Gn-RH

and

Gn-RH receptors

Dr. Vincenzo Volpicelli

Fertility Center Cardito

Hypothalamic anatomyHypothalamic anatomy

Hypothalamus is a pars of diencephalon

Under thalamusIs floor of the third ventriculus

Hypothalamic anatomyHypothalamic 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 Neurotransductor effects on Gn-RH releaserelease

leptineleptine

Modulates NY

Gn-RH secretion

LeptineLeptine

leptine serum levels

0

2

4

6

8

10

12

14

16

1 3 6 9 12 15 18 21 24 27 30

cycle days

ng

/ml

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

hypothalamic nucleihypothalamic nuclei

Region Medial Area Lateral Area

Anterior

Medial preoptic nucleus

Supraoptic nucleus

Paraventricular nucleus

Anterior nucleus

Suprachiasmatic nucleus

Lateral preoptic nucleus

Lateral nucleusPart of supraoptic nucleus

TuberalDorsomedial nucleusVentromedial nucleus

Arcuate nucleusArcuate nucleus

Lateral nucleusLateral tuberal nuclei

Posterior Mammillary nuclei (part of

mammillary bodies)Posterior nucleus

Lateral nucleus

Hormones of the hypothalamusHormones 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-RHGn-RH

Gn-RH neurons are inside the medium-Gn-RH neurons are inside the medium-basal hypothalamus (arcuate nucleus and basal hypothalamus (arcuate nucleus and median eminence)median eminence)

Lately scientists showed Gn-RH syntesis Lately scientists showed Gn-RH syntesis in pituitary gland too.in pituitary gland too.

Human Luteinizing hormone-releasing Human Luteinizing hormone-releasing hormone gene (LH-RH) is located on short hormone gene (LH-RH) is located on short arm of chromosome 8 (region 8p11.2 → p21) 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-RHGn-RH

Gn-RH is considered a neurohormoneGn-RH is considered a neurohormone

produced in a specific neural cell and released at its neural terminalproduced in a specific neural cell and released at its neural terminal A key area for production of Gn-RHA key area for production of Gn-RH11 is the preoptic area of the is the preoptic area of the

hypothalamus, that contains most of the Gn-RHhypothalamus, that contains most of the Gn-RH11-secreting neurons. -secreting neurons.

Gn-RHGn-RH11 is secreted in the hypophysial portal bloodstream at the is secreted in the hypophysial portal bloodstream at the median eminence median eminence

The portal blood carries the Gn-RHThe portal blood carries the Gn-RH11 to the pituitary gland, which to the pituitary gland, which contains the gonadotrope cells, where Gn-RHcontains the gonadotrope cells, where Gn-RH11 activates its own activates its own receptor, gonadotropin-releasing hormone receptor (Gn-RH-R), receptor, gonadotropin-releasing hormone receptor (Gn-RH-R), located in the cell membranelocated in the cell membrane

Gn-RHGn-RH

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

Gn-RH frequencyGn-RH frequency

•Low frequencyLow frequency Gn-RH pulses lead to Gn-RH pulses lead to FSHFSH release release

•high frequencyhigh frequency Gn-RH pulses Gn-RH pulses stimulate stimulate LH LH releaserelease

Gn-RH-rGn-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 prostateas well as lymphocytes, breast, ovary, and prostate

Gn-RH-r FunctionGn-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-RHGn-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-RH1 is the preoptic areapreoptic area of the

hypothalamus, that contains most of the GNRH1-secreting neurons.

GN-RH1 is secreted in the hypophysial portal bloodstream at the median eminence

The portal blood carries the GN-RH1 to the pituitary gland, which contains the gonadotrope cells, where GN-RH1 activates its own receptors, gonadotropin-releasing hormone receptors (GN-RH-R), located in the cell membrane

Gn-RHGn-RH

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

Males/females Gn-RH secretion Males/females Gn-RH secretion

• in males, Gn-RHin males, Gn-RH11 is secreted in pulses at a is secreted in pulses at a constant frequencyconstant frequency • in females the frequency of the pulses varies in females the frequency of the pulses varies during the menstrual cycleduring the menstrual cycle

• there is a large surge of GNRHthere is a large surge of GNRH11 just before just before ovulationovulation

ProteolysisProteolysis

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 LHControl of FSH and LH

At the pituitary, Gn-RH1 stimulates the synthesis and secretion of FSH and LH

These processes are controlled by: the size and frequency of Gn-RH1 pulses, feedback from androgens and estrogens

Gn-RH-R FunctionGn-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.* as well as lymphocytes, breast, ovary, and prostate.

Gn-RH biochemistry Gn-RH biochemistry

a decapeptide (10 amino acids) in mammals. This chain is represented by: pyroGlu-His-Tyr-Ser-Gly-Leu-Arg-Pro-Gly-NH2

previously called LH-RH

The identity of GN-RH1 was clarified by the 1977 Nobel Laureates Roger Guillemin and Andrew V. Schally

Gn-RHGn-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-RH1 is the preoptic area of the hypothalamus, that contains most of the Gn-RH1-secreting neurons.

Gn-RH1 is secreted in the hypophysial portal bloodstream at the median eminence.

The portal blood carries the Gn-RH1 to the pituitary gland, which contains the gonadotrope cells, where Gn-RH1 activates its own receptor (Gn-RH-R), located in the cell membrane.

Gn-RH neural junctionsGn-RH neural junctions

Gn-RH neurons are closely connected with:

noradrenergic, dopaminergic, serotoninergic, oppioid ones.

