GnRH

Question 1

What is GnRH?

Answer 1

• Master controller of reproduction
• Characterised in 1971
  (R.Guillemin & A. Schally – Nobel prize in Physiol/Med in 1977)
• Gene that codes for GnRH is present on chromosome 8
• 23 isoforms in vertebrates
• Most vertebrates- GnRH I (GnRH) and GnRH II
• Roles include:
  1) Neuroendocrine - HPG
  2) Paracrine (placenta/gonads)
  3) Autocrine (prostate/breast cancer)
  4) Neurotransmitter (Regions of the brain)
• In the 70’s, there was an explosion in discovery of different hormones.
• GnRH is highly conserved across vertebrate species. There is usually one amino acid substitution that differentiates the different forms of GnRH across vertebrates (highly conserved)
• There are two variants of GnRH expressed in most vertebrated = GnRH I (classic form that is expressed in humans) and GnRH II.
• As well as the neuroendocrine role in the HPG axis, GnRH is also believed to have paracrine and autocrine roles because evidence shows the placenta, gonads and cancer cells etc. all have GnRH receptors present on those cells and tissues. Also regarded as a neurotransmitter because there are GnRH receptors present in other regions of the brain, e.g. gonadotroph cells of the anterior pituitary and GnRH receptor mRNA is known to be expressed in the hippocampal region of brain; evidence suggests that it could be having effects/actions on other regions of the brain as well.

Question 2

What are the functions of GnRH?

Answer 2

• Roles include:
  1) Neuroendocrine - HPG. As well as the neuroendocrine role in the HPG axis, GnRH is also believed to have paracrine and autocrine roles because evidence shows the placenta, gonads and cancer cells etc. all have GnRH receptors present on those cells and tissues. Also regarded as a neurotransmitter because there are GnRH receptors present in other regions of the brain, e.g. gonadotroph cells of the anterior pituitary and GnRH receptor mRNA is known to be expressed in the hippocampal region of brain; evidence suggests that it could be having effects/actions on other regions of the brain as well.
  2) Paracrine (placenta/gonads)
  3) Autocrine (prostate/breast cancer)
  4) Neurotransmitter (Regions of the brain)

Question 3

Describe the structure of GnRH.

Answer 3

• GnRH is a decapeptide = 10 amino acid sequence.
• A lot of research has been carried out in the past on GnRH-associated peptide. Some suggested that it had similar properties to GnRH, while others believed that it was linked to prolactin secretion but, at the moment, no one really knows what it does (no longer a popular area of research).
• Initially synthesised as a pre-pro hormone; undergoes proteolytic cleavage
• Cleavage steps-= Mature GnRH and GAP
• GAP peptide- co-secreted with GnRH, unknown function

Question 4

What is the migratory path of GnRH neurones?

Answer 4

1) Embryonic period = Originate outside CNS, in medial olfactory placode. Placode=area of thickening of the embryonic epithelial later from which the organ/structure later develops. GnRH-producing cells migrate through the nasal system into the forebrain.

2) Cells migrate =
Nasal region → brain → medio-basal hypothalamus. Numerous genes involved

• GnRH neurons do not originate in the hypothalamus; they actually originate in the nasal region (olfactory placode) during embryonic development. Then they migrate to the hypothalamus. While this migration is going on through the olfactory bulb, they respond to a series of genetic cues and the expression of certain genes that regulate this migration process and ensure that it is successful before it makes its way to the hypothalamus.

Question 5

What regulatory gene mutation causes hypogonadotrophic hypogonadism and anosmia?

