HPG Axis Flashcards
1
Q
How does basic negative feedback occur in the hypothalamus?
A
- Feedback is crucial
- Begins with a positive drive from the hypothalamus (synthesises and secreted GnRH to anterior pituitary)
- Gonadotroph cells in the anterior pituitary synthesise and secrete LH and FSH; positive drive of FSH and LH to the gonads
- Cellular compartments of the gonads in turn synthesise and secrete steroid hormones (progesterone, oestrogen and testosterone), which feedback at different points on the anterior pituitary AND hypothalamus.
- In males, the feedback is always negative; in females, the feedback is primarily negative (with the exception of the LH surge required for ovulation).
2
Q
How is GnRH synthesised and secreted?
A
- GnRH neurons in the hypothalamus are expressed in two regions = the arcuate nucleus and the medial preoptic nucleus
- GnRH is synthesised and secreted from these two neurons; GnRH travels through the median eminence (via the hypophyseal portal circulation) to the anterior pituitary.
- The hypophyseal portal system is a network of blood vessels that connect the hypothalamus and the anterior pituitary; allows GnRH to make its way to the gonadotroph cells (of the anterior pituitary)
- Adenohypophyis is another term for anterior pituitary (gonadotroph cells are found here)
3
Q
What is the GnRH pulse generator?
A
- Single pulse generator in the arcuate nucleus with possible modulation of pulse frequency by other brain regions
- Studies of GnRH over the years started to give pieces of evidence as to what might be responsible for this pulsatility.
- Lots of hypotheses were put forward; the intrinsic pulse generator suggested it was a population of GnRH neurons themselves that were responsible for the pulsatility (hypothesis abandoned), the extrinsic pulse generator suggested that the pulsatility is mediated from outside the GnRH neurons indirectly (i.e., another part of the hypothalamus is acting on the GnRH neurons to modulate that pulsatility). As more papers were being published and more studies were being carried out, they started to favour the extrinsic model that was put forward. Up until a decade ago, considering the existing evidence, it was agreed that it had to be a population of neurons in the arcuate nucleus that was mediating this pulsatility with possible input from other regions of the brain. The model above is what was agreed on based on the existing evidence; arcuate nucleus neurons are mediating pulsatility and acting on the GnRH neurons to create pulses with possible input from other regions of the brain.
- Kisspeptin is also pulsatile
- Studies by Clarkson et al. (2017) provided evidence for the identity of the GnRH pulse generator in mice = A sub-population of Arcuate Nucleus Kisspeptin Neurons (ARNKISS). ARNKISS were all found to exhibit intermittent periods of synchronized electrical activity which had a near perfect correlation with pulsatile LH secretion. Inhibition of ARNKISS in the mid-caudal region of the arcuate nucleus suppressed pulsatile LH secretion.
- (Clarkson et al.) provided evidence for the identity of the pulse generator in mice after the scientific community decided to move forward this model. We knew it had to be in the arcuate nucleus, but the identity was not known. This group took it a step further in line with this model and identified the GnRH pulse generator in mice = turned out to be a sub-population of kisspeptin neurons in the arcuate nucleus. In terms of the evidence that they provided, they found that this subpopulation of kisspeptin neurons exhibited intermittent periods of synchronised electrical activity. When looking at electrical activity in brain neurons, it is synonymous with neurotransmitter release. This synchronised electrical activity by these neurons coincided almost perfectly with the pulsatile secretion of LH. As the subset of kisspeptin neurons were transmitting electrical pulses in a synchronised manner, LH was being released as well. Also provided further evidence that the inhibition of those kisspeptin neurons in the arcuate nucleus suppressed pulsatile LH secretion. This was a major breakthrough in that regard, allowing us to identify the pilse generator in mice (this is as far as it has gotten; still hasn’t been identified in humans or any other species).
4
Q
What is kisspeptin?
A
- In the hypothalamus, there is a new player in the regulation of reproductive function (kisspeptin)
- Kisspeptin was originally called metastin because of it’s ability to suppress metastatic spread of human melanomas and breast carcinomas.
- In recognition of it’s discovery at Pennsylvania State University in Hershey, Pennsylvania, it was later named Kisspeptin after Hershey’s Chocolate Kisses®
- Kiss1 gene was discovered in 1996.
- Peptide products (Kisspeptins) that the gene codes for identified in 2001.
- In 2001, there was a huge subset of orphan GPCR with unknown functions/ligands, so they were labelled in numerical order. A series of experiments were carried out to characterise these receptors; found that kisspeptin was the ligand that binds to GPR54 (deorphanized). Kisspeptin receptor (GPR54) identified and de-orphanised in 2001.
- Role as ‘gatekeeper of puberty’ discovered in 2003. Role of kisspeptin was discovered using knockout mice created for GPR54. These mice failed to undergo puberty.
