Fertility and its onset, puberty, are integrated phenomena. A complex network in the central nervous system (CNS), conveying physiological and environmental signals, converges onto neurons secreting gonadotropin-releasing hormone (GnRH). GnRH leads the hormonal cascade, driving gonadotrophs to secrete gonadotropins, which in turn control the gonads, i.e., steroidogenesis and gametogenesis in both sexes and ovulation in females. Thus, GnRH neurons are the output of the CNS for fertility, integrating and encoding cues into a signal readable by gonadotrophs, GnRH. However, GnRH neurons are not an on/off switch but a precise rheostat. GnRH secretion is pulsatile, with changes in amplitude and frequency over time. Yet, the mechanisms by which GnRH neurons generate pulses are unknown. This review summarizes recent data about GnRH neurons with a focus on secretion and the difficulty of answering this fundamental question.

Undoubtedly, cells upstream of GnRH neurons contribute to fertility by helping to provide the homeostatic conditions necessary for survival. However, in this review, the word fertility simply refers to the ability to generate gametes and offspring in optimal breeding conditions.

Gonadotropin-releasing hormone neurons derive from the olfactory placodes (1, 2) and migrate into the hypothalamus during prenatal development [reviewed in Ref. (3, 4)]. GnRH processes then extend toward the median eminence (ME) (5). This embryonic feature shapes the GnRH neuronal distribution (6). In mouse and rat, the distribution, centered around the preoptic area (POA) and the organum vasculosum laminae terminalis (OVLT), respectively (7, 8), is largely confined to the rostral forebrain. In monkey, it expands caudally to the mediobasal hypothalamus (MBH) (9, 10). However, data suggest that the location of the cell bodies is not important to trigger luteinizing hormone (LH) and promote fertility, as long as GnRH nerve terminals reach the hypophyseal portal system. In hypogonadal mice bearing a deletion in the GnRH gene (11), transplantation of fetal POA in the rostral third ventricle restores spermatogenesis (12) and pregnancies (13). Similarly, in female monkeys with lesioned MBH, menstrual cycles are restored with transplantation of olfactory placodes in the third ventricle (14). Notably, the pregnancies in mice receiving transplants are initiated by reflex, not spontaneous, ovulation (15), but still indicate gametogenesis and an ovulatory surge occur (16). Two possibilities, extrinsic to GnRH neurons, might explain the absence of spontaneous ovulation in transplanted mice: the required inputs (1) cannot reach transplanted GnRH neurons in their abnormal location and/or (2) are reduced/absent in hypogonadal mice (17). In contrast, in female monkeys, cyclicity was recovered since the inputs were present, i.e., the hypothalamic–pituitary–gonadal (HPG) axis was functional before its disruption. The next section addresses the distinct mechanisms for GnRH secretion leading to ovulation and gametogenesis.

Gonadotropin-releasing hormone neurons have two modes of secretion: surge triggering ovulation, restricted to females, and pulses regulating gametogenesis and sex steroidogenesis, in both sexes. In rat, 90% of GnRH neurons project outside the blood–brain barrier as indicated by Fluorogold retrograde labeling (18). In mouse, only 64% of GnRH neurons are labeled in intact animals but hormonal manipulation labels 88% (19). Unfortunately, peripheral injection of Fluorogold does not discriminate the uptake site. In addition to the ME (20), GnRH neurons exhibit branched processes beyond the blood–brain barrier into the OVLT (21). Thus, the hypophysiotropic proportion of the GnRH population is unknown. Lectin wheat germ agglutinin applied onto the ME reveals an uptake in up to 59% of GnRH neurons (22). While the majority of GnRH neurons probably connect to the ME, a specific number might be irrelevant since few GnRH neurons are needed to acquire and maintain fertility (12, 13, 23). Some GnRH neurons may project to other brain areas, in addition to or instead of the ME and OVLT, and may control additional functions (24, 25).