Answer 5

• When the migratory process goes wrong, it can result in hypogonadotrophic hypogonadism (not going to have the GnRH neurons where they should be so they would not be secreting GnRH and regulating the HPG axis).
• There is a long list of genes and genetic cues that have been identified to be involved in this migratory process. Mutations in these genes are known to cause hypogonadotrophic hypogonadism, because they would either result in premature termination of the migration or they alter the migratory pattern in some way.
• A classical example of a mutation that is known to cause this is the mutation in the KAL1 gene. This causes Kallman syndrome; the mutation results in the premature termination of GnRH neuron migration. Patients usually present with anosmia (inability to smell because the GnRH neurons are still in the olfactory region) and hypogonadotrophic hypogonadism.
• Known mutations causing hypogonadotrophic hypogonadism = KAL1, FGFR1, FGF8, PROK2, PROKR2, NELF, CHD7,
  GNRH1, GNRHR, GPR54, TAC3, TAC3R, NKB/NK3R, WDR11.
• Kallmann Syndrome =
  Mutation in KAL-1 gene (also known as ANOS1) = Premature termination of migration → anosmia & hypogonadotrophic hypogonadism

Question 6

Describe the gross anatomy of the hypothalamus and anterior pituitary (related to GnRH).

Answer 6

• GnRH neurons are expressed in the parvocellular system which contains the arcuate nucleus and the medial preoptic nucleus.
• Median eminence leads to the anterior pituitary via the hypophyseal portal circulation.

Question 7

How is GnRH released?

Answer 7

• 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”
• This is as a result of the action of the GnRH Pulse generator = collection of hypothalamic neurons producing endogenous secretory rhythms. GnRH Pulse generator is believed to be a subpopulation of kisspeptin neurons in the arcuate nucleus.
• GnRH t1/2 = 2-4 minutes. Short half-life of GnRH (degraded very quickly in the system).
• GnRH stimulates synthesis and secretion of gonadotrophins (FSH and LH). The intermittent hypothalamic GnRH stimulation of pituitary gonadotrophs promotes episodic LH & FSH release, especially wrt LH where a close temporal relationship between hypothalamic GnRH release and pituitary LH secretion has been found in monkeys, ewes and humans.
• Differential frequency and amplitude alter pattern of FSH and LH secretion, therefore impact gonadal response. In other words, the fast vs slow GnRH pulses have differential effects.

Question 8

Describe the GnRH receptor.

Answer 8

• G-protein-coupled receptor (GPCR)
• GnRH-R is crucial regulatory molecule that transduces the action of GnRH (to stimulate synthesis and release of gonadotrophins).
• Has the archetypal properties of a GPCR. However, the unique feature of the GnRH receptor is that it does not have a C-terminal tail. Thought to be an evolutionary benefit in humans (found in species like xenopus and chicken), because it plays a role in making it resistant to desensitization in the classical sense. Classical desensitisation is when the C-terminal tail is phosphorylated, and the receptor is internalized due to desensitization by the ligand. That type of desensitisation doesn’t occur in this case. It is believed that the lack of that C-terminal tail in humans is what renders it resistant to that type of desensitisation. Characteristic 7-transmembrane domain structure but, unlike other members of GPCR, does not have a carboxyl-terminal tail.
• Two variants Type I and II GnRHR in humans
• Type 1- full length (classical receptor found in humans), Type 2 - missense truncation (the type 2 mRNA is expressed, but the protein and actual receptor is not translated and expressed due to the missense truncation in sequence)
• Resistant to desensitisation (some exceptions)
• Expressed on gonadotroph cells of anterior pituitary
• When looking outside the anterior pituitary at other tissues and cells, e.g. breast, ovarian and prostate cancer cell lines, the GnRH receptor was also found to be present there as well.
• 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 a 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.
• Interestingly, GnRH receptors are found in other tissues like the breast, placenta and gonads. It is thought that in evolutionary terms maybe there was a direct regulation of the gonads as an early function and then the neuroendocrinological role in regulating the pituitary came as a later evolutionary development.

Question 9

How do you think functions of GnRH-r was discovered?

Answer 9

• The molecular cloning of GPCRs revealed many aspects of the structure that required ligand binding. Also various mutations of the receptor indicate functionality.

Question 10

How does GnRH regulate Gonadotrophin production?

