Neuroendocrine

Question 1

Describe the basic pathway from GnRH to Gonadotropes:

Answer 1

1. GnRH is synthesised in the anterior region of the hypothalamus (in the preoptic area)
2. GnRH neurons send long dendrons into the median eminence (external zone) where the GnRH is stored in vesicles until it is required
3. GnRH once released from the GnRH neuron dendron terminals enters the portal blood system which transports it to the anterior pituitary
4. At the anterior pituitary GnRH acts on gonadotropes and stimulates the release of LH and FSH

Question 2

Describe the relationship between GnRH pulses and LH pulses:

Answer 2

• Every LH pulse is preceeded by a GnRH pulse
• The pulsatile secretion of LH is dependent and tightly coupled to GnRH pulses
• GnRH must both be secreted in a pulsatile manner to be effective (continuous GnRH secretion does not stimulate LH or FSH secretion)- due to the rapid desensitization of gonadotroph GnRH receptors

Question 3

What are the novel characteristics of GnRH neurons?

Answer 3

1. They are located in the preoptic area of the hypothalamus but they are very sparsely distributed
2. GnRH neurons act in concert to produce pulses
3. GnRH neurons communicate with eachother via a dendritic network (the dendrites wrap around other GnRH neurons)
4. GnRH neurons have dendrons instead of axons that are covered in dendrites to allow for communicate between each other and trigger terminal on terminal interaction

Question 4

Describe the Effect of Progesterone, Estrogen and Testosterone NEGATIVE FEEDBACK on GnRH pulse frequency and amplitude

Answer 4

1. Progesterone: reduced GnRH pulse frequency in luteal phase (in combo with low estrogen)
2. Estrogen: reduces GnRH pulse amplitude in early follicular phase (weaker negative feedback than progesterone)
3. Testosterone: reduced GnRH pulse frequency

Question 5

What is positive feedback in women?

Answer 5

• Occurs during the late follicular phase
• Positive feedback in response to estrogen at the level of the hypothalamus and the anterior pituitary (but only if progesterone levels are low)

Question 6

Describe the Negative and Positive Feedback of Progesterone and Estrogen across the Ovarian Cycle:

Answer 6

• Luteal phase: high levels of progesterone and estrogen; reduced GnRH pulse frequency (due to progesterone negative feedback) and reduced GnRH pulse amplitude (due to estrogen negative feedback)
• Early follicular phase: progesterone and estrogen levels decrease and progesterone levels become extremely low; there is an increase in GnRH pulse frequency (as there is less/no negative feedback by progesterone); GnRH pulsatility is mainly only controlled by the much weaker negative feedback of estrogen
• Late follicular phase:
  progesterone is very low and estrogen levels increase, once estrogen levels reach a threshold (16-24 hours after estrogen treatment)- estrogen switches from negative to positive feedback and stimulates GnRH pulse increases significantly (frequent, high amplitude pulses), whilst progesterone is too low to exert negative feedback
• This causes the LH surge

Question 7

Describe the converging circuit system of GnRH neurons:

Answer 7

• GnRH neurons do not typically express relevant receptors e.g. sex steroid receptors other than ERb (which is not related to reproduction)
• Therefore information regarding steroid hormone levels/nutrition/stress are processed in other brain regions (in upstream neurons) and then this information is relayed to the GnRH neuron
• This system prevents the overload of single neurons and allows them to act in concert

Question 8

What are the 2 cell types that relay steroid feedback to GnRH neurons?

Answer 8

1. Kisspeptin neurons

2. Gonadotropin inhibitory neurons

Question 9

What is kisspeptin?

Answer 9

• Kisspeptin is produced in cells in the arcuate nucleus (KNDy cells) and in the preoptic area (KISS cells)
• Any mutation to the kisspeptin gene, protein or receptor results in infertility due to a failure to undergo puberty

Question 10

What cells are considered the GnRH pulse generators?

How do they generate these pulses?

Answer 10

• The KNDy cells in the arcuate nucleus
• They trigger GnRH pulses via direct terminal-terminal interaction with the GnRH neuron terminals in the median eminence

Question 11

What are the features of the arcuate nucleus kisspeptin (KNDy) neurons?

