Physiology, Gonadotropin-Releasing Hormone

Issues of Concern

GnRH is vital because it has implications in the pathogenesis of central hypogonadism. GnRH and its analogs are used as a treatment modality in infertility, endometriosis, central precocious puberty, and hormone-dependent malignancies like breast cancer and ovarian cancer.

Cellular Level

Genetics:

In humans, the GnRH I gene, consisting of four exons and three introns, resides on the short arm of chromosome 8 (8p21-p11.2). The journey of GnRH begins in the medial olfactory placode. From here, it travels along the olfactory bulb to reach the hypothalamus. GnRH is then secreted, in a pulsatile fashion, into the hypophyseal portal circulation where it reaches its primary destination, the anterior pituitary. Here it binds the gonadotropin-releasing hormone receptor (GnRHR), which is a G-protein coupled receptor, on the pituitary gonadotrophic cells. Binding of GnRH to the GnRHR initiates downstream signaling of the primary gonadotropins: follicle-stimulating hormone (FSH), and luteinizing hormone (LH).[2]

Multiple genes aid in the process of development and differentiation (fibroblast growth factor receptor 1 (FGFR1) and its ligand fibroblast growth factor 8 (FGF8), heparin sulfate 6-O-sulphotransferase 1 (HS6ST1) and nasal embryonic LH-releasing hormone Factor (NELF)), migration (NOS1, semaphorin 3A (SEMA3A), prokineticin 2 (PROK2) and prokineticin receptor 2 (PROKR2)), and neuronal stability (SEMA3E).

Neuroanatomy:

GnRH cell bodies are in the medial preoptic area (POA) and the arcuate/infundibular nucleus of the hypothalamus, forming a neuronal network with projections to the median eminence. GnRH secretion occurs from the median eminence into the fenestrated capillaries of portal circulation and then is carried to the anterior pituitary. In humans, estimates of the number of GnRH neurons range between 1000 to 1500. The co-location of GnRH neurons with other central regulators allows the GnRH network to be influenced by a range of neuroendocrine and metabolic inputs.[3]

Gonadotropin-releasing hormone receptor (GnRHR):

The location of the GnRH receptor (GnRHR) is in the anterior pituitary and belongs to the family of the G protein-coupled receptors. Its seven transmembrane domains describe this receptor class. These receptors, when bound by an activating subunit, undergo conformation change and activate intracellular pathways leading to modulation of genes within a target cell, via phosphorylation events. Activation of the receptors leads to the creation of receptor clusters. These receptor clusters can be shuttled to the surface of the cell or degraded in lysosomes after they become internalized.  A short intracellular carboxy-terminal tail characterizes this particular receptor. This structure helps to prevent desensitization and slow internalization of the receptor. The GnRHR links to a member of the G-protein family called Gq. The Gq protein cleaves a molecule called phosphatidylinositol-4-5-bisphosphate (PIP). PIP cleavage results in the formation of inositol phospholipid (IP3), and cleavage of the molecule PIP2. IP3 stimulated the endoplasmic reticulum to release calcium into the cytosol. DAG activates the protein kinase C (PKC) signaling cascade. Protein kinase C then goes on to stimulates the MAP kinase and ERK1/2 cascades. As a minor action, activation of the GnRHR can activate the cAMP and protein kinase A (PKA) signaling cascades, which occurs via the Gs and the calcium and calmodulin system. Once these pathways are activated, they lead to the biosynthesis and secretion of gonadotropin.[4][5] 

Development

During Embryonic life:

The embryonic development of GnRH neurons closely ties to the olfactory system. Neurons that release GnRH use vomeronasal and olfactory axons as a scaffold to migrate along. Once in the forebrain, they travel to their eventual final position via a branch of the vomeronasal/terminal nerve. GnRH neuronal development takes place between the 5th and 16th embryonic weeks (EW). By the middle of the 5th EW to start the 6th EW, GnRH neurons are detectable in the olfactory placode. By the middle of the 6th EW, these neurons begin to migrate near the terminal nerve, where they enter the forebrain.