Answer 10

• Gonadotrophins = LH and FSH
• Glycoproteins with α & β chains; α-chains identical in FSH & LH, β-chains unique & confer biological actions/functions
• Rhythm & pulsatility of GnRH
  1) These beta chains have a particular response to the rhythm and pulsatility of GnRH (fast vs slow pulses). Relative rates of gene expression for α/β = Slow frequency or low amp GnRH pulse ⇒ ⇧FSHb gene expression, Fast frequency GnRH pulse ⇒ ⇧LHb transcription
  2) Determines dimerisation (alpha and beta coming together to form the active peptide) of subunits
  3) Determines glycosylation (attachment of sugar residues)
• Genes encoding the gonadotrophin sub-units are regulated in a similar pattern to LH & FSH release
  FSH and LH require carbohydrates (CHOs) to be active. In fact when first tried to make synthetic FSH, couldn’t get it to work because it wasn’t glycosylated.
• Clue came from pioneering experiments by JC Marshall on rats. Showed that changing GnRH pulse frequency and amplitude caused switching between FSH and LH synthesis AND secretion. Illustrates how 1 hormone GnRH can control formation and release of the 2 different gonadotrophins.

Question 11

Compare gonadotrophin expression in males and females.

Answer 11

• In males, GnRH pulsatility and gonadotrophin expression is relatively frequent (around every 2hrs) without the cyclical variations that occur in females. In males, the GnRH pulses generally have a constant frequency every 2hrs, but the exact frequency changes over a 24hr period. That is why significant variations are seen when assaying testosterone and LH levels in males over a 24hr period. This is considered to be normal.
• Important to note that while these pulses are of constant frequency, there is still variation between the pulses. Taking an assay of GnRH pulses, gonadotrophins and steroid hormones, it shows variation with each pulse in terms of amplitude (though the pulses are consistent).
• In the presence of a high pulse, both FSH and LH are still being transcribed, but it is LH that is being upregulated. FSH is still being produced at constitutive levels, but it is not necessarily GnRH-driven; the upregulation of LH is GnRH-driven.
• Cyclical differences in females.
• When introducing GnRH at a rate of one pulse per hour, a study showed that FSH secretion was low and there was an upregulation of LH secretion. When reducing the pulse frequency to about one pulse every three hours, there was an upregulation of FSH and LH production was downregulated. When they reverted to the faster pulse, upregulation of LH could be seen and a downregulation of FSH. This paper by Wildt L et al (1981) shows evidence of fast vs slow GnRH pulses.
• Higher frequency GnRH pulse (every 30min) = favours LH secretion
• Lower frequency/amplitude GnRH pulse (every 90-120min) = favours FSH synthesis and secretion

Question 12

How does GnRH differentially regulate FSH and LH production (molecular mechanisms involved in the differential expression and secretion of FSH and LH)?

Answer 12

• Research is being carried out in this regard. One of the most prominent studies that provided really solid evidence on this is a study by a group from Cornell University. They were able to show that the ERK/MAP kinase signalling pathway is crucial for LH beta secretion in females.
• 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
• Pituitary-specific deletion of ERK MAP Kinase
• They demonstrated this by creating ERK KO mice (double KO mice for the MAP kinase pathway) and they were able to monitor reproductive function/activity.

Question 13

Where would the site of steroid feedback be (at what point in the HPG should we be looking at)?

Answer 13

• Either anterior pituitary or hypothalamus
• Because there are oestrogen & progesterone receptors on both the anterior pituitary and hypothalamus
• Inhibin receptors found only in pituitary
• ANTERIOR PITUITRY = Hormones might regulate FSH & LH secretion by direct action on gonadotrophs to decrease/increase their sensitivity to GnRH pulses. Plenty of receptors of E2, P4 and inhibins here. Carried out by regulation of GnRH receptors.
• HYPOTHALAMUS = Ovarian hormones might change GnRH output signal either by directly affecting GnRH neurones in hypothalamus or indirectly by changing activity of other neural systems that influence GnRH release. Plenty of E2 & P4 receptors on ant. Hypothalamic area and arcuate nucleus and median eminence. But NO evidence to support hypothalamic site of action of inhibin.

