REPRO: Hypothalamic-Pituitary-Gonadal Axis I

Question 1

Practically, what do we need to reproduce?

Answer 1

• correct process of sex determination (genotypic sex) and differentiation (phenotypic sex)
• sexual maturation (puberty)
• production and storage of sufficient supply of eggs and sperm
• sexual intercourse, where the egg and sperm have to be transported and meet (or IVF)
• fertilisation, implantation, then embryonic and placental development
• once delivered, we need to nurture the individual until it is capable of ‘independent’ life

Question 2

The HPG axis is known as the master controller of reproduction. How does it control it?

Answer 2

Gonadal function is controlled via feedback by:

• the hypothalamic and pituitary peptide hormones
• the gonadal steroid (and peptide) hormones

The hypothalamus releases hormones that positively affect the pituitary, which then releases hormones that positively affect the gonads. The gonads then release hormones that, mostly negatively, affect the hypothalamus and pituitary. Thus, their function is controlled.

Question 3

What are the hormones of the HPG axis?

Answer 3

HYPOTHALAMUS (Releasing Hormones):

• gonadotrophin-releasing hormones (GnRH)
• kisspeptin (anterior)

PITUITARY (Stimulating Hormones):

• follicle-stimulating hormone (FSH)
• luteinising hormone (LH)

GONADS: (steroid + protein)

• [F] oestradiol (E2)
• [F] progesterone (P4)
• [M] testosterone
• inhibin
• activin

Question 4

Give a brief overview of the hormones in play in the HPG axis.

Answer 4

Kisspeptin neurons in the hypothalamus (ARC + AVPV) send projections of kisspeptin to GnRH neurons, and bind to GPR54 (GnRH receptor) expressed on GnRH neurons (ARC + MPN) . GnRH goes through the primary plexus to the hypophyseal portal vessel to the secondary capillary plexus (this is the hypophyseal portal circulation.) GnRH acts on the gonadotroph cells of the anterior pituitary to coordinate the synthesis and release of gondadotrophins - LH and FSH.

These, in turn, coordinate the secretion of oestrogen, progesterone, and androgens. These feedback, mostly in a negative manner, back to the hypothalamus and pituitary. Always negative in males and primarily negative in females.

Question 5

Describe Kisspeptin.

Answer 5

• it was recently discovered in 2001 (gatekeeper of puberty).
• it’s expressed in two different nuclei in the hypothalamus –> Arcuate (ARC) and Anteroventral Periventricular Nucleus (AVPV)
• it’s upstream of GnRH (ie. it regulates GnRH production) [paracrine regulation]
• the kisspeptin neurons send projections to GnRH neurons, and bind to GPR54 expressed on GnRH neurons.

Question 6

Describe kisspeptin synthesis.

Answer 6

Kisspeptin is initially synthesised as a prepro protein (preprokisspeptin), it then undergoes proteolytic cleavage to kisspeptin-54. Kisspeptin can then undergo further cleavage to kisspeptin-14, kisspeptin-13 and kisspeptin-10.

Kisspeptin-54 is favoured for in-vivo clinical studies because it is most efficient in crossing he blood brain barrier. Kisspeptin-10 is used in in-vitro cell culture studies because its cheaper to make a 10 amino acid protein than 54. Both still have a high biological activity.

Question 7

Describe GnRH.

Answer 7

it’s synthesised and secreted from GnRH neurons

• it’s secreted in a pulsatile fashion
• orchestrated by pulse generators (a special set of neurons)
• it’s expressed in the hypothalamus –> Arcuate (ARC) and Median Preoptic Nucleus (MPN) - collectively known as the parvocelleular system.
• it binds to the GnRH receptor (GnRHR) on gonadotroph cells of the anterior pituitary to stimulate the synthesis and secretion of gonadotrophin hormones: LH and FSH.

GnRH is also initially synthesised as a preproprotein before going proteolytic cleavage to a 10 amino acid and a GAP (GnRH associated protein) protein.

Question 8

Expand on the importance of pulsatility with LH and FSH release.

