (P less than 0.001 vs. ELP). FSH secretion was significantly (P less than 0.05) correlated with LH secretion at time lags of 0-10 min in 82% of the studies. These results indicate the following. 1) In the EFP and ELP, the frequency of gonadotropin pulsations is reduced at night in association with sleep. 2) The frequency of LH secretion increases from the EFP to MFP and LFP. 3) LH pulse amplitude decreases in the MFP, suggesting enhanced negative feedback of estrogen on the hypothalamic-pituitary axis and/or a decrease in GnRH secretion at this stage. 4) A progressive reduction of LH pulse frequency and amplitude occurs during the LP which is correlated with the duration of exposure of the hypothalamic-pituitary axis to progesterone. 5) A close relationship exists between secretion of LH and FSH, suggesting a common stimulatory factor for both gonadotropins.

Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle.

GnRH neurons form the final common pathway for central control of reproduction, with regulation achieved by changing the pattern of GnRH pulses. To help elucidate the neurobiological mechanisms underlying pulsatile GnRH release, we generated transgenic mice in which the green fluorescent protein (GFP) reporter was genetically targeted to GnRH neurons. The expression of GFP allowed identification of 84-94% of immunofluorescently-detected GnRH neurons. Conversely, over 99.5% of GFP-expressing neurons contained immunologically detectable GnRH peptide. In hypothalamic slices, GnRH neurons could be visualized with fluorescence, allowing for identification of individual GnRH neurons for patch-clamp recording and subsequent morphological analysis. Whole-cell current-clamp recordings revealed that all GnRH neurons studied (n = 23) fire spontaneous action potentials. Both spontaneous firing (n = 9) and action potentials induced by injection of depolarizing current (n = 17) were eliminated by tetrodotoxin, indicating that voltage-dependent sodium channels are involved in generating action potentials in these cells. Direct intracellular morphological assessment of GnRH dendritic morphology revealed GnRH neurons have slightly more extensive dendrites than previously reported. GnRH-GFP transgenic mice represent a new model for the study of GnRH neuron structure and function, and their use should greatly increase our understanding of this important neuroendocrine system.

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Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: Characterization of whole-cell electrophysiological properties and morphology

Mammalian reproduction is dependent upon intermittent delivery of luteinizing hormone-releasing hormone (LHRH) to the anterior pituitary. This mode of secretion is required to sensitize maximally the gonadotrophs to LHRH stimulation and to regulate gonadotropin gene expression. While LHRH secretion is pulsatile in nature, the origin of the pulse generator is unknown. In this report, we show that this oscillator could be located within the LHRH neuronal network. When immortalized LHRH neurons are placed into a perifusion system, LHRH is secreted into the medium in a pulsatile fashion under basal conditions. LHRH secretion and the number of LHRH pulses are reduced when calcium is removed from the medium. Perifusion also influences pro-LHRH processing, since the molar ratio of its processed products varies dramatically when the cells are transferred from a static system. Several different cellular mechanisms may underlie these changes in secretion and processing. Lucifer yellow experiments reveal that some cells are dye-coupled. Hence, these cells could be electrically coupled through gap junctions such that secretion from individual cells could be coordinated. Secretion could also be synchronized through the observed synapse-like contacts. These contacts could perform a negative-feedback role to regulate not only the amount of LHRH released but also the molecular forms secreted. The organization of LHRH neurons into interconnected clusters could serve to coordinate LHRH secretion from individual cells and, thereby, orchestrate functions in vivo as diverse as the onset of puberty, the timing of ovulation, and the duration of lactational infertility.

https://www.pnas.org/content/pnas/89/9/4149.full.pdf

Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons.

Ovarian steroids normally exert homeostatic negative feedback on GnRH release. During sustained exposure to elevated estradiol in the late follicular phase of the reproductive cycle, however, the feedback action of estradiol switches to positive, inducing a surge of GnRH release from the brain, which signals the pituitary LH surge that triggers ovulation. In rodents, this switch appears dependent on a circadian signal that times the surge to a specific time of day (e.g., late afternoon in nocturnal species). Although the precise nature of this daily signal and the mechanism of the switch from negative to positive feedback have remained elusive, work in the past decade has provided much insight into the role of circadian/diurnal and estradiol-dependent signals in GnRH/LH surge regulation and timing. Here we review the current knowledge of the neurobiology of the GnRH surge, in particular the actions of estradiol on GnRH neurons and their synaptic afferents, the regulation of GnRH neurons by fast synaptic transmission mediated by the neurotransmitters γ-aminobutyric acid and glutamate, and the host of excitatory and inhibitory neuromodulators including kisspeptin, vasoactive intestinal polypeptide, catecholamines, neurokinin B, and RFamide-related peptides, that appear essential for GnRH surge regulation, and ultimately ovulation and fertility.

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The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges.

Characteristic increases in neuronal activity coincident with the pulsatile release of luteinizing hormone from the pituitary gland have been recorded from electrodes chronically implanted in the medial basal hypothalamus of the rhesus monkey. This electrophysiologic manifestation of the hypothalamic 'pulse generator' which governs the secretion of hypothalamic luteinizing hormone releasing hormone provides, for the first time, direct access to the central component of the neuroendocrine control system which regulates reproductive processes in this higher primate.

Tsuguo Uemura

Papers

Central electrophysiologic correlates of pulsatile luteinizing hormone secretion in the rhesus monkey.