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Department of Neurobiology and Physiology Northwestern University Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Jon E. Levine, Ph.D., Northwestern University, Department of Neurobiology/Physiology, OT Hogan Hall, 2153 North Campus Drive, Evanston, Illinois 60208.
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Introduction |
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— Petulant Poet, with apologies to Peter Piper
The primate reproductive axis is governed by the secretory activity of a mere 1,500–2,000 GnRH neurons (1). The GnRH perikarya are distributed throughout several hypothalamic nuclei, and most extend axonal processes into the lateral zona externa of the median eminence. Here, GnRH release occurs at neurovascular junctions, into the portal vasculature for transport to the anterior pituitary gland. The decapeptide thereafter binds receptors on pituitary gonadotropes, and activates several signal transduction pathways, leading to regulation of gonadotropin release, gonadotropin subunit gene expression, and responsiveness of gonadotropes to subsequent signals. It is remarkable to consider that this entire cascade of events—and hence, virtually all gonadal functions, and fertility in general—are absolutely dependent upon the specialized, coordinated activity of these few and far-flung GnRH neurons. What do GnRH neurons do that is so special, and how do they do it?
A critically important feature of GnRH neurosecretion is pulsatility. In virtually all physiological situations, the decapeptide is secreted intermittently into the portal vasculature. The existence of this rhythmic secretory process was originally inferred from early measurements of peripheral LH pulses, especially in animals where gonadal negative feedback influences were eliminated (2). With the advent of methods to monitor GnRH release directly, temporal associations between pulsatile GnRH and LH release were eventually demonstrated, first in sheep (3, 4), and subsequently in rhesus macaques (5, 6), rats (7, 8), and rabbits (9, 10). Even before these measurements were made, however, Ernst Knobil and colleagues had already deduced the physiological significance of this neurosecretory language: only pulsatile GnRH stimulation can sustain continued secretion of LH and FSH, whereas continuous GnRH administration is either ineffective or even inhibitory in this regard (11). The same group also demonstrated that modulation of the frequency of pulsatile GnRH stimulation can lead to differential LH and FSH secretion (12), a phenomenon that has yet to be fully explained at the molecular and cellular level. The physiological importance of GnRH pulsatility, in any case, has thus been firmly established. The cellular mechanisms that constitute the GnRH pulse generator, however, have proven to be much more difficult to define.
What is the GnRH pulse generator? Electrophysiological correlates of pulse generator activity have been characterized in monkeys (13) and other species (14, 15) and have provided at least a functional definition of the GnRH pulse generator: a set of neurons that periodically fire a high frequency volley of action potentials, eventuating in the neurosecretion of a GnRH pulse into the hypophysial portal vessels. Beyond these electrophysiological features, and the characteristics of GnRH neurosecretion described in the complex in vivo situations, there is little known of the intra and intercellular signals that mediate pulse generation.
It is likely, nevertheless, that there exist two cellular mechanisms that are central to the pulse-generating process: 1) a pacemaker or pseudo-pacemaking process, which endows the system with its rhythmic properties, and 2) a mechanism for electrophysiological synchronization among cells. A simple model of pulsatility holds that pacemaking activity occurs within GnRH neurons themselves, and that slave GnRH neurons are entrained to the rhythm of a dominant pacemaker within the population. Alternatively, the pulsatile rhythm may arise from a stochastic, pseudo-pacemaking process, in which the random activity of any neuron within an interconnected network initiates activity within the group, followed by a refractory period that would impose a prolonged interpulse quiescence. In either scenario, synchronicity among neurons could be achieved through intercellular signaling via GnRH-GnRH synaptic contacts or gap junctions.
Until recent years, there have been few methodological avenues available to explore cellular and molecular determinants of pulsatility. A technical catch-22 could not be overcome: to explore pulsatility at the molecular level, GnRH neurons had to be isolated and cultured, yet no methods were available that preserved GnRH pulsatility by an isolated, uniform GnRH neuronal population. This technical barrier was finally broken by Mellon and colleagues in 1990 (16), when they generated the first immortalized GnRH-producing cell lines. Several groups have since demonstrated that the GT1 cells are capable of producing coherent patterns of GnRH pulsatility in vitro (17, 18, 19). It is thus certain that these cells possess all of the requisite molecular components to exhibit pulsatility; does this mean that normal GnRH neurons in vivo are alone capable of—and solely responsible for—pulsatile GnRH neurons at neurovascular junctions in the median eminence?