Elucidating how pulsatile GnRH secretion occurs is the key to understanding reproductive neuroendocrinology. However, measuring GnRH secretion is difficult. The GnRH neuronal population is small and a subset generates a pulse, therefore the amount of released GnRH is near threshold detection, even with sensitive radioimmunoassay (70). In addition, access to the hypophyseal portal system requires complex surgery and apparatus (53, 120–122), incompatible with the mouse. Finally, a half-life of GnRH is only 2–4 min. Thus, LH secretion, amplifying and diffusing the GnRH signal to the systemic circulation, is commonly used as a mirror of GnRH secretion (121, 123). However, pulsatile GnRH/LH release requires serial sampling and even LH measurements are hardly achievable with mouse blood volume (124). At a cellular level, the first challenge is anatomical: preserving the connectivity with relevant inputs (125), GnRH cell morphology (126), and tracking a neuron within the complexity of the ME (20). The second challenge is technical as methods for detection of quantal secretion are not applicable to GnRH neurons: (1) synaptically coupled neurons are recorded simultaneously in brain slices [reviewed in Ref. (127)], but GnRH neurons lack downstream partners, (2) patch clamp measurement of capacitive current is limited to soma and isolated nerve terminals (128, 129), therefore does not reflect hypophysiotropic GnRH secretion, and (3) fast-scan cyclic voltammetry (FSCV) is restricted to electrochemically active small neurotransmitters [reviewed in Ref. (130)].

Since techniques directly monitoring secretion cannot be applied to GnRH neurons, the alternative is to rely on the relationship between electrical activity, voltage-gated calcium channels, calcium, and secretion [reviewed in Ref. (131)] and use electrophysiology and calcium imaging of the GnRH cell bodies to assess GnRH secretion indirectly. The hypothesis of pulsatile secretion being intrinsic to GnRH neurons led to studies of electrical properties in GnRH neurons {GT1 cells (73); nasal explants (71); ex vivo GnRH neurons [reviewed in Ref. (40, 132, 133)]}. Although most GnRH neurons display autonomous firing of APs, firing is heterogeneous among GnRH neurons (126), far from an oscillatory activity that could trigger pulses every ~20 min (124). Even in vivo GnRH neurons exhibit heterogeneous behavior (114). The search for changes in the firing pattern ex vivo, i.e., increases in firing rate occurring at the same frequency as GnRH pulses, is rather inconclusive (134–136). Notably, in addition to intrinsic properties, it is assumed that each GnRH neuron contributes to consecutive GnRH pulses. Although experimentally activated GnRH neurons can trigger multiple LH pulses (88), this assumption has yet to be proven.

Simultaneous recording from multiple GnRH neurons bypasses this assumption and shows in vitro relationships between synchronized [Ca²⁺]_i oscillations and GnRH pulses (70) or frequency of [Ca²⁺]_i oscillations and GnRH secretion (137). Recently, optogenetic activation of GnRH neurons defined the firing of GnRH neurons triggering LH secretion in vivo (88) (Figure 1). However, the predicament to linking an electrical event to a secretion, at a single-cell level, is the resolution for the detection of GnRH release. Calcium dynamics in GT1 cells correlate with FM1-43 uptake, i.e., secretion (138), but this observation cannot be extrapolated to native GnRH neurons with complex morphology and where GnRH release occurs from cell bodies and fibers (139, 140).