Answer 8

• GnRH is secreted in pulses from the hypothalamus every 30-120 mins
• a GnRH pulse stimulates a pulse of LH and FSH secretion from the pituitary
• pulsatile GnRH secretion is vital for stimulation of LH/FSH secretion
• slow frequency pulses favour FSH release, while rapid frequency pulses favour LH release [this occurs via differential pathway activation]
• continuous release results in the cessation of response

Question 9

Explain the therapeutic exploitation of GnRH.

Answer 9

SYNTHETIC GNRH: has the same structure as endogenous GnRH (but it is produced in cell culture bodies - in-vitro studies)
- its pulsatile administration is stimulatory (for fertility) - affects people who, essentially, haven’t undergone puberty –> stimulatory affect on the HPG axis. (For pubertal disorders)

GNRH ANALOGUES: Modified GnRH peptide structure.

• Has a single bolus
• Increase half-life.
• High receptor affinity to compete with endogenous GnRH.
• The loss of pulsatility. These modifications inhibit the HPG axis.
• Can be agonists or antagonists
• Can be used to treat hormone dependent cancer (due to inhibitiory HPG axis) + IVF treatment

The synthetic GnRH takes advantage of the pulsatile secretion of GnRH and the GnRH analogues take advantage of the continuous secretion of GnRH.

Question 10

Describe the mechanism of action of GnRH.

Answer 10

WITH GNRH:

1. It binds to the receptor, GnRHR
2. It activates the signaling pathway
3. It stimulates gonadotrophin synthesis and secretion (LH + FSH)
4. There is then the dissociation of GnRH from the GnRHR
5. The GnRHR is responsive to the next GnRH pulse.

Question 11

Describe the mechanism of action of synthetic GnRH and GnRH analogues, as agonists.

Answer 11

WITH AGONISTS:

1. It binds to the receptor, GnRHR
2. It activates the signaling pathway
3. It stimulates gonadotrophin synthesis and secretion (LH + FSH)
4. There is then the uncoupling of GnRHR from G-protein signaling (because of long half life)
5. GnRHR is now non-responsive to GnRH

Because of the long half life, it stays attached to the GnRHR for a long time and desensitises it, thus making it unresponsive.

Shuts down HPG axis.

Question 12

Describe the mechanism of action of GnRH analogues, as antagonists.

Answer 12

WITH ANTAGONISTS:

1. It binds to the receptor, GnRHR
2. It blocks the receptor
3. There are no downstream effects

Shuts down HPG axis.

Question 13

List some clinical uses of GnRH analogues.

Answer 13

They inhibit the HPG axis leading to uses in:

• ovulation induction and IVF (shuts down HPG axis allowing selective manipulation of the axis using external FSH to stimulate follicles)
• Prostate cancer (shutting down HPG axis - terminating synthesis/secretion of androgens the cancer thrives on)
• GnRHR/GnRH and ovarian and endometrial cancers
• ER+ breast cancer in pre-menopausal women
• Endometriosis
• Uterine fibroids
• PCOS

Question 14

Describe the structure of gonadotrophin hormones.

Answer 14

• LH, FSH (hCG) - they’re heterodimeric peptides - with a common α subunit and hormone-specific β subunit (they are of different length and sequence).
• they are glycoprotein hormones, so the N-linked carbohydrate side chains are required for biological function. Glycolsylation is needed for biological activity.
• Free subunits have no biological action. Dimerisation is required for activation.
• The α-subunits are synthesised in excess with the β-subunit limiting the hormone concentration. The β-subunit is dependent upon GnRH activity. Fast pulse of GnRH favours the LH β-subunit and a slow pulse of GnRH favours the FSH β-subunit but the α-subunit is unaffected.
• Pulsatile secretion is due to pulsatile GnRH release from the hypothalamus, but the pulsatile secretion is not necessary for biological activity. Meaning GnRH need pulsatile activity to function whereas LH and FSH don’t ‘need’ pulsatility to function.

Question 15

What are the functions of LH in the gonads?

Answer 15

In the testis:
- Stimulation of Leydig cell (express LH receptor) androgen synthesis.

In the ovary:

• Theca cell androgen synthesis
• triggers ovulation
• contributes to progesterone production of corpus luteum (for maintaining pregnancy)

Question 16

What are the functions of FSH in the gonads?