Question 14

How would this feedback occur? Is it direct/indirect action?

Answer 14

• It could be in anterior pituitary by direct regulation of GnRHR
• Could be in the hypothalamus either by directly affecting GnRH neurones or indirectly by changing activity of other neural system that influence GnRH release (affecting other regions of the hypothalamus which, in turn, affect the GnRH neurons, e.g. kisspeptin).

Question 15

What evidence supports steroid feedback in the anterior pituitary?

Answer 15

• Looking at the evidence for steroid feedback in the anterior pituitary. In a study carried out in Rhesus monkeys, lesions were created in the hypothalamus, rendering it inactive. They were also ovariectomised (ovaries were removed). As a result, endogenous production of GnRH was switched off. Only the anterior pituitary was left untouched; expressing low levels of FSH and LH, because there is no GnRH being produced. However, when pulses of GnRH were introduced exogenously, it was characterised by pulsatile secretions of LH and FSH. This showed that the anterior pituitary is involved in feedback (at least in part). This was further shown when oestrogen was injected exogenously and there was negative feedback because there was a characteristic drop in FSH and LH production. When the levels of oestrogen introduced increased, there was an LH and FSH surge. This experiment showed positive and negative feedback, proving the anterior pituitary is (at least in a part) involved in feedback on the HPG axis.
• Hence E2 exerting both negative and positive feedback on gonadotrophin secretion and this can only be via the anterior pituitary since hypothalamus is lesioned.

Question 16

What are the mechanisms of feedback in the pituitary?

Answer 16

1) Positive feedback
- E2 induces & maintains GnRHR expression by increasing GnRHR mRNA expression in pre-ovulatory phase, enabling it to deal with the LH surge since a lot of GnRH comes through at that point (the more receptors available, the better)
- E2 sensitises (“self-priming” effect), i.e. enhances interaction between GnRH & GnRHR
2) Negative feedback
- E2 ??
- Inhibin??
- Progesterone ↓ GnRHR mRNA, also evidence of P4 response element in GnRHR gene
- In the case of negative feedback, not much is known. In terms of oestrogen and inhibin, there is evidence to suggest that they may have a role to play in the stability of the GnRH receptor. It is also known that progesterone has a modulatory effect on the expression of GnRH receptor mRNA. There is also evidence of activity in the expression of the receptor gene as well, so there is evidence of progesterone involvement.

Question 17

What evidence supports steroid feedback in the hypothalamus?

Answer 17

• Looking at evidence for feedback in the hypothalamus, these studies in this case were carried out in sheep. They ovariectomised the sheep and injected large boluses of oestrogen. They were able to cannulate the hypophyseal portal circulation (inserted a cannula in the hypophyseal portal circulation). As a result, they were able to assay directly the GnRH levels in the brain (without harming the animals). They saw that the introduction of the large boluses of oestrogen was accompanied by increased GnRH pulses. Because they were assaying the hypophyseal portal GnRH levels directly, they were able to see that rise in GnRH pulses. This was also accompanied by a rise in LH levels as well. This showed that the hypothalamus also has a role in that negative feedback.
• 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.

Question 18

What are the mechanisms of feedback in the hypothalamus (regarding oestrogen)?

Answer 18

• GnRH neurones only express ERβ. In terms of what we know about mechanisms of feedback in the hypothalamus, it is known that GnRH neurons only express the oestrogen beta receptor. However, the alpha subunit is needed for positive feedback and is also crucial for reproduction.
• Need ERα for positive feedback
• Erα is crucial for reproductive function (seen using Erα knock-out mice). Using KO mice for the oestrogen alpha receptor, the mice were found to be anovulatory (did not ovulate), because it is crucial for positive feedback. It is believed that oestrogen must act on other afferents that project onto GnRH neurons (there is no alpha receptor on the hypothalamus). This is where kisspeptin comes into play.
• E2 must act on other afferents that project onto GnRH neurones
• E2 indirectly stimulates GnRH neurones via other neuronal inputs:
  1) Kisspeptin. As mentioned previously, kisspeptin holds answers to questions about feedback. Kisspeptin expresses the alpha variant of the oestrogen receptors.
  2) Other hypothalamic neurotransmitters have been implicated as well - e.g. GABAnergic neurons

