GnRH

Question 1

What is the function of GnRH

Answer 1

Master controller of reproduction- characterised in 1971

Question 2

Where is the gene for GnRH located?

Answer 2

Chromosome 8

Question 3

How many isoforms of GnRH are there?

Answer 3

23

Question 4

GnRH is highly conserved in vertebrate species. What does this mean?

Answer 4

There is usually one amino acid substitution that differentiates the different forms of GnRH across vertebrates

Question 5

What are the 2 variants of GnRH expressed in vertebrates?

Answer 5

GnRH 1 and GnRH 2

Question 6

List some roles of GnRH

Answer 6

Neuroendocrine - HPG
- Paracrine (placenta/gonads)
- Autocrine (prostate/breast cancer)
(Has paracrine and autocrine role as when you look at placenta/gonad/cancer cells, there are GnRH receptors present on those tissues)
- Neurotransmitter (Regions of the brain)
(There are GnRH receptors present in other regions of the brain eg gonadotroph cells of the anterior pituitary and in the hippocampus region of the brain.)

Question 7

Describe the structure of GnRH

Answer 7

• It is initially synthesised as a Pre-pro hormone
• Will undergo Proteolytic cleavage steps-= Mature GnRH and GAP (GnRH associated protein)

GnRH is a decapeptide
GAP peptide is co-secreted with GnRH but its function is unknown

Question 8

Describe the migratory path of GnRH neurones

Answer 8

• GnRH neurons originate in the olfactory region (nasal region) during embryonic development
• then undergo migration to the hypothalamus
• Whilst the migration is occuring through the olfactory bulb, they respond to a series of genetic queues and the expression of certain genes that regulate the migration process, ensuring that it is successful.

Question 9

Embryonic period?

Answer 9

Originate outside CNS, in medial olfactory placode

Question 10

Cells migrate

Answer 10

Nasal region–> Brain–> Medio-basal hypothalamus
There are numerous genes involved

Question 11

Problems in the migratory process can lead to:

Answer 11

Hypogonadotropic Hypogonadism as the GnRH neurons will not be where they should be in the hypothalamus

Question 12

Name some known mutations causing Hypogonadrotropic Hypogonadism

Answer 12

KAL1, FGFR1, FGF8, PROK2, PROKR2, NELF, CHD7,
GNRH1, GNRHR, GPR54, TAC3, TAC3R, NKB/NK3R, WDR11.

Question 13

What is Kallman Syndrome caused by?

Answer 13

Mutation in the KAL-1 gene
Premature termination of migration: anosmia & hypogonadotrophic hypogonadism

Question 14

What is anosmia?

Answer 14

inability to smell as GnRH neurons are still in the olfactory region

Question 15

GnRH release

Answer 15

• 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.
• GnRH t1/2 2-4 minutes
• GnRH stimulates synthesis and secretion of gonadotrophins.
• Differential frequency and amplitude alter pattern of FSH and LH secretion, therefore
  impact gonadal response.

Answer 16

• Rhythmic pulses- every 30-120 minutes – “circhoral pulses”

Question 17

What does the GnRH pulse generator do

Answer 17

GnRH Pulse generator: collection of hypothalamic neurons producing endogenous secretory rhythms

Question 18

Describe GnRH receptor structure

Answer 18

G-protein-coupled receptor (GPCR) expressed on gonadotroph cells of anterior pituitary
* Two variants Type I and II GnRHR
* Type 1- full length, Type 2 - missense truncation (humans)

NO C-terminal tail* (evolutionary benefit- resistant to receptor desensitisation)

Question 19

How doe GnRH regulate Gonadotrophin production?

Answer 19

Glycoproteins have alpha and beta chains:
- alpha chains identical in FSH and LH
- beta chains are unique and confer biological actions

Rhythm and 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
- Determines dimerisation of subunits
- Determines glycosylation

Question 20

In males, what are the GnRH pulses like?

Answer 20

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 21

In females, what are the GnRH pulses like?

Answer 21

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

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

Question 22

How does GnRH differentially regulate FSH and LH
production?

Answer 22

Research: One of the pathways activated by GnRH activity is the MAP Kinase pathway via Gq and Gs activation. In this study, pituitary-specific (gonadotroph-specific) deletions/knock-outs of ERK/MAP kinase were generated and their reproductive function was characterised.
ERK=Extracellular-signal-regulated pathway. MAP kinase is another name for ERK.
MAP=Mitogen activated protein
They were able to show that the ERK/MAP kinase pathway was crucial for LHb secretion in females.