Modified FSCV, applicable to GnRH, is a step forward, providing secretion data from one to few GnRH neurons (140). It supports, at a smaller scale, the relationship between APs and secretion: increased firing rate evoked by hormonal status, recorded at the cell body (142), correlates with increased secretion, at the ME (140), highlighting the regulation of firing activity. Most importantly, it allows subcellular measurements and shows a site-specific regulation of GnRH release (141). Different regulation of somatodendritic and nerve terminal release is known in magnocellular neurons (143), but a new insight in GnRH neurons. In the POA (at bundles of proximal processes), increases in [Ca²⁺]_i evoked by sarco/endoplasmic reticulum calcium-ATPase blocker evoke GnRH release. While in the ME (at nerve terminals), APs must accompany such increases to evoke GnRH release (141). In addition, locally applied inositol triphosphate receptor blocker prevents kisspeptin-evoked GnRH release in the ME but not in the POA. In contrast, locally applied calcium channel blocker prevents kisspeptin-evoked GnRH release in the POA but not in the ME, where calcium and sodium channel blockers are necessary (141).

Subcellular electrophysiology and calcium imaging identify different functions at different locations in GnRH neurons (20, 115, 144) (Figure 1). APs initiate in the proximal process (144) and patterning occurs at the cell soma (145). APs propagate along the process (144) and elicit temporally restricted calcium rises at the nerve terminals (115). The activation of GnRH neurons in the POA triggering a GnRH/LH pulse in vivo illustrates this phenomenon (88). However, the straightforwardness stops with electrical stimuli. GnRH neurons become versatile when exposed to ligands. GnRH projections exhibit unique properties allowing local depolarizations to reshape APs along the way to the ME (20). Applied at the cell body, kisspeptin evokes a calcium rise, accompanied by APs (115). Although the calcium rise at the cell body is independent of firing (48, 115, 141), APs will travel and evoke a spike-dependent calcium rise at the nerve terminals (115). Applied at the nerve terminals, kisspeptin evokes a local calcium rise, independent of APs (115), and triggers GnRH secretion, even when sodium channel blockers are present (141).

Until today, the conundrum was with kisspeptin producing both a massive surge and timely restricted pulses. However, subcellular regulation in GnRH neurons provides new hypotheses for GnRH secretion (Figure 2). RP3V kisspeptin neurons innervating the GnRH cell body (118) probably initiate different response than ARC kisspeptin neurons innervating the nerve terminals (118), thus regulating GnRH secretion differently. For example, kisspeptin evokes a long-lasting calcium rise in nerve terminals (>60 min) (115) but FSCV detects GnRH release for ~1 min (141). Possibly, non-secreting calcium-dependent vesicle dynamics might follow calcium-evoked GnRH secretion at the nerve terminals (115, 129, 146). FSCV indicates kisspeptin-evoked secretion at the ME is specifically regulated and an increase in [Ca²⁺]_i is not the only requirement (141). Exocytosis involves many protein–protein interactions regulated by second messengers and phosphorylation (147). Kisspeptin triggers a complex signaling pathway (48, 148) that might allow it to define the relationship between calcium and secretion at GnRH nerve terminals.

Our knowledge of the physiology of GnRH neurons is ever evolving and we should remain as naïve as possible when studying them. As in many other fields, the knowledge is limited by techniques and none of the “classical” tools available in neuroscience are readily usable for GnRH neurons. Even nowadays, the knowledge of GnRH neurons still suffers from the anatomical intricacy of the system. However, with creativity and tenacity, knowledge about GnRH neurons builds up and common assumptions fall: the simple bipolar GnRH neuron displays arborized distal processes, the scattered cell bodies are reunited with entwined dendrites, GnRH is released at the cell body, the processes become dendrons with merged features of axon and dendrite, and neuronal inputs relocate into differentially regulated GnRH neuron segments. I am positive the list will continue to grow as we try and understand the mechanism(s) underlying pulsatile GnRH secretion.

What do we need to unravel the mystery behind GnRH secretion? I believe the next step is to tailor genetic tools to target genetically encoded sensors such as GCaMP6s and pHuji, to GnRH neurons for simultaneous imaging of calcium dynamics and secretory vesicle fusion. This should allow for the deciphering of their precise relationship and the investigation of how intracellular signaling pathways downstream of GPCRs and other receptors can modulate this relationship.

The author confirms being the sole contributor of this work approved it for publication.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.