Normal mammalian sexual maturation and reproductive functions require the integration and precise coordination of hormones at the hypothalamic, pituitary, and gonadal levels. The hypothalamic GnRH (Gonadotropin Releasing Hormone), also called LHRH (Luteinizing Hormone Releasing Hormone), is a key regulator in this system (the hypothalamic-pituitary-gonadal axis), that plays a decisive role in the neuroendocrine regulation of human reproduction. GnRH is a decapeptide, released in an episodic manner from the hypothalamic GnRH neurons. The pulsatile delivery of GnRH to the anterior pituitary gland is essential to maintain the circulating gonadotropin profiles. It acts via a specific GPCR (G-Protein Coupled Receptor): GnRHR (GnRH Receptor) and triggers the synthesis of the common Alpha- and Beta-chains of the gonadotropins, which in turn, control the function of the gonads and induce steroidogenesis. The primary site of action of GnRH in the pituitary gland is the gonadotropes, the cells which express GnRHR and secrete gonadotropins, namely LH (Luteinizing Hormone) and FSH(Follicle Stimulating Hormone). These hormones in turn regulate most of the reproductive functions in both sexes through the production of gonadal steroids and regulation of their gametogenic and hormonal functions. GnRH not only causes the production de novo of the gonadotropins but also induces their secretion from pituitary gonadotrophs, allowing them to regulate the synchronization of the reproductive cycle (Ref.1, 2 & 3).
GnRH stimulates the synthesis and release of pituitary gonadotropins (LH and FSH), acting through the GnRHR located on the plasma membrane of pituitary gonadotropes. GnRHR transmits its signals mainly through heterotrimeric G-proteins for its downstream signaling and is capable of activating multiple signaling pathways. The mammalian GnRHR is unique among GPCRs in that it lacks the common carboxyl-terminal cytoplasmic domain and possesses a relatively short intracellular third loop. The distinct structure of the pituitary GnRHR in mammals is likely to have functional relevance related to the development of mammalian reproductive strategies (Ref.4 & 5).
The signaling events by GnRH include: actvation of the G-Proteins ; enhanced Phosphoinositide turnover; activation of PLC (Phospholipase-C) (apparently PLC-Beta); Ca2+ mobilization and influx; translocation and activation of PKC (Protein Kinase-C); and formation of bioactive lipoxygenase products that culminate in gonadotropin production and release. Binding of GnRH to GnRHR activates G-AlphaQ-GN-AlphaI- and G-AlphaS-dependent signaling pathways (Ref.5 & 6). Activation of G-AlphaS further activates the AC (Adenylyl Cyclase)-->cAMP (Cyclic Adenosine Monophosphate)-->PKA (Protein Kinase-A) pathway, which positively influences transcriptional activity of the GnRHR, via CREB (cAMP Responsive Element Binding Protein) (Ref.7). GN-AlphaI activation by GnRH has both positive and negative effects on the transcriptional activities of the GnRHR. GN-AlphaI counteract the G-AlphaS mediated CREB activation by direct inhibtion of AC. On the other hand, GN-AlphaI also helps in the synthesis of LH and FSH via activation of the transcription factor, NF-KappaB (Nuclear Factor-KappaB). However, the major signal transduction pathways stimulated by GnRH are initiated by activation of G-AlphaQ protein that contributes to the activation of PLC-Beta (Ref.8). The consequent PIP2 (Phosphatidylinositol 4,5-Bisphosphate) activation, Ca2+ (Calcium) mobilization, production of DAG (Diacylglycerol) and activation of PKCs, switch on the MAPK (Mitogen Activated Protein Kinase) cascade. GnRHR occupancy is linked with an increase in intracellular Calcium through mobilization of two distinct pools. Extracellular calcium enters the cell through VGCCs (Voltage-Gated Ca2+ Channels) in the plasma membrane while IP3 (Inositol Triphosphate) releases calcium from the ER (Endoplasmic Reticulum). The IP3-released Calcium is one of the critical signals required for secretion of the gonadotropic hormones: LH and FSH . This secretory function is accomplished by the activation of CalmKII (Calcium/Calmodulin-Dependent Protein Kinase Type-II) by Calcium, that further activates the Trnscription Factor CREB (Ref.9).