Answer 11

1. All kisspeptin cells in the ARC express ERa (only 50% of KISS cells in the POA express ERa)
2. Commonly referred to as KNDy cells as they also express neurokinin B and dynorphin (neurokinin B acts in a paracrine manner to increase kisspeptin secretion and dynorphin inhibits LH secretion)
3. ARC KNDy cells mediate both positive and negative feedback effects of estrogen (POA cells are only involved in positive feedback)
4. Activated at the time of a GnRH pulse
5. Increase in activity during the follicular phase
6. They do not project directly to GnRH cell bodies- they initiate positive feedback and act on the GnRH neurons via an intermediary neuron and by direct terminal-terminal interaction
7. Regulate GnRH secretion at the site of teh terminal

Question 12

Describe the 2 Step Process of Kisspeptin cell activation in Estrogen positive Feedback:

Answer 12

1. KNDy cells are activated by estrogen in the late follicular phase and initiate positive feedback
2. KISS cells in the POA are activated just preceding the GnRH/LH surge and facilitate this positive feedback as they project directly onto GnRH neurons

Question 13

What is GnIH?

Answer 13

• Gonadotropin inhibitory hormone
• A type of RFRP peptide
• A neuropeptide that negatively inhibits reproduction
• GnIH neurons are located in the dorso-medial hypothalamus

Question 14

What is the function of GnIH neurons?

Answer 14

• GnIH neurons act directly on GnRH neurons and inhibit GnRH secretion (disinhibition must occur for the GnRH surge to occur)
• The activity of GnIH neurons on GnRH neurons in the POA increases in the luteal phase
• During the late follicular phase there is a decrease in GnIH activity and thus a disinhibition of the GnRH neurons which is permissive of the LH surge
• GnIH neurons also secrete GnIH into the median eminence portal blood system which acts to blunt GnRH-induced signalling in anterior pituitary but this does not change throughout the ovarian cycle (involved in seasonal breeding)

Question 15

Describe the difference between LH and FSH secretion:

Answer 15

1. LH secretion is:
   - Active
   - Regulated
2. FSH secretion is:
   - Passive
   - Facilitated

Question 16

Describe the action of GnRH on Gonadotropes:

Answer 16

1. GnRH binds to the GnRH receptor
2. GnRH receptor activates second messenger systems that act on genes to: 2.1. increase gonadotropin production
3. 2. modify calcium stores (Ca2+ is needed for secretion of LH and FSH)
4. 3. the modification of regulatory proteins such as Ras that fuse LH vesicles to the membrane so they are ready to be exocytosed

Question 17

What is the hypothalamo-pituitary disconnection (HPD) model?

• What does it show?

Answer 17

The HPD Model:

• Surgical separation of the hypothalamus and pituitary gland; it removes an area of the brain between the optic chiasm and the median eminence and thus prevents the GnRH neurons in the hypothalamus from being able to secrete GnRH into the hypophyseal blood system
• Allows for the study of pituitary gland function without the input of the hypothalamus in vivo

What did the HPD model show?

• withdrawal of GnRH pulsatile secretion immediately ceased LH secretion but FSH secretion still continued as it is under the control of inhibin
• exogenous administration of GnRH restores LH and FSH secretion

Question 18

Describe the relationship between H pulse frequency and amplitude:

Answer 18

• If there are rapid high frequency GnRH pulses there will be rapid high frequency low amplitude LH pulses
• If there are infrequent GnRH pulses there will be slow frequency high amplitude LH pulses (time to synthesise more LH)
• Thus is it is important that positive feedback acts on the pituitary also making it more receptive to GnRH as if it acted on the hypothalamus only it would not cause the LH surge as the LH surge requires high frequency, high amplitude pulses

Question 19

What are the 2 fundamental intracellular pathways involved in gonadotropin secretion?

