GnRH

Question 1

What is the main function of GnRH?

Where is it coded for

Answer 1

This is the main control of reproduction

The gene which codes for it is found on chromosome 8 and there are 23 isoforms in vertebrates

(The difference being that it is one amino acid substitutions between the different isoforms in vertebrates)

Most vertebrates contain GnRH1 and GnRH 2

Question 2

What are some of the roles of GnRH?

Answer 2

Neuroendocrine -HPG axis
Paracrine -Placenta/gonads)
Autocrine(Prostate/breast cancer )
Neurotransmitter (regions of the brain-can have effects on other regions of the brain

Question 3

Outline the structure of GnRH

Answer 3

It is synthesised as a pre-prohormone and undergoes cleavage which forms Mature GnRH and GAP.

GAP peptide -co-secreted with GnRH

Question 4

What is the migratory path of GnRH neurones?

Answer 4

It does not originate in the hypothalamus , it actually originates in the nasal region of the olfactory node outside the CNS in the medial olfactory placode.
Undergo migration to the hypothalamus and it will respond to different genes which make sure this is successful

Question 5

How does an error in the migration process cause hypogonadotrophic hypogonadism

Answer 5

This is because we will not have GnRH neurons where it is meant to be meaning there is no secretion GnRH and regulating the hypothalamus.

The genes in which mutations can take place are :

KAL1,FGFR1

Question 6

What can a mutation in the KAL1 gene cause

Answer 6

Kallman syndrome
This can result in premature termination of migration.

Anosmia -the inability to smell as the GnRH neurons are in the olfactory region
and hypogonadotropic hypogonadism

Question 7

Describe the release of GnRH and the nature of this.

Answer 7

GnRH is processed and packaged into storage granules which are transported down the axosn to the external zone of the median eminence.
GnRYH is released in synchronised pulses from the GnRH nerve endings into the hypophyseal portal system.

Question 8

The release of GnRH?

Answer 8

It is released in rhythmic pulses every 30-120 minutes and these are called circhoral pulses.

This is regulated by the GnRH pulse generator
(A collection of hypothalamic neurons producing endogenous secretory rhythms )

It has a half life of around 2-4 minutes so it is degraded very quickly

Question 9

What does GnRH stimulate when released ?

Answer 9

It will stimulate the synthesis and secretion of gonadotrophins

Differential frequency and amplitude alter pattern of FSH and LH secretion therefore this will impact gonadal response

Question 10

Describe the structure of the GnRH receptor

Answer 10

This is a g-protein coupled receptor (GPCR)

Contains two variants type 1/2 GnRHR

Type 1 =full length
Type 2 =Missense truncation (humans)
(genetic variant which results in a shorter version of the protein being produced)

Question 11

Describe the characteristic structure of the GnRHR which makes it different to GPCRS

Answer 11

It has a characteristic 7-transmembrane domain structure, but unlike other members of GPCR does not have a carboxyl-terminal tail.
This means that it is unlikely to be desensitised by c terminal tail being phosphorylated and the receptor becoming internalised .

Question 12

What cell types has the GnRHR been found to be expressed in?

Answer 12

GnRH receptor mRNA was found to be expressed in human pituitary, breast, breast tumor, ovary, ovarian tumor, prostate, prostate tumor and in breast tumor cell lines (MCF-7 and MDA-MB 468) and prostate tumor cell lines (PC-3 and LNCaP). These findings demonstrate that mRNA representing the pituitary form of the GnRH receptor (which shows high affinity binding with GnRH) is also expressed in certain normal tissues and in hormone related human tumors and tumor cell lines derived from them.

Question 13

How does GnRH regulate gonadotrophin production?

Answer 13

Glycoproteins with α & β chains…
α-chains identical in FSH & LH
β-chains unique & confer biological actions

Rhythm & pulsatility of GnRH…
Relative rates of gene expression for α/β

Slow frequency or low amp GnRH pulse ⇒ ⇧FSHb gene expression
Fast frequency GnRH pulse ⇒ ⇧LHb transcription
α subunit is tonically produced.
Determines dimerization of subunits.
Determines post-translational modifications – rate of glycosylation.