By the 9th EW, these neurons will reach the hypothalamus. Between weeks 13 and 16 of gestation is when migration is considered complete. GnRH levels are detectable at the 10th gestational week, but LH and FSH levels are detectable only after 13th gestational week, the reason of delayed appearance of LH and FSH is the formation of vascular connections between pituitary and hypothalamus around 10 to 13 weeks, after which GnRH can reach pituitary and cause the release of FSH and LH.

The levels of GnRH gradually increase and reach a peak level at the mid gestational age, after which they gradually fall toward the end of the gestational period due to the negative feedback effect of circulating placental steroids. At birth development of GnRH neurons is complete, but the functional maturation of synaptic connectivity is attained later in life, especially at puberty. After birth, these levels remain elevated for two years in girls while for six months in boys. The mechanism of suppression of GnRH after birth is still unknown, but certain neurotransmitters like GABA and Neuropeptide Y seem to play an important role in the suppression of GnRH before puberty.

At Puberty:

This temporary pause in GnRH release ends at puberty, and recent studies have shown that Kisspeptin neurons are responsible for activation of hypothalamic-gonadotropic axis activation causing the GnRH release at puberty. Initially, at puberty, GnRH is released in low-frequency pulses during the night, but after the maturation of synaptic connections, it matches the adult pattern. In males, the GnRH pulses occur after 2 hours, while in a female, it changes according to the phases of the menstrual cycle. It is clear that the episodic release of GnRH is a general phenomenon. Also, fluctuation in the amplitude and frequency of GnRH bursts plays a vital role in initiating hormonal charges that ultimately regulate the menstrual cycle. The frequency of GnRH bursts is decreased by testosterone and progesterone and increased by estrogens. The frequency (1 pulse of GnRH/60 to 90 mins) during the late follicular phase of the menstrual cycle is increased, culminating in the LH surge. In the secretory phase of the menstrual cycle, the action of progesterone decreases the frequency (1 pulse of GnRH/200mins). At the end of the menstrual cycle, when progesterone and estrogen secretion decreases, the frequency increases. The sensitivity of gonadotropes increases significantly during the midcycle LH surge; this is due to the exposure to pulses of GnRH at a specific frequency, which describes a critical self-priming effect of GnRH that produces a maximum LH response. Thus, changes in GnRH frequency and amplitude alters the synthesis of gonadotropin synthesis and LH and FSH release.

At menopause:

GnRH release changes during the perimenopausal period. As the number of follicles recruited during each menstrual cycle decreases near menopause, so does the amount of estrogen produced, resulting in the reduced negative feedback of estrogen on GnRH release leading to an increase in GnRH release frequency (every 55 mins) and amplitude. As women age from 50 to 80 years of age, the frequency of GnRH pulses decreases by 35%.  

Organ Systems Involved

GnRH is a central regulator of the hypothalamic-pituitary-gonadal (HPG) axis. The neurons that produce GnRH are in the hypothalamus, specifically in the infundibular nucleus. Once secreted, GnRH acts on the anterior pituitary where follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are secreted and modulate sex steroid production from the gonads.[6]

Function

As the GnRH is a peptide hormone, its receptors are located in the cell membrane.

The GnRH receptor (GnRHR), is part of a receptor superfamily of rhodopsin-like G protein-coupled receptors. These receptors have a hydrophilic extracellular domain, intracellular domain, and finally, a hydrophilic transmembrane domain.  This transmembrane domain spans the cell member a total of seven times.  These receptors, once bond to the hormone, undergo conformational changes that activate an intracellular signal cascade, and through several phosphorylation events, eventually leads to transcriptions modulation of genes within the target cell. Activation of these receptors induces the formation of receptor clusters that become internalized into the cell, where they are then shuttled back to the surface of the cell or degraded in lysosomes. The relatively short intracellular carboxy-terminal tail of the GnRH Receptor makes it differ from other G-protein coupled receptors. This tail slows the internalization of the receptor and prevents rapid desensitization. GnRH receptors are Gq-protein coupled. This process occurs in the following steps.