Question 23

How was the research able to show that the EK/MAP kinase pathway was crucial for LHb secretion in females?

Answer 23

To do this they created ERK knockout mice, they monitored its reproductive function.
They injected PBS into the cervix of the mice and took the epithelial cells of the cervix and monitored it throughout the menstrual cycle.
They observed that in the control mice there was a consistent change in the epithelial cell number with each menstrual cycle. BUT in the KO mice for the ERK/MAP kinase pathway, there was a disruption in the oestrous cycle.
CLs were observed which is a sign that ovulation has occurred. In the knock-out, the follicles were not making it past the antral follicle stage = no ovulation.

Question 24

What was the research able to show at a molecular level?

Answer 24

When looking at the molecular level they were able to show that there was a reduction in LHb expression in the KO mice. BUT FSH was not affected.

Question 25

What showed that ERK-MAPK activation is essential for LHβ transcription and translation?

Answer 25

Looking at protein expression, in the kOs there was a complete depletion of LHβ expression, while this was not the case for FSHβ. This shows that the MAP kinase pathway is essential for LH expression in females...not so much for FSH. ERK/MAP kinase pathway doesn’t appear to play a major role in gonadotroph expression for males.

Question 26

Mechanism of the ERK/MAP kinase pathway

Answer 26

GnRH will bind to the GnRHR Gs and Gq signalling is activated This in turn will activate PKA (Gs) and PKC (Gq) – Protein kinase These will activate the ERK/MAP kinase pathway ERK1/2 Following the activation of the ERK/MAP kinase pathway there is an upregulation of Egr1 (early growth restriction factor 1) The upregulation of EGR1 will cause an upregulation of LHb transcription. Studies have shown that when the ERK/MAP kinase pathway is blocked, it is accompanied by a drop in LHb transcription and translation. This pathway is for GnRH 1 (type 2 is non-functional in humans)

Question 27

Describe the differential GnRH receptor signalling when there is a HIGH GnRH pulse (LH)

Answer 27

GnRH binds to the GnRHR The Gq pathway is activated (GPCR) This activates the ERK/MAP kinase pathway This causes an upregulation of EGR 1 EGR 1 binds to the promotor on the LHb gene There is an upregulation in the transcription and translation of LHb

Question 28

Describe the differential GnRH receptor signalling when there is a HIGH GnRH pulse (FSH)

Answer 28

GnRH binds to the GnRHR The Gq pathway is also activated (GPCR) HOWEVER ICER is upregulated ICER is an inhibitor, so when it binds to the promoter it prevents the upregulation of FSH. In order for FSH upregulation to occur you need CREB binding to the promoter, but once ICER binds it inhibits the activity of CREB. ICER inbibits CREB from binding to it’s response element on the FSHβ gene. SO… ICER downregulates FSH In the presence of a high pulse both FSH and LH are being transcribed, but LH is upregulated.

Question 29

Describe the differential GnRH receptor signalling when there is a LOW GnRH pulse (LH)

Answer 29

GnRH binds to the GnRHR The Gq pathway is activated (GPCR) This activates the ERK/MAP kinase pathway (Low pulse frequency = Not as much MAP kinase activated). BUT as there is not sufficient frequency of GnRH pulses, there will not sufficient expression of EGR 1 Due to the small amount of EGR 1 there is no upregulation of LH SO… Low EGR1 levels = low level of LH transcription

Question 30

Describe the differential GnRH receptor signalling when there is a LOW GnRH pulse (FSH)

Answer 30

GnRH binds to the GnRHR Both Gs and Gq are activated Gs = ERK/MAP kinase pathway Gq = PKA and CREB pathway Both pathways result in the expression of CREB and the binding of CREB to FSHbeta gene promoter. As there is no ICER there to inhibit CREB, CREB is successfully able to bind to the FSHb gene promoter and upregulate FSHb transcription.

Question 31

What happens at the end of the luteal phase?

Answer 31

At the end of the luteal phase you have an increase in GnRH pulses -> FSH release

Answer 32

BUT just before this (after ovulation) you will have slow pluses (1 every 3-5 hrs) -> FSH is being produced but NOT released. At the end of the luteal phase the pulses will increase and the FSH will be released. This is due to the regression of the corpus luteum, therefore you do not have estrogen and progesterone that normally creates a negative feedback. So now the negative feedback is lifted, there will be an increase in GnRH pulses and then and increase in the expression and release of FSH.

