GnRH

Question 1

What is GnRH known as?

Answer 1

Master controller of reproduction

Question 2

How many isoforms of GnRH exist?

Answer 2

23 isoforms in vertebrates – it’s highly conserved

Question 3

What GnRH are found in vertebrates?

Answer 3

Most vertebrates have GnRH I (Classic GnRH) and GnRH II

Question 4

What are the roles of GnRH?

Answer 4

o Neuroendocrine - HPG
o Paracrine (placenta/gonads)
o Autocrine (prostate/breast cancer – GnRH receptors in the cancer tissue)
o Neurotransmitter (Regions of the brain)

Question 5

What is the structure of GnRH?

Answer 5

• Synthesised as a pre-pro hormone
• Cleavage steps-= Mature GnRH and GAP
• GAP peptide- co-secreted with GnRH, unknown function

Question 6

What is the migratory path of GnRH neurones?

Answer 6

During the embryonic period: originate outside CNS, in medial olfactory placode

Then cells migrate:
Nasal region -> brain -> medio-basal hypothalamus (forebrain)

o Numerous genes involved

Question 7

What is the placode?

Answer 7

area of thickening of the embryonic epithelial later from which the organ/structure later develops.

Question 8

What happens when the migrations goes wrong?

Answer 8

leads to hypogonadotropic hypogonadism – Less/no GnRH released due to lack of GnRH neurones

Question 9

What is Kallmann syndrome?

Answer 9

• Mutation in KAL-1 gene

• Premature termination of migration
o Patients present with anosmia & hypogonadotropic hypogonadism

Question 10

Where are GnRH neurones found?

Answer 10

GnRH neurones are expressed in the parvocellular system – includes medial preoptic nucleus and arcuate nucleus

Question 11

How is GnRH released?

Answer 11

• GnRH is processed and packaged into storage granules that are transported down the axons to the external zone of the median eminence. GnRH released in synchronized pulses from the GnRH nerve endings into hypophyseal portal system.
• Released in Rhythmic pulses - every 30-120 minutes – “circhoral pulses”

Question 12

What is the GnRH pulse generator?

Answer 12

collection of hypothalamic neurons producing endogenous secretory rhythms

Question 13

What is GnRH’s half life?

Answer 13

2-4 minutes

Question 14

What does GnRH do?

Answer 14

• GnRH stimulates synthesis and secretion of gonadotrophins.

* Differential frequency and amplitude alter pattern of FSH and LH secretion, therefore impact gonadal response

Question 15

What is the structure of the GnRH receptor?

Answer 15

• G-protein-coupled receptor (GPCR)
• 7-transmembrane domain structure, but unlike other members of GPCR does not have a carboxyl-terminal tail – for evolutionary purpose in humans, plays a role in making it resistant to desensitisation

o In desensitisation: c terminal tail is phosphorylated, receptor is internalised

Question 16

How many variants of the GnRHR exist?

Answer 16

• Two variants Type I and II GnRHR:

o Type 1- full length, classical receptors, in humans
o Type 2 - missense truncation, mRNA is expressed but the protein and receptor is not translated and expressed

Question 17

Where is the GnRHR expressed?

Answer 17

• Expressed on gonadotroph cells of anterior pituitary

* GnRH receptors are also found in other tissues like the breast, placenta and gonads

Question 18

How does GnRH regulate Gonadotrophin (FSH/LH) production?

Answer 18

• Glycoproteins (LH/FSH) with α & β chains - β-chains unique & confer biological actions
• Beta chains have a particular response to rhythm & pulsatility of GnRH
• Slow frequency or low amp GnRH pulse ⇒ ⇧FSHb gene expression
• Fast frequency GnRH pulse ⇒ ⇧LHb transcription

o It also determines dimerisation of subunits
o And it also determines glycosylation

Question 19

How are the GnRH pulses in males?

Answer 19

• GnRH pulses – constant frequency every 2hr
• 15% of normal men with mean testosterone levels in normal range had considerable variability in LH pulses & low serum T recordings

Question 20

How are GnRH pulses in females?

Answer 20

• Higher frequency GnRH pulse (every 30min) = favours LH secretion
• Lower frequency/amplitude GnRH pulse (every 90-120min) = favours FSH synthesis and secretion
• cyclical variations occur in females

Question 21

How does GnRH differentially regulate FSH and LH production?

Answer 21

• One of the pathways activated by GnRH activity is the ERK/MAP Kinase pathway via Gq and Gs activation.
• ERK=Extracellular-signal-regulated pathway. MAP kinase is another name for ERK.
• MAP=Mitogen activated protein
• Pituitary-specific deletion of ERK MAP Kinase

Question 22

What happens with the ERK/MAP kinase pathway is knocked out?

Answer 22

• the follicles do not make it past the antral follicle stage – no ovulation
• drastic reduction in LH expression in females, complete depletion of LHβ expression
• Did not have an effect on FSH production.
• ERK/MAP kinase pathway doesn’t play a major role in gonadotroph expression for males.

Question 23

What is the mechanism of the ERK/MAP kinase pathway?

Answer 23

• GnRHR activated – Gs and Gq signalling activated.
• That activated protein kinase A and protein kinase C pathways which diverge to activate the ERK/MAP kinase pathway – it upregulates Egr1 (early growth restriction factor 1)
• Egr1 upregulation accompanied by LH-beta transcription upregulation
• When this pathway is blocked there’s a drop in LH-beta transcription and translation

Question 24

How does high GnRH frequency cause high production of LH?