- Controls synthesis and secretion of GnRH in GnRH neurons.
- Upstream of GnRH.
- Kisspeptin is expressed in both the arcuate nucleus and the anteroventral periventricular nucleus of the hypothalamus.
(species dependent). - Expression regulated by gonadal steroids (regions in kisspeptin neurons that express ER, PR). Before the discovery of kisspeptin, it was known that there was positive and negative feedback in the HPG axis, but it was not known how because the hypothalamus and anterior pituitary do not express all forms of the ER and progesterone receptor so this discovery provided a lot of answers.
5
Q
Describe the structure of kisspeptin.
A
- Initially synthesised as a preproprotein before undergoing further proteolytic cleavage into different kisspeptins.
- Proteolytic cleavage produces kisspeptin-54. Can be further cleaved into kisspeptin-14, 13 and 10.
- When looking at in vivo human and animal studies, kisspeptin-54 is usually the kisspeptin of choice in these studies. This is because kisspeptin-54 is able to cross the blood-brain barrier very efficiently. When looking at in vitro cell culture studies, kisspeptin-10 is more the kisspeptin of choice in these studies. This is because kisspeptin-10 is a very active ligand of the GPR54 receptor and it is also cheaper to make a 10 peptide sequence than a 54 peptide sequence.
- Kisspeptin 54 is a 54 peptide sequence.
- Kisspeptin 54 is able to cross the blood-brain barrier.
6
Q
What is the kisspeptin receptor?
A
- KISS1R de-orphanised in 2001, physiological relevance to reproduction identified in 2003 via human and rodent discovery (studies). Older papers refer to the receptor as GPR54
- Expressed on GnRH neurons.
- The Kisspeptin/KISS1R system is a component trigger of puberty. Kisspeptin must bind to its receptor to trigger puberty. This has been discovered through studies of activating and inactivating mutations.
- Inactivating mutations of KISS1R and Kisspeptin = failure to undergo spontaneous puberty.
- Activating KISS1R mutations can lead to precocious (early) puberty.
7
Q
How does KISS1 regulate GnRH secretion?
A
- Hypothalamic explants (slices that were propagated into cell culture) from mice shown kisspeptin administration stimulates GnRH synthesis and secretion.
- GnRH neurons have kisspeptin receptors. Kisspeptin neurons send projections to GnRH neurons, and binding to KISS1R expressed on GnRH neurons (acts upstream of GnRH).
- When carrying out these experiments, introducing a bolus of kisspeptin in turn produces a peak of LH secretion. LH is used to clinically measure GnRH activity, because it is known that LH secretion is very much GnRH driven (some elements of FSH expression are constitutive, but LH does whatever GnRH does). Also, LH is measured due to practicalities and ethical considerations (to take GnRH samples, the hypophyseal portal circulation in the brain would have to be accessed or CSF samples would have to be used). Hence, LH is used as a determinant of GnRH activity. A bolus of Kisspeptin correlates with a peak of LH secretion.
- In terms of how kisspeptin regulates GnRH activity, there is a difference between males and females.
Sexual dimorphic responses = cyclical differences. - In rodents, 2 populations of Kisspeptin expressing and secreting neurons in the arcuate and AVPV have been documented. The population of kisspeptin expressing neurons in the arcuate nucleus respond to the negative feedback endured by E2 and are responsible for tonic GnRH and gonadotrophin hormone synthesis and secretion. Kisspeptin neurons in the AVPV however, are responsive to E2 and P4, mediating the change in GnRH secretion to set up the LH surge and increasing responsiveness that mediates ovulation. This appears to be clear in rodents, however in sheep and primates this is not so clear. In sheep, two populations of Kisspeptin neurons are found in the arcuate nucleus, however in humans and primates, just kisspeptin neurons that respond to negative feedback have been identified to date.
- When the sex steroids are produced by the gonads downstream of the hypothalamus and pituitary, the oestrogen, progesterone and testosterone mediate their feedback via the kisspeptin neurons located on the arcuate nucleus for negative feedback. In turn, GnRH is downregulated, hence the expression of LH and FSH.
- When oestrogen threshold levels have been reached, causing the LH surge, and there is a switch from negative to positive feedback, this feedback is mediated via the kisspeptin neurons on the AVPV. This, in turn, results in the upregulation of GNrH and hence the LH surge. It is important to note that this is how it works in rodents, such as mice or rats. This is how kisspeptin-mediated regulation of GnRH works in rodents, but there is still more to be understood in humans and other animals. In sheep, it is known that there are two different populations of kisspeptin neurons in the arcuate nucleus. In humans, it is known that there is a population of kisspeptin neurons that respond to both positive and negative feedback. There are a lot of pieces of evidence that we have for humans and other animals, but it has not been figured out how it all comes together; this diagram gives an idea of how things work from a mammalian standpoint (in rodents).
