Chapter 1
The Framework Upon Which Current Research on the GnRH Neuron and its Control is Built

Tony M. Plant and 1Allan E. Herbison2

1Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee Womens Research Institute, Pittsburgh, PA, USA

2Centre for Neuroendocrinology and Department of Physiology, University of Otago School of Biomedical Sciences, , Dunedin, New Zealand

Abstract

The ovarian cycle and ovulation in the female, and spermatogenesis in the male, are dependent upon the brain, and specifically upon a diffusely distributed network of peptidergic neurons in the hypothalamus that synthesize the neurohormone, gonadotropin-releasing hormone (GnRH). The decapeptide was isolated in 1971 from bovine and ovine hypothalami by the laboratories of Andrew Schally and Roger Guillemin, respectively (Matsuo et al., 1971; Amoss et al., 1971), and initially termed luteinizing hormone releasing hormone (LH-RH) or luteinizing hormone releasing factor (LRF). This review provides a brief historical account of the development of the concepts underpinning current research on the GnRH neuron and its control.

1.1 Introduction

The idea that the gonads might be governed by the central nervous system (CNS) via a neurohormone had emerged well before the isolation of GnRH. It had been apparent for centuries that reproduction is closely related to environmental cues. For example, sheep in the northern hemisphere generally breed during the months of September-November, but if these animals are relocated to the southern hemisphere, a 180° phase shift in this behavior occurs and breeding is observed from February-April. This and other observations were taken by F.H.A. Marshall, at the University of Cambridge, to champion the view during the first half of the 20th century that reproduction was governed by the CNS. While Marshall was formulating the ideas of the CNS control of reproduction, the foundations of endocrinology were also being laid. In the present context, it was established that extracts of the anterior pituitary, now known to contain the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), were able to stimulate the gonads. The nerve supply of the ovary and testis, on the other hand, was recognized as scant and limited primarily to vasomotor control. These two views naturally led to the idea that environmental cues, such as those that regulate seasonal breeding, were registered by the CNS and relayed to the gonads by hormonal signals from the pituitary. That the hypothalamus was the critical region of the brain in the regulation of pituitary function was established between 1925 and 1950 by classic physiological experimentation using lesions, electrical stimulation, and pituitary transplantation (see Harris, 1955).

The question of how the hypothalamus communicates with the anterior pituitary was heatedly debated during the 1940s and early 50s. One school, championed by Geoffrey Harris, proposed a humoral link utilizing the specialized portal vasculature between the hypothalamus and anterior pituitary. A second, led by Solly Zuckerman, focused on the more traditional pathway used for control by the CNS; that is, a direct neural link. For an account of this important era of neuroendocrine research, the reader is referred to the classic monograph written by Harris, entitled "The Neural Control of the Pituitary Gland" (Harris, 1955), where he reiterated his view, developed earlier in collaboration with J.D. Green, "that nerve fibres of the hypothalamus liberate some humoral substance into the primary plexus of the hypophysial vessels and that these vessels transmit the substance to the adenohypohysis where it exerts an activating effect on the gland cells." However, it was not until 1971, with the isolation and characterization of GnRH, that the neuro-humoral hypothesis for the control of the gonadotropin secreting cells of the anterior pituitary was finally accepted by all. The last act in the discovery of GnRH involved a protracted "race" between the laboratories of Guillemin and Schally to isolate the gonadotropin releasing neurohormone from the hypothalamus. This race was at times acrimonious, although its successful conclusion, together with the earlier isolation of thyrotropin releasing hormone (TRH), was responsible for the award to these two scientists of half the Nobel Prize for Physiology or Medicine in 1977 (the other half was awarded to Ross Yalow for "the development of radioimmunoassays of peptide hormones"; see later). A riveting account of the discovery of GnRH has been provided in a series of three articles written by Nicolas Wade and published in Science in April/May 1978.