GnRH or GnRH agonist binds to GnRH receptors located at pituitary cells.

1. It results in phosphorylation of GDP and formation of GTP, GTP binds to the alpha subunit of Gq protein, and the alpha subunit along with bound GTP gets detached from G protein and attaches itself to phospholipase C.
2. Phospholipase C breaks down PIP (Phosphoinositol diphosphate) into IP (inositol triphosphate) and DAG (Diacyl Glycerol).
3. DAG causes further activation of Protein kinase C.
4. Protein kinase C causes phosphorylation of intracellular proteins.
5. Inositol triphosphate binds to receptors on the endoplasmic reticulum membrane and causes the release of calcium into the cytoplasm.
6. The proteins phosphorylated by Protein kinase C, serve as the intracellular signaling pathway for mitogen-activated protein kinases (MAPK) pathway, including ERK, JNK, and p38 MAPK.
7. Calcium released into cytosol binds to calmodulin, causing further phosphorylation of proteins via activation of another set of protein kinases, performing functions of intracellular transport, enhancement of cell metabolism, and exocytosis of vesicles storing hormones. 

Another point worth remembering is that although most GnRH receptors are Gq-protein coupled, few of them are also Gs protein-coupled. This Gs/cAMP pathway is involved in the initial response of pituitary gonadotroph to GnRH or GnRH analogs and further regulation of synthesis and secretion of pituitary gonadotroph.

Differential regulation of LH and FSH:

Although GnRH acts on pituitary cells to enhance gonadotropes release, its action is not equal to both FSH and LH. The frequency of GnRH pulses selectively upregulates gonadotrope gene transcription. Rapid pulses promote LH formation and secretion, while slow pulses promote FSH formation and secretion. Researchers have observed that even blockade of GnRH agonist by giving GnRH antisera also has a differential effect on FSH and LH, resulting in the absence of LH pulses in 24 hours while FSH levels remain detectable for a longer period.   

Mechanism

GnRH secretion and pulsatility:

The secretion of GnRH into hypophyseal blood occurs in two modes:

• Surge mode: refers to the high frequency or continuous release of GnRH in the preovulatory phase of the menstrual cycle.
• Pulsatile mode: refers to the release of GnRH after regular intervals and occurs both in males and females, but the frequency of release is different in men and women. In women, the frequency of GnRH release changes throughout the menstrual cycle. One pulse every 1 to 2 hours in the follicular phase, continuous in the preovulatory phase, and then decreasing to 1 pulse every 4 hours in the secretory phase. 

The actual location and source of GnRH pulsatile release are not entirely understood. Episodic multi-unit electrical activity in the medial basal hypothalamic (MBH) is correlated with LH release, suggesting that ‘GnRH pulse generator’ is anatomically located in the MBH or closely linked to it neuro hormonally. GnRH neurons themselves show intrinsic neuronal activity. Physiologically, the pulsatile release of GnRH depends upon a complex interaction between Glutaminergic neurons, GnRH neurons, and KNDy neurons.

Regulation of GnRH release:

GnRH neurons respond to sex steroids, glucocorticoids, inflammation, stress, Drugs, body metabolism, and nutrition modulating their release in response to these stimuli. GnRH neurons do not directly respond to these stimuli. Instead, another set of neurons called KNDy neurons acting as a bridge between GnRH neurons and environmental and Internal stimuli are mainly responsible for this communication and thus regulation of GnRH release. The discovery of KNDy neurons has led to a new understanding of GnRH release regulation.

KNDy Neuronal System: 

KNDy neuron is a combination of neurons secreting kisspeptin, neurokinin B, and dynorphin. These neurons are afferent to GnRH neurons and act as a bridge between various modulators of GnRH release and GnRH neurons. Input fed by KNDy neurons to GnRH neurons is physiologically essential for the adequate functioning of GnRH neurons. 