GnRHR signaling through the MAPK cascades provides an important link for the transmission of signals from the cell surface to the nucleus. Activation of ERKs (Extracellular-Signal-Regulated Kinases), one of the MAPK cascades by GnRH depends mainly on the phosphorylation of Raf by PKC, supported by a pathway involving Src, Dynamin, and Ras (Ref.10). Thus, the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf. The main pathway involves a direct activation of Raf by PKC , and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a Dynamin-dependent manner, downstream of Src (Ref.11). ERK1 and ERK2 are markedly activated upon GnRH stimulation of GnRHR-expressing cell. However, ERK activation by GnRH does not lead to the well-known effects of ERK, namely enhanced proliferation or differentiation (Ref.12). It is rather involved with the transcription of gonadotropins and cytoskelatal rearrangements. Apart from activating the ERK cascade, GnRH also stimulates the activity of other stress-related MAPK cascades (JNK, BMK1, and p38). The mechanism of BMK1 (Big MAP Kinase-1) and p38 activation by GnRH is PKC-dependent (Ref.11). p38 is activated by PKC through the activation MEKKs (MAP/ERK Kinase Kinases) and MKK3 (MAP Kinase Kinase-3) and MKK6. GnRH--->JNK (c-Jun N-terminal Kinase) cascade is activated by a pathway that involves PKC, Src, CDC42 (Cell Division Cycle-42), MEKKs (MEKK1 and MEKK2) , MKK4 and MKK7, which leads to JNK activation and to the induction of c-Jun in response to GnRH (Ref.7 & 13). GnRHR signals to ERKs can also be transmitted by activating the EGFR (Epidermal Growth Factor Receptor). The EGF (Epidermal Growth Factor)-activated EGFR is transactivated by the GnRH-induced Src kinase. The activated EGFR provides docking sites for several signaling proteins, including SOS (Son of Svenless) and GRB2 (Growth Factor Receptor-Bound Protein-2), which leads to activation of the Ras-->Raf-->MEKs -->ERK pathway (Ref.14 & 15).
Activation of BMK1, JNKs and ERKs provides a route for activation of the transcription factors: c-Jun and c-Fos; to form the Jun-Fos dimer that activates the Activator Protein-1 responsive element present in both LH and FSH promoters. Gonadotropins are composed of two Alpha and Beta subunits, which are noncovalently linked. Beta subunits as well as the common Alpha subunit are encoded by different genes on separate chromosomes. GnRH induces expression of the gene encoding FSH-Beta via c-Jun and c-Fos. The gene encoding the common Alpha subunit is regulated by c-Jun and ATF2 (Activating Transcription Factor-2) at the CRE (cAMP Responsive Element) site, and by Elk1 at the Ets
site of the PGBE (Pituitary Glycoprotein Hormone Basal Element) domain. Although GnRH induction and basal control of the Alpha subunit gene appear to occur through the ERK and p38 pathways, induction of the LH-Beta gene is dependent on JNKs in a c-Jun-dependent mechanism, suggesting the differential stimulation of transcription of LH subunit genes by GnRH (Ref.1, 3, 4 & 12).
In addition to the synthesis of the Gonadotropins, cell motility and
migration also gets affected by the GnRH signaling. The GnRH-activated Src
Kinases bring about phosphorylation of the focal adhesion tyrosine kinase, FAK, which is then recruited to the plasma membrane focal adhesion complex. These protein signaling complexes assemble on Integrin heterodimers following integrin engagement of ECM (Extracellular Matrix) proteins. The transmembrane Integrin family of cell adhesion molecules mediates cellular contacts to the ECM. These cell surface receptors are vital for the control of regulating cell motility, polarity, growth and survival. Integrin signaling, via actin reorganization, at cell leading edges can activate CDC42 and Rac1 monomeric G-Proteins, resulting in cytoskeletal rearrangement and membrane extension. Cytoskeletal rearrangement can also be brought about by ERK1 and ERK2. Rac1 activates PAK (p21/CDC42/Rac1-Activated Kinase) which further leads to the activation of MEK1 and/or MEK2; followed by ERK1 and/or ERK2 (Ref.16).
Male fertility is regulated through the hypothalamic-pituitary-gonadal axis, through the actions of GnRH and the gonadotropins secreted by it. In men LH stimulates Leydig cells to produce Testosterone and stimulates activity of the interstitial cells of the testes. It also causes growth of the Corpus Luteum of the Ovary. FSH acts on Sertoli cells, thereby stimulating spermatogenesis and stimulates growth of Graafian Follicles of the Ovary. In addition to regulating pituitary gonadotropin release, GnRH has extrapituitary actions in neural and nonneural tissues and in several types of tumor cells. The expression of GnRH and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary and endometrium. GnRH and GnRH analogues have extensive application in the treatment of human diseases (Ref.17). For example, they are utilized to treat infertility, precocious puberty, polycystic ovarian syndrome and breast cancer in women, delayed puberty in boys, prostatic cancer in men, in the protection of gonadal tissue during radiotherapy and chemotherapy and show promise as new contraceptive agents for both men and women. Attempts to apply GnRH-based technology to manage fertility have focussed on the development of GnRH agonists, antagonists and vaccines. GnRH agonists and vaccines are the only two GnRH-based fertility control methods that have successfully been used in a wide range of domestic animal species and are approaching the commercialization stage (Ref.18, 19 & 20).