Question 14

Outline the GnRH pulsatility in males

Answer 14

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 15

Outline the GnRH/Gonadotrophin pulsatility in females

Answer 15

Higher frequency GnRH pulse (every 30min) = favours LH synthesis and secretion

Lower frequency/amplitude GnRH pulse (every 90-120min) = favours FSH synthesis and secretion

Question 16

Where is GnRH secreted from once it migrates ?

Answer 16

Once GnRH neurones settle in Hypothalamus Parvocellular system (comprised of medial preoptic nucleus and arc nucleus)
GnRH is produced and the terminal axons release it into median eminence
Via Hypophyseal circulation it is released into the gonadotrophic cells of anterior pituitary

Question 17

At a low GnRH pulse what mechanism occours to form LH ?

Answer 17

1. GnRH will bind to its receptor GnRHR which wills stimulate G alpha s and G alpha q/11 signalling is activated.
2. Gs will stimulate protein kinase A and Gq/11 will stimulate protein kinase c.
3. These will diverge and activate the ERK1/2 kinase pathway which will lead to an upregulation of Egr1 (early growth restriction factor 1)-fission factor.

EgR1 will bind to the promoter on the LH gene and upregulates transcription/translation of LH beta .

4.This is also accompanied with the the expression of LH (beta)

This pathway is mainly for GnRH1 as type 2 is non-functional in humans

Question 18

What is the difference in the mechanism where there is a high pulse of GnRH?

Answer 18

The process will remain the same however there will be an upregulation of ICER instead of the Egr1 and as a result this will bind to the FSH gene and downregulate the transcription and translation.
This is because this pathway requires CREB is required and this is inhibited by ICER.

Question 19

At a high GnRH pulse what mechanism will take place and what gonadotrophin will this form instead?

Answer 19

1. The process for LH will remain the same however as there isn’t a sufficient pulse , this will not result in sufficient Egr1 being formed meaning there isn’t enough to result in the upregulation of LH.

2.FSH
When GnRH binds to the receptor both Gs and Gq/11 will be activated.
There is also a Creb cycle taking place as well which means that it will eventually bind to the FSH gene and as there is no ICER available to inhibit it FSH production will be upregulated

Question 20

What takes place in the Creb pathway ?

Answer 20

When GnRH binds to its receptor this will activate both Gs and Gq/11.
Gs will activate AC in the membrane and this will cause ATP to be converted into cAMP.
This will upregulate PKA which phosphorylates CREB.
The CREB will go on and bind to the promoter of FSH and upregulate the transcription of FSH beta.

Question 21

What happens in GnRH levels in the menstrual cycle

Answer 21

There is an intercycle rise in FSH and that occurs because there is an increase in GnRH pulse frequency as a result in a decline in progestogen
As the corpus luteum dies there’s a decrease in P production which negatively feedback to GnRH neurones
So there is an increase in GnRH pulses which allows FSH secretion to increase and cause intercycle rise

Early Follicular Phase
Steady state FSH occurring as FSH increases there is a rise in inhibin B
This feeds back to pituitary to inhibit FSH secretion
Likewise, increase in Oestrogen feeds back to reduce FSH

Mid-late Follicular Phase
Decrease in FSH which allows for dominant follicle selection to occur
Increase in LH responsiveness (as granulosa cells make LH receptors)
Oestrogen starts to be produced (once produced for 48 hrs then feedback switches from negative to positive)
mid-cycle rise in LH which corresponds with increase in oestrogen

After Ovulation
The pulse frequency slows in response to feedback of the increase in progestogen (negative feedback to H and P)
Slower GnRH pulses in response

Question 22

How is feedback mediated by steroids in males?