Question 19

What is the expected nature of gonadotropin expression in response to a bolus of exogenous GnRH administered every 30 minutes to a sheep within ablated hypothalamus?

Answer 19

• LH upregulated, less FSH.
• The GnRH pulse every 30 minutes is a fast pulse; administering a bolus of GnRH every 30 minutes should result in upregulation of LH secretion and less expression of FSH.
• 90-120 minutes is considered slow (high FSH, low LH). 30 mins to 1 hour is considered fast.

Question 20

What evidence was used to show that that the ERK/MAP kinase signalling pathway is crucial for LH beta secretion in females?

Answer 20

• Premise = ERK1/2 was thought to be the major pathway mediating GnRHR-dependent control of LHb and FSHb- is this the case?
• Pituitary specific deletion of ERK1/2 to delete ERK1/2 from pituitary gonadotrophs and investigate reproductive consequence in male and female mice.
• ERK=Extracellular-signal-regulated Kinase. MAP kinase is another name for ERK.
• MAP=Mitogen activated protein
• Created ERK KO mice (double KO mice for the MAP kinase pathway) and they were able to monitor reproductive function/activity.
• How they did it:
  1) They injected PBS (phosphate buffer saline) into the cervix of these mice and they took the epithelial cells of the cervix, smeared it on slides and monitored it throughout the menstrual cycle. The menstrual cycle in mice is roughly a four to five day cycle for context.
  2) Showed consistent change in the control mice in the cervical epithelial cell number with each menstrual cycle (As indicated by block pattern). In the KO mice, obvious disruption can be seen in oestrous cycle. They were able to show that the cycles in these knockout mice were anovulatory (did not ovulate). When the ovaries are examined, ovulation can be identified by the presence of the CL and mice tend to have more than one (multiple ovulatory species). In the KO mice, there is no sign of ovulation. High level evidence; this pathway is crucial for regulating reproduction.
  3) In control, 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. Anovulatory phenotype in female DKO (ERK1/2-/-) mice
• At the transcription and translational level, the absence of ERK was shown to result in a drastic reduction in LH expression in females. Did not have an effect on FSH production.
• 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.
• They were able to show that there was a drastic reduction in LH beta expression in the KO mice and also that FSH wasn’t so much affected. Clear that the ERK/MAP kinase pathway is crucial for LH beta transcription and translation.
• The western blots show that in the female KO mice, LH beta expression is almost non-existent but a bit of FSH expression is ongoing. Just to emphasise, this was characterised in females.
• ERK-MAPK activation is essential for LHβ transcription and translation

Question 21

How do the gonadal steroids feedback in males?

Answer 21

1) LH
- Testosterone from Leydig cells reduces LH secretion (Rhesus monkey experiments)
- Experiment; rhesus monkeys were immunised against testosterone (stopped producing it; testosterone was conjugated with a protein which gave it antigen-like properties so that the immune system would treat it as a foreign entity). Testo inhibits mainly LH secretion. Expt: In ♂ rhesus monkeys neutralize T by immunization → ↑↑ circulating LH….Administration of exogenous T → ↓ circulating LH. Acts on both hypothalamus and pituitary. CONSTANT NEGATIVE FEEDBACK.
- This showed that gonadal steroids, in males in this case, testosterone modulated feedback and was definitely involved in feedback, whether it was endogenously or exogenously.