1.2 Approaches taken to study the GnRH neuron

Two fundamental approaches were taken to understand the GnRH neuron and its control following the isolation and characterization of GnRH in 1971. The first was a direct "neurobiological" approach, which employed anatomical, cellular, and electrophysiological methodologies that examined the location and morphology, birth, migration, and cellular and molecular biology of the GnRH neuron. The second was an indirect "endocrinological" approach that treated the GnRH neuron as a "black box" and investigated its regulation and output (tracked indirectly by measuring LH in peripheral blood) in the context of the control system that governs various aspects of gonadal function. It is worth noting that both approaches greatly benefited from an appreciation of immunology that enabled specific antibodies to be generated against GnRH and LH, which were then used to develop sensitive immunohistochemical (IHC) procedures and radioimmunoassays (RIAs) for these two peptide hormones.

1.2.1 Neurobiological approach

The IHC localization of hypothalamic GnRH, pioneered by Julien Barry and his colleagues in Lille, France in the early 1970s (Barry et al., 1973), soon revealed three important features of GnRH neurons in the mammalian hypothlamus: first, there were only a few hundred of these peptide neurons; second, they were diffusely distributed throughout the hypothalamus; and third, their far-reaching projections were striking. The diffuse distribution of GnRH neurons in the hypothalamus is a characteristic that, to this date, has frustrated cellular investigations of these cells, including those relating their electrophysiological properties to their secretory activity. Later IHC studies of GnRH neuron location in the embryonic brain by Donald Pfaff and Susan Wray led to the recognition of another peculiar feature of the GnRH neuron, namely that unlike other neurons, it is not born in the ependymal lining of the cerebral ventricles, but rather outside the brain in the nasal placode (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989). This means that before the GnRH neuron can subserve a hypophysiotropic function, it has to enter the brain and migrate through the forebrain to the hypothalamus: a complex process that takes place during early embryonic development. Most recently, contemporary transgenic, electrophysiology, and imaging techniques have led to the view that projections from the GnRH cell body to the median eminence, where the primary plexus of the hypophysial portal circulation is located, exhibit the unique feature of possessing properties of both axons and dendrites; these projections are now termed "dendrons" (Herde et al., 2014).

Parenthetically, the GnRH gene (GnRH1) was cloned from human and rat by the Seeburg laboratory in the mid-1980s, and our understanding of the regulation of expression of GnRH1 was greatly facilitated by the creation of an immortalized GnRH cell line from the mouse brain using targeted tumorigenesis (Mellon et al., 1990).

1.2.2 Endocrinological approaches

The endocrinological approach has invariably involved studies of the female, because ovulation is a key and easily identifiable event of the ovarian cycle, and, historically, one that provided the only reliable surrogate marker of acute hypothalamic activation (i.e., GnRH discharges). Application during the early 1970s of LH and FSH RIAs to various species, particularly the monkey and human, had indicated that the pattern of gonadotropin secretion during the ovarian cycle could be conceptualized as comprising two modes of secretion: a basal or tonic mode, observed during the follicular and luteal phases of the cycle, which was interrupted at mid-cycle by an abrupt and large discharge or surge of LH and FSH, known as the pre-ovulatory gonadotropin surge; this was what was responsible for ovulation.

Before the structure of GnRH was reported, Ernst Knobil and his colleagues in Pittsburgh had observed that circulating LH concentrations measured at frequent intervals exhibited a striking saw-tooth pattern with peak levels at approximately hourly intervals in the ovariectomized rhesus monkey (Dierschke et al., 1970). They proposed that this pulsatile or episodic mode of gonadotropin secretion is likely due to intermittent signals from the brain that are relayed to the anterior pituitary by an "LRF." It was not until 1982, however, that the pulsatile mode of this LRF (i.e., GnRH) release into the portal circulation was empirically demonstrated by the group of Iain Clarke (Clarke and Cummins, 1982). By this time, the notion that an intermittent pattern of GnRH stimulation was required to sustain gonadotropin secretion had...