Answer 22

Testosterone is secreted by Leydig cells, stimulated by LH
Mainly inhibin that decreases FSH production, in males one of the clinical markers for spermatogenesis is the inhibin levels (lower the inhibin, better the spermatogenesis)
Activin causes increase in FSH secretion from pituitary.

Question 23

How is feedback mediated by steroids in females?

Answer 23

Throughout MC it is primarily negative feedback 🡨 Progesterone & low plasma oestrogen will maintain that
This causes a net reduction of LH & FSH
However, the sustained high level of oestrogen for 48hr causes an enhancement in LH production 🡪 switch to positive feedback

Progesterone = decreases GnRH pulse frequency
Oestrogen = decreases GnRH pulse amplitude

Threshold is 300 pico mols for two days

Question 24

Where would the site of steroid feedback be?

Answer 24

Either anterior pituitary of hypothalamus
Oestrogen and progesterone receptors on anterior pituitary and hypothalamus
Inhibin receptors are only found in the pituitary

Question 25

How does the feedback occour in anterior pituitary /hypothalamus ?

Answer 25

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

Question 26

Evidence for site of steroid feedback

Answer 26

Study in rhesus macaques:
made hypothalamus non-functional and removed ovaries
this removed GnRH (so LH and FSH decreased)
then they added back pulsatile GnRH
this then lead to pulsatile release of LH and FSH
They then mimicked what would happen at the ovary and injected a bolus of oestrogen
(pituitary still intact so only site of feedback)
initially they saw a decrease in LH and FSH (this showed the oestrogen was able to exert negative feedback on the pituitary)
they also saw if they gave sustained high oestrogen they saw a large increase in LH and FSH production – mimicking follicle selection (showed the anterior pituitary was also a site for positive feedback)

Question 27

How does oestrogen exert positive and negative feedback at the pituitary?

Answer 27

It is all done via the GnRH receptor
POSITIVE FEEDBACK
Is mediated by increasing GnRHR mRNA in the pre-ovulatory phase
So more receptors responsive to the changing of GnRH pulse frequency
Oestrogen sensitises “self-priming” setting up that positive feedback loop by enhancing the interaction between GnRH and GnRHR

NEGATIVE FEEDBACK
We don’t actually know
It may be via direct (oestrogen) or indirect mechanisms (inhibin)
They may inhibit the stability of GnRHR
Progesterone may downregulate GnRHR mRNA as there is evidence of a progesterone response element in the GnRHR gene

But overall, we don’t really know how this feedback works at the pituitary

Question 28

How does oestrogen feedback to the hypothalamus ?

Answer 28

GnRH neurones only express ERβ
Need ERα for positive feedback
Erα is crucial for reproductive function
Erα knock-out mice
E2 must act on other afferents that project onto GnRH neurones

E2 indirectly stimulates GnRH neurones via other neuronal inputs:
Kisspeptin
Other hypothalamic neurotransmitters- e.g., GABAnergic neurons

Interesting that GnRH neurones only express ER-beta, but to have E2 positive feedback need ER-alpha. Hence realised that E2 must act on other afferents that project onto GnRH neurones. Kisspepetin protein binds & activates GPR54 receptors which is co-localised with GnRH neurones in mammalian brains, and virtually all Kisspeptin neurones in mice express ER-alpha mRNA. Hence E2 acts on kisspeptin neurones, which project onto GnRH neurones to increase or decrease Kisspeptin and hence GnRH. Similarly for noradrenaline which directly stimulates GnRH or indirectly does it by inhibiting GABA.

Question 29

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

Answer 29

Inject large bolus of E2 to induce LH surge and sample directly from portal blood – see increased frequency of GnRH pulses.
For hypothalamus would have to measure levels of GnRH in portal blood after giving exogenous E2, but impossible in humans and very difficult in animals. Also, whilst easy to show increase in GnRH, it would be difficult to show negative feedback because the levels of GnRH are already low.