Gn-ReceptorsGn-Receptors

Upon binding Hormone externally to the Upon binding Hormone externally to the membrane, a transduction of the signal membrane, a transduction of the signal takes place that activates the takes place that activates the G proteinG protein

that is bound to the receptor internallythat is bound to the receptor internally

Gn-ReceptorsGn-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 SystemG Protein System

Alfred G. Gilman Alfred G. Gilman

and and

Martin Rodbell Martin Rodbell

received the 1994 received the 1994 Nobel PrizeNobel Prize in in Medicine and Physiology for the Medicine and Physiology for the discovery of the G Protein Systemdiscovery of the G Protein System

Gn-RH-R FunctionGn-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 Gonadotropin-releasing hormone receptorhormone 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.* as well as lymphocytes, breast, ovary, and prostate.

The seven transmembrane α-helix structure of The seven transmembrane α-helix structure of

a G protein-coupled receptora G protein-coupled receptor

hormone 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 bindsA protein on the surface of a cell to which a specific hormone binds

hormone receptorshormone receptors

a subunit activates

intracellular effectors (enzymes) adenyl-ciclases cAMPguanil-cyclase cGMPphospholipase DAG*, IP3 * ionic ducts (K+ , Ca++)

*Diacilglicerolo, Inositolo trifosfato

(II° messenger)

POPO44 by cAMP-dependent PKA by cAMP-dependent PKA

II° messengerII° messenger

activates Protein kinasi (PKA)activates Protein kinasi (PKA)

activates Phosphorylation (POactivates Phosphorylation (PO44) of:) of:

•citoplasmatic protein citoplasmatic protein •nuclear transcription factors (cAMP nuclear transcription factors (cAMP Responsive Element Binding Protein, Responsive Element Binding Protein, CREB).CREB).

DNA transcription DNA transcription

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

hormone receptorshormone receptors

Control of FSH and LHControl of FSH and LH

At the pituitary, Gn-RH1 stimulates the synthesis and secretion of FSH and LH

These processes are controlled by: the size and frequency of GN-RH1 pulses feedback from androgens and estrogens Gn-receptors wholeness

Control of FSH and LHControl of FSH and LH

•Low frequencyLow frequency Gn-RH pulses lead to Gn-RH pulses lead to FSHFSH release release

•high frequencyhigh frequency Gn-RH pulses Gn-RH pulses stimulate stimulate LH LH releaserelease

Gn-RH secretion males/females

in males, GN-RHin males, GN-RH11 is secreted in pulses at a is secreted in pulses at a constant frequencyconstant frequency • in females the frequency of the pulses varies in females the frequency of the pulses varies during the menstrual cycleduring the menstrual cycle

• there is a large surge of GN-RHthere is a large surge of GN-RH11 just before just before ovulationovulation

Gn-RH proteolysisGn-RH proteolysis

Proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called

protease or by intramolecular digestion.

Gn-RH is degraded Gn-RH is degraded by proteolysis by proteolysis within a few within a few minutesminutes

Gn-RH-aGn-RH-a

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

Gn-RH-aGn-RH-aAminoacid sequenceAminoacid sequence

namename actact 11 22 33 44 55 66 77 88 99 1010 forfor

Gn-RHGn-RH 11

Pyro-Pyro-gluglu HisHis TrpTrp

sese

roro

toto

ninnin

TyrTyr

LeuLeu

LeuLeu ArgArg ProPro

Gly-Gly-NHNH22

iviv

LeuprorelineLeuproreline** 1515 D-LeuD-LeuN-N-

EtNHEtNH22sc, imsc, im

Buserelin Buserelin * ** * 2020 D-SerD-SerN-N-

EtNHEtNH22sc, imsc, im

triptor triptor * ** * ** D-TripD-Trip sc, imsc, im

GoserelinGoserelin* * ** * * ** 100100 D-SerD-SerAzGly-AzGly-

NHNH22depot scdepot sc

* * Enantone 3.75, 11.25 mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.; Enantone 3.75, 11.25 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) Suprefact 5.5 ml fl s.c.; Suprefact spray nasale 10 gr (1 buff = 200 mg)

* * ** * * Decapeptyl 3.75, 11.25 mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c. Decapeptyl 3.75, 11.25 mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c.

* * * * * * * * ZoladexZoladex 3.6, 10.8 mg fl s.c. im3.6, 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 DownregulationGn-RH-a Downregulation

after their initial stimulating action – termed a “flare” effect -, eventually caused a paradoxical and sustained drop in gonadotropin secretion.

This second effect was termed “downregulation” and can be observed after about 10 daysafter about 10 days.

While this phase is reversible it can be maintained with further GnRH agonist use for a long time.

Gn-RH-aGn-RH-a Downregulation 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-aGn-RH-a Desensitization 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 antagonistGn-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

Typically endogenous FSH and LH activity returns FSH and LH activity returns about 40 hours after cessationabout 40 hours after cessation of GnRH antagonist administration, although with a higher dose the return to normal pituitary function will be postponed for longer.

Unlike the GnRH agonists, long actinglong acting or depot forms of the agent are not currently availablenot currently available, thus GnRH antagonists are not used in the long term therapy

receptors activationreceptors activation

a receptor molecule exists in a a receptor molecule exists in a conformational equilibrium between active conformational equilibrium between active and inactive states. and inactive states.

The binding of LH (or FSH) to the receptor The binding of LH (or FSH) to the receptor shifts the equilibrium between active and shifts the equilibrium between active and inactive receptors. inactive receptors.

Gn-RH-a shift the equilibrium in favor of Gn-RH-a shift the equilibrium in favor of active states; active states;

Gn-RH antagonists shift the equilibrium in Gn-RH antagonists shift the equilibrium in favor of inactive states.favor of inactive states.

THE END