Gn-RH and Gn-RH receptors Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università degli Studi di Napoli Dipartimento di Scienze della Vita SUNfert

Hypothalamic anatomy  Hypothalamus is a pars of diencephalon  Under thalamus  Is floor of the third ventriculus

Hypothalamic anatomy  Optic Chiasm  Mammillary bodies  Median eminence  Infundibulum

Legenda: b- END = b-endorfine; CRF = Corticotropin Releasing Factor; OT = Somatostatina; DA = Dopamina; GABA = Acido g-Amino- Butirrico; ACh = Acetilcolima; NPY = Neuropeptide Y; NA = Noradrenalina Neurotransductor effects on Gn-RH release

leptine  Modulates NY    Gn-RH secretion

Leptin bioactivity

Leptine

Hypothalamic nuclei medial eminence (ME) ventromedial nucleus (VM) arcuate nucleus (AR)

hypothalamic nuclei RegionMedial AreaLateral Area Anterior Medial preoptic nucleus Supraoptic nucleus Paraventricular nucleus Anterior nucleus Suprachiasmatic nucleus Lateral preoptic nucleus Lateral nucleus Part of supraoptic nucleus Tuberal Arcuate nucleus Dorsomedial nucleus Ventromedial nucleus Arcuate nucleus Lateral nucleus Lateral tuberal nuclei Posterior Mammillary nuclei (part of mammillary bodies) Posterior nucleus Lateral nucleus

Hormones of the hypothalamus o Corticotropin-releasing hormone (CRH) o Dopamine (DA) o Gonadotropin-releasing hormone (Gn-RH) o Growth hormone releasing hormone (GH-RH) o Somatostatin (ST) o Thyrotropin-releasing hormone (TRH) o Antidiuretic Hormone (ADH)

Gn-RH  Gn-RH neurons are inside the medium- basal hypothalamus (arcuate nucleus and median eminence)  Lately scientists showed Gn-RH syntesis in pituitary gland too. Human Luteinizing hormone-releasing hormone gene (LH-RH) is located on short arm of chromosome 8 (region 8p11.2 → p21) * Teresa L. Yang-Feng, Peter H. Seeburg and Uta Francke: Somatic Cell and Molecular Genetics Volume 12, Number 1 / January, 1986

Gn-RH  Gn-RH is considered a neurohormone  produced in a specific neural cell and released at its neural terminal  A key area for production of Gn-RH 1 is the preoptic area of the hypothalamus, that contains most of the Gn-RH 1 -secreting neurons.  Gn-RH 1 is secreted in the hypophysial portal bloodstream at the median eminence  The portal blood carries the Gn-RH 1 to the pituitary gland, which contains the gonadotrope cells, where Gn-RH 1 activates its own receptor, gonadotropin-releasing hormone receptor (Gn-RH-R), located in the cell membrane

Gn-RH  Gn-RH neurons are closely connected with noradrenergic, dopaminergic, serotoninergic, oppioid ones.

Gn-RH frequency Low frequency Gn-RH pulses lead to FSH releaseLow frequency Gn-RH pulses lead to FSH release high frequency Gn-RH pulses stimulate LH releasehigh frequency Gn-RH pulses stimulate LH release

Gn-RH-r  Gn-RH-r is a member of the seven-transmembrane, G-protein coupled receptor (GPCR) family  It is expressed on the surface of pituitary gonadotrope cells* * as well as lymphocytes, breast, ovary, and prostate

Gn-RH-r Function  Following binding of Gn-RH activate a phosphatidylinositol (PtdIns)-calcium second messenger system.  Activation of GNRH-r ultimately causes the release of FSH and LH

Gn-RH  Gn-RH is considered a neurohormone  produced in a specific neural cell and released at its neural terminal preoptic area  A key area for production of GN-RH 1 is the preoptic area of the hypothalamus, that contains most of the GNRH1-secreting neurons.  GN-RH 1 is secreted in the hypophysial portal bloodstream at the median eminence  The portal blood carries the GN-RH 1 to the pituitary gland, which contains the gonadotrope cells, where GN-RH 1 activates its own receptors, gonadotropin-releasing hormone receptors (GN-RH-R), located in the cell membrane

Gn-RH  Gn-RH neurons are closely connected with noradrenergic, dopaminergic, serotoninergic, oppioid ones.

