The Hypo-pituitary Ovarian Axis

Question 1

Describe GnRH.

Answer 1

GnRH is a small hormone of only 10 amino acids. It is very easily modified and so we have a number of GnRH analogs. GnRH is used to refer to a factor that releases LH and FSH.

Question 2

Describe gonadotrophins.

Answer 2

Gonadotrophins are quite large proteins consisting of common alpha and similar beta subunits.

FSH has a longer half life than LH due to glycosylation making FSH harder to break down.

Question 3

What cells are gonadotrophins secreted by?

Answer 3

Gonadotrophins are secreted by cells in the pituitary called gonadotrophs. These cells are characterised by having GnRH receptors .

Some gonadotrophs secrete either LH and FSH whole others secrete both.

Question 4

Describe how LH and FSH are secreted from gonadotrophs.

Answer 4

LH is organised into secretory granules and LH secretion is pulsatilla in a 1:1 fashion with GnRH.

FSH has been reported as been pulsatile in humans but is not in ruminant circulation.

Question 5

How can we visualise gonadotrophins in the pituitary?

Answer 5

We can use IHC for immunolocalisation of LH and FSH in the pituitary. We can see secretory granules of LH in the gonadotroph.

Question 6

What does the portal vein canulatiom model demonstrate?

Answer 6

The portal vein canulation model demonstrates that GnRH and LH are pulsatile in a 1:1 fashion (utilised in sheep). The model involved collecting blood from the blood vessels between the hypothalamus and the pituitary. Blood is then assayed for LH and GnRH.

Question 7

What are the two different ways that GnRH secretion is controlled?

Answer 7

1) . The pulse generator - responsive to endocrine, metabolic, immune and behavioural cues. Small pulses of GnRH leads to the release of small pulses of LH.
2) . The surge generator - another region of the hypothalamus that is responsive to oestrodiol. This causes a massive release of GnRH leading to a massive release of LH into the peripheral circulation. The LH surge causes ovulation and final maturation of the oocyte which is caused by a big surge of oestrodiol into the circulation.

Question 8

LH concentration is pulsatile in line with GnRH release in both the hypophyseal blood and the peripheral blood. FSH concentration is pulsatile in the hypophyseal blood but remains level in the peripheral blood. What is the reason for this?

Answer 8

This is caused by the relatively long half life of FSH. Because the levels of FSH don’t vary markedly over time we see a relatively level profile.

Question 9

Why is GnRH pulsatile? Does it need to be pulsatile?

Answer 9

We don’t really know why GnRH is pulsatile but we do know it is needed. When we change the GnRH to a continuous non pulsatile signal we get down regulation of the receptor.

Question 10

Why do we get down regulation of the receptor if we stimulate cells constantly with GnRH.

Answer 10

Down regulation of the receptor seems to be related to the way the GnRH receptors are actually processed.

The GnRH receptors are G-protein coupled receptors. Following ligand binding the receptor is commonly internalised into the cell cytoplasm by a vesicle. Commonly these endocytic vesicles will have either the GnRH stripped off and the receptor recycles to the surface, or the receptor and the GnRH will be lysed and destroyed within the cell.

Therefore we can see that if we have very high levels of GnRH stimulation to a particular cell then the number of GnRH receptors at the cell surface will be much lower. Therefore the cell will become unresponsive to GnRH.

This seems to be what happens if we stimulate the gonadotrophs with constant GnRH.

Question 11

How can GnRH receptor down regulation be utilised in assisted reproduction?

Answer 11

We use this phenomenon quite commonly to cause the cessation in reproductive function. It is one of our basic tools in the manipulation of reproductive cycles.

Question 12

What will a GnRH agonist do?

Answer 12

GnRH agonists interact with a GnRH receptor and activates it.

Question 13

What do GnRH antagonists do?

Answer 13

GnRH antagonists block the GnRH receptor and prevent normal pulsatile LH being secreted.

Question 14

FSH mainly acts in two ways in the ovaries. Describe its actions.