Answer 16

In the testis:
- regulation of Sertoli cell metabolism (spermatogenesis)

In the ovary:

• contributes to follicular maturation
• acts on granulosa cell for oesterogen synthesis (from the androgens produced in the theca cells)

Question 17

Male gonadal steroid production

Answer 17

In Testis:

• Leydig cells (in interstitial spaces) express LH receptors –> Leydig cells produce androgens such as testosterone.
• Sertoli cells (within tubules) express FSH receptor –> FSH acts on the sertoli cells to regulate spermatogenesis.

Question 18

Female gonadal steroid production

Answer 18

Mature antral follicle with oocyte inside. Theca cell express LH receptor. LH acts on Theca cells to produce androgens. Androgens make their way further into the follicle to the granulosa cells (immediately surround oocyte as well as inner lining of the follicle). Granulosa cells have FSH receptors, the FSH acts on the granulosa cell to produce oestogens (from the androgens) catalysed by aromatase enzyme. After ovulation, the corpus luteum expresses both LH and FSH receptors and is thus able to produce progesterone and oestrogen in the luteum phase.

Question 19

What are the gonadal steroid hormones and what are their key roles?

Answer 19

Oestrogens:
- Oestradiol and also oestrone –> crucial in folliculogenesis –> positive feedback of oestrogen triggers LH surge which is responsible for ovulation.

Androgens: - Androstenedione, testosterone, dihydrotestosterone –> crucial for spermatogenesis.

Progesterone:

• Prepares the uterus for implantation
• Maintains conditions required for the early stages of pregnancy.

Question 20

Summary of the Hypothalamic-Pituitary-Gonadal Axis in females.

Answer 20

In response to kisspeptin, the GnRH neurones secrete GnRH in the hypothalamus. The GnRH travels through the hypophyseal portal circulation to the gonadotroph cells of the anterior pituitary. The gonadotroph cells secrete LH and FSH.

At the gonads (ovaries): In females, LH acts on the Theca cells producing androgens. FSH acts on the granulosa cells where the androgens are converted to oestrogens. In the luteul phase of the menstrual cycle, progesterone is produced as well. These androgens, oestrongens and progesterones regulate the hypothalamus and pituitary via negative feedback. However, there is also one occasion where oestrogens regulate the hypothalamus and pituitary via positive feedback (in ovulation - triggering the LH surge).

Question 21

Summary of the Hypothalamic-Pituitary-Gonadal Axis in males.

Answer 21

In response to kisspeptin, the GnRH neurones secrete GnRH in the hypothalamus. The GnRH travels through the hypophyseal portal circulation to the gonadotroph cells of the anterior pituitary. The gonadotroph cells secrete LH and FSH.

In gonads (testis): LH acts on Leydig cells which produce Testosterone. FSH acts on sertoli cells producing oestrogens. Testosterone regulates the hypothalamus and pituitary via negative feedback (always negative feedback).

Question 22

Glossary

Answer 22

• Gonadotrophin releasing hormone (GnRH): a hypothalamic decapeptide hormone, released in a pulsatile fashion that controls the synthesis and secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary.
• Luteinsing hormone (LH): a heterodimeric gonadotrophin hormone, comprised of a common alpha glycoprotein hormone subunit and hormone specific beta subunit. LH is synthesised and secreted by the gonadotroph cells of the anterior pituitary. It is released into the portal circulation and binds to its receptor (LHR) on the theca cells of the ovary and Leydig cells of the testis. It is essential for androgen production, and progesterone production.
• Follicle stimulating hormone (FSH): a heterodimeric gonadotrophin hormone, comprised of a common alpha glycoprotein hormone subunit and hormone specific beta subunit. FSH is synthesised and secreted by the gonadotroph cells of the anterior pituitary. It is released into the portal circulation and binds to its receptor (FSHR) on the granulosa cells of the ovary and Sertoli cells of the testis. It is essential for oestrogen production.
• Steroid hormones: Derived from cholesterol, steroid hormones (androgens, oestrogens and progesterone) are produced by the theca and granulosa cells of the ovary and Leydig and Sertoli cells of the testes, by the actions of the gonadotrophin hormones.