Males/females Gn-RH secretion in males, Gn-RH 1 is secreted in pulses at a constant frequency in males, Gn-RH 1 is secreted in pulses at a constant frequency in females the frequency of the pulses varies during the menstrual cycle in females the frequency of the pulses varies during the menstrual cycle there is a large surge of GNRH 1 just before ovulation there is a large surge of GNRH 1 just before ovulation

Proteolysis  Proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called protease or by intramolecular digestion. Gn-RH is degraded by proteolysis within a few minutes

Control of FSH and LH At the pituitary, Gn-RH 1 stimulates the synthesis and secretion of FSH and LH These processes are controlled by:  the size and frequency of Gn-RH 1 pulses,  feedback from androgens and estrogens

Gn-RH-R Function Following binding of Gn-RH activate a phosphatidylinositol(PtdIns)-calcium second messenger system. Activation of Gn-RH-R ultimately causes the release of FSH and LH

receptors  Hormones and active metabolites bind to different types of receptors. Water-soluble molecules (i.e., insulin) cannot pass through the lipid membrane of a cell and thus rely on cell surface receptors to transmit messages to the interior of the cell. In contrast, lipid-soluble molecules (i.e., certain active metabolites) are able to diffuse through the lipid membrane to communicate messages directly to the nucleus

Gn-RH-R Gn-RH-R is a member of the seven- transmembrane, G-protein coupled receptor (GPCR) family. It is expressed on the surface of pituitary gonadotrope cells* * as well as lymphocytes, breast, ovary, and prostate.

Gn-RH biochemistry a decapeptide (10 amino acids) in mammals. This chain is represented by: pyroGlu-His-Tyr-Ser- Gly-Leu-Arg-Pro-Gly-NH 2 previously called LH-RH The identity of GN-RH 1 was clarified by the 1977 Nobel Laureates Roger Guillemin and Andrew V. Schally

Gn-RH Gn-RH is considered a neurohormone produced in a specific neural cell and released at its neural terminal. A key area for production of Gn-RH 1 is the preoptic area of the hypothalamus, that contains most of the Gn-RH 1 -secreting neurons. Gn-RH 1 is secreted in the hypophysial portal bloodstream at the median eminence. The portal blood carries the Gn-RH 1 to the pituitary gland, which contains the gonadotrope cells, where Gn-RH 1 activates its own receptor (Gn-RH-R), located in the cell membrane.

Gn-RH neural junctions  Gn-RH neurons are closely connected with: noradrenergic, dopaminergic, serotoninergic, oppioid ones.

Gn-Receptors  Upon binding Hormone externally to the membrane, a transduction of the signal takes place that activates the G protein  that is bound to the receptor internally

Gn-Receptors  With Hormone attached, the receptor shifts conformation  mechanically activates the G protein, which detaches from the receptor and activates the cAMP system.

G Protein System Alfred G. Gilman and Martin Rodbell received the 1994 Nobel Prize in Medicine and Physiology for the discovery of the G Protein System

Gn-RH-R Function  Following binding of Gn-RH activate a phosphatidylinositol (PtdIns)-calcium second messenger system.  Activation of Gn-RH-R ultimately causes the release of FSH and LH

Gonadotropin-releasing hormone receptor  Gn-RH-R is a member of the seven- transmembrane, G-protein coupled receptor (GPCR) family.  It is expressed on the surface of pituitary gonadotrope cells* * as well as lymphocytes, breast, ovary, and prostate.

The seven transmembrane α-helix structure of a G protein-coupled receptor

hormone receptor*  I° messenger: hormone  Hormone + Receptor  G-Protein activation: Adhesion H-R complex Scission H-R complex Subunit a production * A protein on the surface of a cell to which a specific hormone binds

hormone receptors  a subunit activates  intracellular effectors (enzymes)   adenyl-ciclases  cAMP   guanil-cyclase  cGMP   phospholipase  DAG*, IP3 *   ionic ducts  (K +, Ca ++ )  *Diacilglicerolo, Inositolo trifosfato (II° messenger)

PO 4 by cAMP-dependent PKA II° messenger  II° messenger  activates Protein kinasi (PKA)  activates Phosphorylation (PO 4 ) of: citoplasmatic proteincitoplasmatic protein nuclear transcription factors (cAMP Responsive Element Binding Protein, CREB).nuclear transcription factors (cAMP Responsive Element Binding Protein, CREB).