2) FSH
- Inhibin ↓FSH secretion from pituitary
- Activin ↑FSH secretion from pituitary
- In the case of FSH, we know that there are inhibin receptors on the anterior pituitary. Intuitively, we expect that these modulate downregulation of FSH secretion from the pituitary via negative feedback and, conversely, activin (the exact opposite of inhibin in terms of action), upregulates FSH secretion from the anterior pituitary. In terms of feedback in males, as it relates to LH and FSH, we know that testosterone is definitely involved in the feedback for LH production. We also know that inhibin and activin are involved in the feedback for FSH production.

• Hypothalamo-pituitary control of spermatid production. The hypothalamus secretes gonadotropin releasing-hormone (GnRH) which stimulates synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). LH binds to specific G-protein coupled receptors, stimulating testosterone production from the Leydig cells. FSH binds to receptors on the Sertoli cells and stimulates the production of a number of Sertoli cell proteins, including inhibin B, activin, androgen binding protein, and transferrin. Inhibin B and activin feedback to the pituitary, regulating the production and secretion of LH and FSH. The importance of FSH and testosterone for the initiation and maintenance of spermatogenesis is clearly documented. Further maturation of sperm to spermatid occurs in the epididymis and through contributions from the prostate and seminal vesicle.
  testosterone-3(o-carboxymethyl)oxime-BSA

Question 22

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

Answer 22

• When GnRH activates the GnRH receptor (binds to it), Gs and Gq signalling is activated. In turn, activates protein kinase A and PKC pathways which diverge to activate the ERK/MAP kinase pathway. Following the activation of the ERK/MAP kinase pathway, there is an upregulation of Egr1 (early growth response 1). The upregulation of Egr1 is accompanied by an upregulation of LH beta transcription.
• Studies have shown that blocking this pathway causes a drop in LH beta transcription and translation too.
• The ERK/MAP kinase pathway is crucial for LH beta transcription and translation.

Question 23

Summarise the differential GnRH receptor signalling pathway in response to a high GnRH pulse.

Answer 23

• GnRH differentially regulates FSH and LH expression in the presence of a high GnRH pulse as well as in the presence of a low GnRH pulse.
• When the pulses are high or fast, GnRH binds to the receptor, the Gq pathway is activated which, in turn, activates the ERK/MAP kinase pathway and there’s an upregulation of EGR1. EGR1 binds to the promoter on the LH beta gene. There is an upregulation in the transcription and translation of LH beta.
• Looking again in the presence of high pulses, but looking at FSH, GnRH binds to the receptor, the Gq pathway is also activated. However, in this case, the relevant bit that is upregulated is ICER. EGR1 is upregulated at the same time but there is also upregulation of ICER.
• ICER is an inhibitor. When it binds to the promoter, it prevents the upregulation of FSH. In order for FSH transcription to proceed and be upregulated, CREB is needed to bind to the promoter. However, once ICER gets there, it inhibits the activity of CREB and, as a result, there is not much expression of FSH.
• In the presence of a high pulse, both FSH and LH are still being transcribed, but it is LH that is being upregulated. FSH is still being produced at constitutive levels, but it is not necessarily GnRH-driven; the upregulation of LH is GnRH-driven.

Question 24

Summarise the differential GnRH receptor signalling pathway in response to a low GnRH pulse.

Answer 24

• GnRH differentially regulates FSH and LH expression in the presence of a high GnRH pulse as well as in the presence of a low GnRH pulse.
• In the presence of a low pulse, GnRH binds to the receptor. In the case of LH, it is pretty much the same; the Gq signalling is activated, ERK/MAP kinase pathway. However, as there is not sufficient amplitude of GnRH pulses, there is not sufficient expression of EGR1. There is a reduction in EGR1 expression and the transient effects of this small amount of EGR1 is not enough to result in that upregulation of LH. There are low EGR1 levels and, as a result, low level of LH transcription.
• In the case of FSH, for the low pulse, GnRH binds to the receptor and both Gs and Gq are activated. On one end, there is the ERK/MAP kinase pathway taking place and there is also the PKA/CREB pathway taking place. Both pathways result in the expression of CREB and the binding of CREB to the FSH beta gene promoter. As there is no ICER there to inhibit it, CREB is successfully able to bind to the gene promoter and upregulate FSH beta transcription.
• Low pulse frequency = Not as much MAP kinase activated.