Answer 24

• GnRH binds to GnRHR
• Gq pathway activated, it activates ERK/MAP kinase pathway which causes upregulation of egr1
• it binds to promoter on the LH-beta gene and there’s an upregulation in the transcription and translation of LH-beta

Question 25

How does low GnRH frequency cause low levels FSH to be produced?

Answer 25

- Gq signalling activated and along with egr1 ICER is also upregulated. - ICER binds to the promotor, prevents upregulation of FSH FSH transcription needs CREB binding to the promoter. - ICER inhibits the activity of CREB - Leads to low FSH expression

Question 26

How does low GnRH pulse cause low levels of LH?

Answer 26

- after GnRHR is activated Gq signalling is activated - ERK/MAP kinase pathway activated but because there isn’t enough GnRH pulses there will also be low expression of Egr1. - Not enough to upregulate LH transcription so low levels of LH

Question 27

How does low GnRH pulse cause high levels of FSH?

Answer 27

- Both Gq and Gs activated - ERK/MAP kinase pathway activated along with protein kinase A/CREB pathway being activated. - Increased CREB expression and CREB binding to FSH gene promotor. - Lack of ICER means CREB can upregulate FSH transcription and translation.

Question 28

What happens at the end of the luteal phase?

Answer 28

the CL has regressed, no oestrogen/progesterone negative feedback leads to increase in GnRH pulses.

Question 29

GnRH pulses in the early follicular phase?

Answer 29

Slow - FSH is produced

Question 30

GnRH pulses in the mid-late follicular phase?

Answer 30

Pulse frequency increases - LH made

Question 31

GnRH pulses after ovulation?

Answer 31

Slow - FSH is made

Question 32

GnRH pulses at the end of the luteal phase?

Answer 32

There's an increase in GnRH but FSH is still made

Question 33

What happens for the negative feedback to stop?

Answer 33

In the follicular phase: - FSH secretion - Increase in estrogen levels - negative feedback resulting in FSH decline - dominant follicle selection - switch from FSH pulses to LH pulses - E2 levels continue to rise-48hr period where there is sustained E2 expression at high levels reaching the threshold of 300pg/mL - switch to positive feedback, rapid pulses. - LH surge causes ovulation

Question 34

Feedback by gonadal steroids in males?

Answer 34

LH: Testosterone from Leydig cells reduces LH secretion FSH: • Inhibin ↓FSH secretion from anterior pituitary by negative feedback • Activin ↑FSH secretion from anterior pituitary

Question 35

Feedback by gonadal steroids in females?

Answer 35

* Progesterone & low plasma oestrogen -> negative feedback on anterior pituitary and thalamus * Net effect = reduced LH & FSH * High sustained (48h) plasma [E2] = enhanced LH & FSH >> positive feedback * P4 ↓GnRH pulse freq via negative feedback * E2 ↓GnRH pulse amplitude via negative feedback

Question 36

Where would the site of steroid feedback be?

Answer 36

* Either anterior pituitary or hypothalamus * Because oestrogen & progesterone receptors are on anterior pituitary and hypothalamus * Inhibin receptors found only in pituitary

Question 37

How steroid feedback on GnRH occur?

Answer 37

* In anterior pituitary by direct regulation of GnRHR * In hypothalamus by directly affecting GnRH neurones or indirectly by changing activity of other neural system that influence GnRH release

Question 38

What would happens if the hypothalamus and ovaries were removed?

Answer 38

- no GnRH production at all - Pituitary made low levels of FSH and LH - When GnRH was introduced exogenously – there was pulsatile secretion of LH and FSH - When oestrogen was inserted exogenously there was a drop in LH and FSH – negative feedback - Increased the oestrogen being inserted – LH and FSH surge - Hence E2 exerting both negative and positive feedback on gonadotrophin secretion and this can only be via the anterior pituitary.

Question 39

Mechanisms of positive feedback in the pituitary?

Answer 39

* E2 induces & maintains GnRHR by increasing GnRHR mRNA in pre-ovulatory phase * E2 sensitises “self-priming” effect i.e. enhances interaction between GnRH & GnRHR

Question 40

Mechanisms of negative feedback in the pituitary?

Answer 40

* E2 may affect the stability of GnRHR, don’t know that much * Inhibin may affect the stability of GnRHR, don’t know that much * Progesterone ↓ GnRHR mRNA, also evidence of P4 response element in GnRHR gene

Question 41

What evidence is there that the hypothalamus is a site of steroid feedback?

Answer 41

- Studies carried out in sheep - Their ovaries were removed, and large doses of oestrogen injected - Directly were able to measure GnRH levels - Introduction of large amounts of oestrogen seen with increased frequency of GnRH pulses

Question 42

Mechanisms of feedback in the hypothalamus?

Answer 42

* GnRH neurones only express Erβ (oestrogen beta receptor) * Need ERα for positive feedback * Erα is crucial for reproductive function * E2 must act on other afferents that project onto GnRH neurones • E2 indirectly stimulates GnRH neurones via other neuronal inputs: o Kisspeptin o Other hypothalamic neurotransmitters- e.g., GABAnergic neurons