• Kisspeptin: is a polypeptide (145 amino acids) that binds to G-protein-coupled receptor 54 (GPR54). Approximately 50 to 75% of GnRH neurons express the kisspeptin receptor. Neurons that secrete kisspeptin are in the arcuate and anteroventral periventricular (AVPV) nuclei of the hypothalamus. AVPV neurons are more abundant than arcuate kisspeptin neurons. However, the arcuate neurons are closer in proximity to the GnRH neurons located in the median eminence. Kisspeptin acts at the level of the hypothalamus, which stimulates GnRH secretion. In recent studies, it is also shown that kisspeptin can also bind to Pituitary cells to cause an increase in LH release. 

Kisspeptin neurons also have receptors for sex steroids and thus modulate GnRH release from the hypothalamus. A very interesting phenomenon shown by kisspeptin neurons is sexual dimorphism, Expression of KISS-1 gene is under the control of both estrogen and androgens.  A male’s arcuate nuclei contain more kisspeptin neurons and are under the influence of androgens, while a female’s AVPV has more kisspeptin neurons and is mainly under the influence of estrogen. It is the reason mainly responsible for the inability of estrogen to cause a GnRH surge in males.

• Neurokinin B: Neurokinin B belongs to the tachykinin family of peptides and stimulates TACR3 receptors, neurokinin stimulates kisspeptin neurons and further leading to GnRH release. This neurotransmitter is also physiologically important as some patients with hypogonadotropic hypogonadism have shown missense mutation involving TAC3 (gene encoding neurokinin B) and TACR3 (Gene encoding neurokinin B receptor).
• Dynorphin: Opioids are involved in the regulation of the hypothalamic-pituitary-gonadal axis as they inhibit kisspeptin neurons and thus modulate GnRH release.

All three neurons communicate via neuron-neuron and neuron-glia gap junctions to modulate the release of GnRH release. Kisspeptin neurons have receptors for neurokinin (stimulatory) and dynorphin (inhibitory). While kisspeptin receptors are located on GnRH neurons, stimulation of these receptors results in the release of GnRH. However, continuous stimulation of GnRH neurons by kisspeptin results in desensitization and down-regulation of kisspeptin receptors.

Related Testing

It is challenging and difficult to take blood samples from the hypophyseal portal blood because of its short half-life (2 to 4 minutes) and confinement only to hypophyseal blood. Luteinizing hormone levels are therefore measured as a surrogate to measure the GnRH concentration.  

Pathophysiology

Diseases related to defective GnRH neurons migration:

Kallmann Syndrome: Characterized by a combination of hypogonadotropic hypogonadism and anosmia

Idiopathic hypogonadotropic hypogonadism: Characterized by hypogonadotropic hypogonadism without anosmia due to mutations of prokinetic genes (PROK 1 and PROK 2)

Clinical Significance

Multiple feedback mechanisms in humans regulate GnRH activity and have implications on sexual development, breastfeeding, menstruation, and fertility. Prolactin is a hormone that exhibits inhibitory effects on GnRH, thus inhibiting FSH and LH production by the anterior pituitary. In females, this ovulatory inhibition leads to lactational amenorrhea, which acts as a physiologic contraceptive. In addition to lactation in females, prolactin inhibition of GnRH can result in infertility in males due to decreased spermatogenesis.[7] 

Pharmacologically, GnRH agonists are associated with a decrease in uterine fibroma volume and have been used as presurgical treatment.[8] Leuprolide, a GnRH receptor agonist, is used in the treatment of prostate cancer, which has replaced the former treatment of surgical castration. Leuprolide is also used in the treatment of endometriosis, infertility, and precocious puberty. Given that this medication and other GnRH analogs cause hypogonadism some common side effects are hot flashes, erectile dysfunction, loss of libido, depression, nausea, diarrhea, and weight gain.[9] GnRH antagonists have been shown to effective in controlling ovarian stimulation, making them useful in reproductive medicine.[10]