Answer 19

The pCREB pathway:

1. GnRH binds and activates the GnRH receptor
2. The Gs subunit of the GnRH receptor activated adenyl cyclate (AC) protein kinase
3. AC activates protein kinase A (PKA)
4. pCREB then binds to the gene and alters transcription of gonadotropin protein subunits
   * Sex steroid hormones e.g. estrogen, can modulate the phosphorylation of CREB

The FOS pathway:

1. GnRH binds and activates GnRH receptor
2. The Gq subunit of the GnRH receptor then activates PLC
3. PLC activates phosphokinase C (PKC) that then activates the ERKs
4. The ERK molecules then activate Egr1 and Sf1 that lead to the induction of FOS
5. FOS then binds to the gene and alters transcription of gonadotropin protein subunits

Question 20

Describe the process of negative feedback of estrogen at the pituitary:

Answer 20

• Acute estrogen treatment at the pituitary causes negative feedback:
• LH secretion is inhibited by blocking GnRH-induced calcium mobilisation in gonadotropes (occurs rapidly and has no effect on gene transcription)

Question 21

Describe the process of positive feedback of estrogen at the pituitary:

Answer 21

• Allows the pituitary gonadotropes to become more responsive to GnRH
  1. Estrogen stimulates the movement of LH vesicles to the cell membrane (but not FSH vesicles)
  2. Estrogen increases the level of GnRH receptor expression of the cell surface of gonadotropes

Question 22

What creates the LH surge?

Answer 22

• Coordination between the hypothalamus and pituitary gland:
• Hypothalamus: increased GnRH pulse frequency and amplitude
  Pituitary: Increased GnRH receptor expression, LH is moved to the cell membrane (gonadotrope is primed)
• Not dependent on the GnRH surge, rather it always occurs 16 hours after estrogen injections

Question 23

What is the action of GnIH on the pituitary?

Answer 23

• GnIH is secreted into hypophyseal portal blood (it is not a hormone that is released into general peripheral circulation):
• Within the gonadotropes, treatment of these cells with GnIH inhibits GnRH induced LH secretion, this occurs both in vitro and in vivo:
• GnIH does this by inhibiting the increase in intracellular calcium and inhibiting the increase in ERK signalling that helps regulate LH secretion
• The level of GnIH secreted into the anterior pituitary during the ovarian cycle does not change, rater it increases during the non-breeding season of seasonal breeding animals

Question 24

Describe the key neuroendocrine changes in the luteal phase?

Answer 24

• Estrogen and progesterone levels are high
• Strong negative feedback to hypothalamus and pituitary
• Reduced GnRH secretion
• Reduced KNDy activity in ARC
• No change in KISS cells in POA
• Increased GnIH expression in the Brain
• This causes low GnRH pulse frequency
• Which subsequently causes low LH pulse frequency

Question 25

Describe the key neuroendocrine changes in the early follicular phase?

Answer 25

• Reduced progesterone levels
• Reduced progesterone negative feedback
• Only estrogen negative feedback (much weaker than progesterone negative feedback)
• Lack of progesterone negative feedback increases GnRH pulse frequency (due to a small increase in KNDy cell activity)
• Increase in GnRH pulse frequency causes an increase in LH pulse frequency
• Pituitary is still receiving some negative feedback from estrogen

Question 26

Describe the key neuroendocrine changes in the late follicular phase (just preceding the LH surge):

Answer 26

• Progesterone levels are low (they must be low to allow estrogen to exert positive feedback)
• Estrogen levels are high and exert positive feedback at level of hypothalamus and pituitary
• Increase in KNDy cell activation in ARC (acts on GnRH via intermediary neuron and also acts on GnRH terminals)
• KISS cells in preoptic area become activated after KNDy cells to facilitate positive feedback of estrogen on GnRH neurons
• GnIH is reduced to allow disinhibition to occur
• Increase in GnRH pulse frequency and amplitude
• Positive feedback to pituitary gland causes increased expression of GnRH receptors, moves LH vesicles to surface via upregulating intracellular proteins such as Ras (gonadotrope is primed)
• Positive feedback to pituitary allows dissociation of LH pulse frequency and amplitude relationship and thus high frequency and high amplitude LH pulses leading to the LH surge