DNA transcription CREB modulates transcription of genes interacting directely with specific DNA string

hormone receptors

Control of FSH and LH  At the pituitary, Gn-RH 1 stimulates the synthesis and secretion of FSH and LH  These processes are controlled by: the size and frequency of GN-RH 1 pulses feedback from androgens and estrogens Gn-receptors wholeness

Control of FSH and LH Low frequency Gn-RH pulses lead to FSH releaseLow frequency Gn-RH pulses lead to FSH release high frequency Gn-RH pulses stimulate LH releasehigh frequency Gn-RH pulses stimulate LH release

Gn-RH secretion males/females in males, GN-RH 1 is secreted in pulses at a constant frequency in males, GN-RH 1 is secreted in pulses at a constant frequency in females the frequency of the pulses varies during the menstrual cycle in females the frequency of the pulses varies during the menstrual cycle there is a large surge of GN-RH 1 just before ovulation there is a large surge of GN-RH 1 just before ovulation

Gn-RH proteolysis  Proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called protease or by intramolecular digestion. Gn-RH is degraded by proteolysis within a few minutes

Gn-RH-a  A gonadotropin-releasing hormone agonist (GnRH agonist) is a synthetic peptide modeled after the hypothalamic neurohormone GnRH that interacts with the gonadotropin-releasing hormone receptor to elicit its biologic response, the release of the pituitary hormones FSH and LH

Gn-RH-a Aminoacid sequence nameact for Gn-RH1 Pyro- glu HisTrpserotoninTyr Leu LeuArgPro Gly- NH 2 iv Leuproreline * 15D-Leu N- EtNH 2 sc, im Buserelin * * 20D-Ser N- EtNH 2 sc, im triptor * * * D-Trip sc, im Goserelin * * * * 100D-Ser AzGly- NH 2 depot sc * Enantone 3.75, mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.; * Enantone 3.75, mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.; * * Suprefact 5.5 ml fl s.c.; Suprefact spray nasale 10 gr (1 buff = 200 mg) * * * Decapeptyl 3.75, mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c. * * * * Zoladex 3.6, 10.8 mg fl s.c. im Triptorelin is an agonist with only a single substitution at position 6.

Gn-RH-a administration mode  These medications can be administered  intranasally,  by injection,  or by implant.  Injectables have been formulated for daily, monthly, and quarterly use; and implants can last from 1 to 3 months.

Gn-RH-a Downregulation  after their initial stimulating action – termed a “flare” effect -, eventually caused a paradoxical and sustained drop in gonadotropin secretion. after about 10 days  This second effect was termed “downregulation” and can be observed after about 10 days.  While this phase is reversible it can be maintained with further GnRH agonist use for a long time.

Gn-RH-a Downregulation  refers to the decrease in the number of receptor sites.  This can be accomplished by metabolizing bound LH-R sites.  The bound LCGR complex is brought by lateral migration to a “coated pit” where such units are concentrated and then stabilized by a framework of clathrins.  A pinched-off coated pit is internalized and degraded by lysosomes. Proteins may be metabolized or the receptor can be recycled. Use of long-acting agonists will downregulate the receptor population.

Gn-RH-a Desensitization  The LH-Rs become desensitized when exposed to LH for some time.  A key reaction of this downregulation is the phosphorylation of the intracellular receptor domain by protein kinases  This process uncouples Gs protein from the LH-R  Another way to desensitize is to uncouple the regulatory and catalytic units of the cAMP system.

Gn-RH antagonist  GnRH antagonists are also derivates of the natural GnRH decapeptide with multiple amino acid substitutions  A gonadotropin-releasing hormone antagonist (GnRH antagonist) is an analogue that blocks the GnRH receptor resulting in an immediate drop in gonadotropin (FSH, LH) secretion  The Gn-RH antagonist is primarily used in IVF treatments to block premature surge of LH

Gn-RH antagonist  These substitutions modify the agent so that it blocks the receptor and decreases FSH and LH secretions within hours  In contrast to GnRH agonists, antagonists have no flare effect, thus their therapeutic effect is immediately apparent  However, their action is short-lived and daily injections are necessary to maintain their effect

Gn-RH antagonist FSH and LH activity returns about 40 hours after cessation  Typically endogenous FSH and LH activity returns about 40 hours after cessation of GnRH antagonist administration, although with a higher dose the return to normal pituitary function will be postponed for longer. long acting not currently available  Unlike the GnRH agonists, long acting or depot forms of the agent are not currently available, thus GnRH antagonists are not used in the long term therapy

receptors activation  a receptor molecule exists in a conformational equilibrium between active and inactive states.  The binding of LH (or FSH) to the receptor shifts the equilibrium between active and inactive receptors.  Gn-RH-a shift the equilibrium in favor of active states;  Gn-RH antagonists shift the equilibrium in favor of inactive states.

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