Question 33

What happens in the EF phase?

Answer 33

In the early follicular phase, the pulses are slow (ever 90-120min) -> FSH is released and is needed for follicular recruitment.

Question 34

What happens at the mid-follicular phase?

Answer 34

In the mid-late follicular phase, the GnRH pulse frequency increased (1 every hour). -> switch from FSH production to LH. So there is an increase in LH and a decrease in FSH. This is where there is selection of the dominant follicle. The dominant follicle must acquire more LH and FSH receptors.

Answer 35

During ovulation, once the threshold has been reached, there is a switch from negative to positive feedback resulting in a LH surge.

Answer 36

Then after ovulation the pulses switch to slow (every 3-5 hours). FSH is produced and packaged into secretory granules, but NOT released. This is due to the negative feedback caused by the CL and the high levels of progesterone and low levels of estrogen being produced, this causes negative feedback on the HPG axis.

Question 37

Feedback by Gonadal Steroids (in MALES)

Answer 37

LH Testosterone from Leydig cells reduces LH secretion (Rhesus monkey experiments) FSH Inhibin ↓ Downregulate FSH secretion from pituitary (via negative feedback) (there are inhibin receptors on the anterior pituitary) Activin ↑upregulates FSH secretion from pituitary

Question 38

Feedback by Gonadal Steroids (in FEMALES)

Answer 38

Progesterone & low plasma oestrogen modulate negative feedback on the anterior pituitary and the hypothalamus. Net effect = reduced LH & FSH production High sustained (48h) plasma [E2] (>300pmol?)= enhanced LH & FSH = positive feedback (LH surge) The negative feedback from progesterone (P4) modulates the ↓GnRH pulse frequency The negative feedback from estrogen (E2) affect the ↓GnRH pulse amplitude

Question 39

How do we know that GnRH is being affected by steroids during the menstrual cycle?

Answer 39

After ovariectomy or the menopause plasma conc of circulating FSH and LH increase markedly……this is because no E2 is present. So low E2 suppresses gonadotrophin release i.e. negative feedback. It’s the steroids that are doing this – Prog acts to decrease GnRH pulse frequency and E2 to decrease GnRH pulse amplitude.

Question 40

Where is the site of steroid feedback for GnRH?

Answer 40

- Either anterior pituitary or hypothalamus As there are Oestrogen & progesterone receptors on both the anterior pituitary and hypothalamus Inhibin receptors are found only in the anterior pituitary

Question 41

How does the steroid feedback occur?

Answer 41

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

Question 42

Evidence of the steroid feedback in the anterior pituitary

Answer 42

Lesions created in the hypothalamus (making it inactive). Also removed ovaries. = no GnRH As a result the anterior pituitary is expressing low levels of LH and FSH (as there is no GnRH being produced) BUT when pulses of GnRH were introduced exogenously, it resulted in pulsatile secretion of LH and FSH. This shows that the anterior pituitary is involved in feedback When estrogen was injected exogenously there was negative feedback = reduced LH and FSH. When they increased estrogen there was a FSH and LH surge

Question 43

Describe the positive feedback in the anterior pituitary

Answer 43

Positive feedback – More E2 = Less FSH/LH E2 induces & maintains GnRHR expression by increasing the GnRHR mRNA in the pre-ovulatory phase E2 sensitises “self-priming” effect i.e. enhances interaction between GnRH & GnRHR

Question 44

Describe the negative feedback in the anterior pituitary

Answer 44

Negative feedback - E2 and Inhibin may have a role in the stability of the GnRHR Progesterone has a modulatory effect on the expression↓ GnRHR mRNA, also evidence of P4 response element in GnRHR gene

Question 45

Evidence of steroid feedback in the hypothalamus

Answer 45

Study in sheep Removed the ovary and injected large bolus of E2. They monitored via hyoposeal portal circulation and were able to directly assay GnRH levels With the introduction of the large bolus of E2, there was increased GnRH pulses which led to a rise in LH levels. Shows that the hypothalamus also has a role in the negative feedback Inject large bolus of E2 to induce LH surge and sample directly from portal blood – see increased frequency of GnRH pulses