- For negative feedback, the sex steroids act on the kisspeptin neurons
8
Q
What is GnRH?
A
- Synthesised and secreted from specialised neurons of hypothalamus (GnRH neurons). About 800 to 1000 GnRH neurons within the human hypothalamus.
- Secreted in a pulsatile fashion (pulse generator orchestrated). Pulsatility is crucial; the pulse generator is a series of specialised neurons that modulate the pulsatility of GnRH.
- Stimulates the synthesis and secretion of pituitary gonadotrophin hormones - LH and FSH.
9
Q
Describe the structure of GnRH.
A
- Just like kisspeptin, GnRH is initially synthesised as a prepropeptide before undergoing proteolytic cleavage into GnRH (a decapeptide; 10 peptide sequence) and GAP (GnRH-associated protein is a produce expressed alongside it)
- GAP used to be heavily researched because it was believed to have GnRH-like properties, but that has since been abandoned
10
Q
What evidence confirms the importance of GnRH pulsatility?
A
- GnRH is secreted in pulses from hypothalamus every 30-120min
- A GnRH pulse stimulates a pulse of LH and FSH secretion from the anterior pituitary
- Pulsatile GnRH secretion is vital for stimulation of synthesis and secretion of LH/FSH
- Slow frequency pulse favours FSHb transcription and FSH release, rapid pulse frequency favours LHb transcription and LH release. Important to remember that a slow frequency pulse of GnRH stimulates FSH release, but a rapid GnRH pulse stimulates LH release. When there’s a slow GnRH pulse, it is the expression of the FSH beta gene that is more favoured (like the default transcription process). When there is an increase in the pulse frequency, the signalling transduction process is switched to favour the transcription and expression of the LH beta gene.
- Continuous release results in the shutdown of the HPG axis; cessation of FSH and LH response.
The above study was carried out in Rhesus monkeys in 1970s (Knobil et al.). They use hypophysectomised monkeys, where the hypothalamus isn’t functional (just the anterior pituitary), and they introduced pulsatile GnRH which immediately corresponded to a rise in expression of LH and FSH. After a few days, they switched to continuous administration of GnRH which lead to a drop in LH and FSH levels. When they maintained that state for a few days, they switched back to pulsatile administration and the levels increased again.
Common alpha subunit transcription is not strictly GnRH dependent. The synthesis occurs independent of GnRH pulse frequency and in excess of that required for FSH and LH production and secretion.
11
Q
How does GnRH action?
A
1) GnRH binding to GnRHR
2) Signal transduction
3) gonadotrophin gene expression → gonadotrophin synthesis → gonadotrophin secretion
- Signal transduction leads to the expression of the FSH and LH beta genes, followed by the synthesis and secretion of FSH and LH
12
Q
How are the gonadotrophin hormones (LH, FSH and hCG) similar?
A
- Glycoprotein hormones
- A glycoprotein has a sugar residue attached from post translational modifications (glycosylation).
- Heterodimeric
- Heterodimers consist of two subunits that are different
- LH, FSH and hCG all share a common alpha subunit. It is the beta subunit that gives them distinct, unique properties/functions. It is also the beta subunits that are crucial to their pulsatility. The beta subunits are synthesised and expressed in response to GnRH pulsatility; the alpha subunit is produced constitutively and the beta subunit is usually produced in response to GnRH pulsatility.
- Pulsatile
13
Q
Describe the structures of the gonadotrophins.
A
- When looking at the structures of FSH and LH, they have a heterodimeric configuration that is made up of an alpha and beta subunit. The alpha subunits are the same in FSH and LH. The alpha and beta subunits are held together by disulphide bonds (the N structures). The Y structures are the sugar residues that are attached to the peptide sequence (the glycosylation). Two things make the beta subunit unique = the length of the sequence and the type of glycosylation that occurs (the type of sugar that attaches and where in the sequence it does).
14
Q
Describe the structure of the gonadotrophin receptors.
A
- The FSH and LH receptors are characterized by large extracellular regions. This is the region where FSH and LH tend to bind to; common site of activating and inactivating mutations.
- They also have transmembrane and intracellular regions.
15
Q
What are the functions of gonadotrophins?
A
1) Follicle-stimulating hormone (acts via FSHR)
• Testis:
- regulation of Sertoli cell metabolism
• Ovary:
- follicular maturation and dolliculogenesis
- granulosa cell estrogen synthesis
2) Luteinising hormone (via LHR)
• Testis:
- stimulation of Leydig cell androgen synthesis
• Ovary:
- theca cell androgen synthesis (conversion of oestrogens into androgens)
- ovulation (LH surge is crucial for ovulation)
- progesterone production of corpus luteum (via LHR)
- Overall, they support germ cell growth, development and maturation to produce gametes (oocyte and sperm) that are competent for fertilisation.