Doubting the physiological relevance of pulsatility in the GT1 cells might seem like looking a gift horse in the mouth, but some legitimate concerns have been raised (20). Early in embryonic development, GnRH neurons are concentrated in the olfactory placode and commence a peculiar migration along the nervus terminalis, into the central nervous system (21, 22, 23). They eventually become permanently positioned at various hypothalamic locations, and extend axonal processes into the median eminence. The GT1 cells were generated by targeted tumorigenesis in transgenic mice, using a construct containing the GnRH promoter fused to the SV40 T-antigen gene. The resulting tumor cells are highly differentiated, yet they likely represent an early embryonic GnRH neuronal phenotype—one that is almost certainly assumed before the migration of GnRH neurons into the central nervous system. It is thus fair to consider whether or not the pulsatility of these cells is truly reflective of any in vivo physiological GnRH pulsatility. For example, why would embryonic GnRH neurons release the decapeptide, much less exhibit pulsatility, long before they make their trek into the hypothalamus and form viable neurovascular junctions in the median eminence? One answer to that question could have been because the GT1 cells are tumor cells that don’t behave like their normal counterparts in vivo.
The most recent paper of Dr. Teresawa and her colleagues at the University of Wisconsin (24) appearing in this issue puts this idea to rest. Teresawa’s group previously demonstrated in Endocrinology (25) that cultures of embryonic olfactory placode explants, which contain mostly GnRH neurons, can release GnRH into culture medium; moreover, they are capable of starting their migration toward the CNS within the tissue explant. In their current work, they have demonstrated for the first time that embryonic GnRH neurons exhibit spontaneous pulsatility. Remarkably, the pulsatile release patterns from these explant cultures are comparable to the most robust pulsatile patterns that have been measured in monkeys in vivo. Moreover, this activity resembles that seen in a variety of in vitro (26, 27) preparations in several important ways, as it depends upon mobilization of intracellular calcium from ryanodine-sensitive stores, and it is susceptible to activation and blockade with sodium channel openers and blockers. Moreover, the frequency of pulses from the explants compares closely with those seen in vivo gonadectomized adult macaques (5, 6). Similar results were obtained in tissues comprised of the GnRH migratory track. Thus, Teresawa and colleagues have confirmed the finding in GT1 cells that GnRH neurons are intrinsically equipped to support pulsatility. Additionally, Teresawa’s work clearly demonstrates for the first time that primary GnRH neurons do exhibit pulsatility both before and during their migration into the central nervous system.
The reasons for prenatal GnRH pulsatility, if indeed there are any, may involve some role for GnRH release in the migration process. Teresawa’s findings beg the larger question, however, as to how and why GnRH pulsatility may be abated during the transition from the neonatal to the juvenile period. Measurements of LH release in postnatal, juvenile, and pubertal monkeys have revealed that GnRH pulsatility likely occurs before birth and continues for approximately 20 weeks (28). At that time, LH pulsatility all but disappears in males and is likely reduced in females. The available evidence, including the only extant GnRH data in a pubertal animal (also from the Teresawa laboratory, 29), indicate the GnRH pulse generator is reawakened as puberty proceeds, and is likely the major determinant of the pubertal process. Terasawa’s current observations provide a key new piece of the ontological puzzle: Pulsatility is already operative in embryonic GnRH neurons; it is then either tonically inhibited or even actively disassembled during infancy and throughout the juvenile period. Why does GnRH pulsatility decelerate or even disappear during the juvenile period? Studies in primates (30) and rodents (31) have suggested that sufficient GnRH synthesis occurs in GnRH neurons of the juvenile, making it unlikely that any lack of releasable GnRH may limit the expression of pulsatility. Rather, it would appear that some facet of the pulse generating process, perhaps the number or patency of key ion channels in the GnRH neuron, is altered in infancy, either by factors produced locally in the hypothalamus, or by circulating factors that convey somatic signals. The net result is a dormant pulse generator that awaits rousing at puberty by neuroendocrine signals.
The GnRH pulse generator is regulated by a variety of neural and endocrine signals, including those mediating homeostatic and ovulatory control within the reproductive axis, as well as reproductive responses to stress, metabolic cues, photoperiod, pheromones, and mating. For many, if not all of these vital regulatory processes, a new physiological state or endpoint is achieved through alteration of the frequency and/or amplitude of GnRH pulses. How are these signals registered by the GnRH pulse generator, and how are changes in frequency and amplitude affected? It is clear that we must attain a complete understanding of the molecular and cellular components of the GnRH pulse generator, before these questions can be fully answered. The current work of Teresawa et al. (24) provides important new information on the physiology and development of GnRH neurons, and additionally establishes the fetal explant system as an important avenue to further explore the molecular and cellular determinants of pulsatility, and perhaps address questions of pulse generator regulation at the cellular level.
Is GnRH pulsatility in vivo solely a function of GnRH neurons? Probably. Is GnRH pulsatility manifest before and during embryonic migration? Positively. Is GnRH pulsatility diminished in infancy, and restored at puberty? Presumably. So, how is pulsatility turned off, and how is it turned back on? This is presently an unanswered question for future research.
Received December 30, 1998.
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