Question 25

How do GnRH pulses change frequency during menstrual cycle?

Answer 25

1) Early Follicular phase – pulses slow (every 90-120mins) »FSH. When looking at the early follicular phase, now that there is the increased FSH release, the pulses are slow pulses (the pulses increase enough to start producing FSH but they are still slow in the context of fast vs slow; slow pulses are required for FSH production). In the early follicular phase, pulses are about every 90 minutes to two hours and this is essential for FSH release and follicular recruitment. In the mid to late follicular phase, the GnRH pulse frequency increases to once every hour. There is a switch from FSH production to a more dominant LH expression. As LH begins to rise, is also accompanied by a drop in FSH expression. In the menstrual cycle, in terms of events in the ovary, selection of the dominant follicle is taking place. The dominant follicle has acquired more LH and FSH receptors. Those FSH receptors are still operating at a lower threshold, so they can still respond to the low levels with FSH. As this is going on, there is a rise in oestrogen levels as well. During ovulation, once the threshold levels have been reached, there is a switch from negative to positive feedback. This is why the LH surge occurs (the peak). Then, after ovulation, when starting to approach the luteal phase, the pulses switch again back to slow pulses (every 3 to 5 hours). In this case, FSH is produced but not released. FSH is produced and packaged in secretory granules until the end of the luteal phase where there is now an increase in GnRH pulses. The FSH is then released.
2) Mid-late F phase – pulse freq increases (every hr.) »LH
3) After ovulation – pulses slow (every 3-5h) »FSH production. During the luteal phase, FSH is not released because of the negative feedback. At this point, the corpus luteum is active. There are high levels of progesterone and low levels of oestrogen being produced. These feedback negatively on the HPG axis and inhibit that expression of FSH.
4) End of luteal phase – increase in GnRH pulse secretion » FSH release. Looking at GnRH and the differential pattern of the pulses during the menstrual cycle in females. At the end of the luteal phase, there is an increase in GnRH pulses and FSH release. Immediately before that, there are slow pulses once every three to five hours. At that point, FSH is being produced but not released. At the end of the luteal phase, the pulses increase and there is FSH release. Why is there this switch from non-release to release of FSH (from a feedback perspective)? Because the corpus luteum has regressed, there is no oestrogen and progesterone creating negative feedback. As the negative feedback has lifted, there is an increase in GnRH pulses and then increase in expression and release of FSH.

Q(4). What do you think happens for the negative feedback to stop?
Follicular phase – FSH secretion - Increase in estrogen levels – negative feedback resulting in FSH decline – dominant follicle selection (due to follicle expressing LH receptors) – 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 – ovulation

Question 26

How do the gonadal steroids feedback in females?

Answer 26

• When looking at feedback by gonadal steroids in females, this is where there are cyclical changes. It is known that progesterone and low plasma oestrogen modulate negative feedback on the anterior pituitary and the hypothalamus. The result of that is reduced FSH and LH production (negative feedback) Net effect = reduced LH & FSH. It is also known that, in the case of positive feedback, when oestrogen threshold levels are reached and are sustained for a 48 hour period, there is a switch from negative to positive feedback and there is the LH surge.
• High sustained (48h) plasma [E2] = enhanced LH & FSH = positive feedback
• The negative feedback from progesterone is known to modulate the GnRH pulse frequency. That negative feedback from progesterone affects more of the frequency and the negative feedback from oestrogen affects more of the amplitude. Just talking about the threshold levels for the switch from negative to positive feedback, 300pM is required for 48 hours.
• P4 ↓GnRH pulse freq
  E2 ↓GnRH pulse amplitude
• Negative feedback over most of cycle. Positive feedback on days 12-14 of cycle.

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