Answer 14

Stimulation of follicle development by stimulating:

1) . proliferation - at low doses of FSH.
2) . differentiation - at high doses of FSH.

In addition the sensitivity of ovarian somatic cells is modulated at a local level by a range of local factors which can also serve to amplify the gonadotrophic signal.

Question 15

Where are FSH receptors found in the ovarian follicles?

Answer 15

FSH receptors are only found on the granulosa cells of ovarian follicles. They are found from the primary stage all the way through to the pre-ovulatory phase.

Question 16

What roles does LH serve?

Answer 16

LH stimulates steroid secretion from the follicle/corpus luteum. LH is the major hormone that stimulates ovarian steroid secretion.

The LH surge also stimulates ovulation and nuclear maturation of the oocyte.

Question 17

Where are LH receptors found the in the ovarian follicle?

Answer 17

LH receptors are mainly restricted to the Theca cells in most follicles.

In addition, LH receptors also develop on the granulosa cells of oestrogenic and preovulatory follicles.

Question 18

What cells of the follicle produce progesterone?

Answer 18

Granulosa cells and Theca cells in the follicle and also the luteal cell.

Question 19

Where are androgens produced in the follicle?

Answer 19

The Theca cells.

Question 20

Where are oestrogens produced in the follicles?

Answer 20

The granulosa cells in the ovarian follicles.

Question 21

Describe how oestrogen is formed from cholesterol.

Answer 21

1) . Cholesterol undergoes conversion to progestagen under the influence of side chain cleavage enzyme.
2) . Progestogen is converted to androgen under the influence of 17-alpha-hydroxylase.
3) . Androgen is converted to oestrogen under the influence of aromatase.

Question 22

Where does steroidogeneis occur within the cell?

Answer 22

The localisation of steroidogeneis in the cell occurs mainly in the mitochondria and the smooth endoplasmic reticulum.

Question 23

Where does the cholesterol required for steroid synthesis come from?

Answer 23

Most steroids are derived from cholesterol and most cholesterol used comes from that circulating in the peripheral blood. However, it can also be synthesised by ovarian somatic cells directly from acetate.

Question 24

Where do the various stages of steroidogeneis take place within the cell?

Answer 24

1) . Cholesterol is transferred into the mitochondria under the influence of an enzyme called StAR.
2) . Within the mitochondria the first stage of steroidogeneis occurs. Cholesterol is converted to pregnenalone and then progesterone.
3) . Progesterone moves to the SER where it is converted to androgen and then to oestrogens (dependent on cell type).

Question 25

Describe the 2-cell, 2-gonadotrophin hypothesis.

Answer 25

It states that the Theca cells under the influence of LH produce androgens from cholesterol but lack the aromatase enzyme to convert this androgen to estradiol. This androgen is then transferred across the basement membrane of the follicle to a granulosa cell where it is converted to estradiol under the influence of FSH (which stimulates aromatase production). It is also very likely that a lot of the progesterone that the Theca cells use for androgen production comes from the granulosa cells in a transfer the opposite way across the basement membrane.

Question 26

What subunits is Inhibin A made up of?

Answer 26

Alpha and beta A.

Question 27

What subunits is Inhibin B made up of?

Answer 27

Alpha and Beta B.

Question 28

What size are Inhibin A and Inhibin B?

Answer 28

Approximately 32KD.

Question 29

What subunits is activin made up of? What does activin do?

Answer 29

Beta A or Beta B + Beta A or Beta B. Can therefore have 3 different forms of activin. Approximately 25KD. Activin has emerged as the primary regulator of differentiation during foetal development.

Question 30

Describe FSH feedback in the hypo-pituitary ovarian axis.

Answer 30

FSH feedback is a synergistic negative feedback activity at the level of the pituitary by oestrodiol and Inhibin A. When we have the secretion of oestrodiol or Inhibin A from the ovary we have a negative effect on FSH release from the pituitary. The number of ovarian follicles secreting oestrodiol and Inhibin A determines how much FSH will be released to stimulate follicle development. This is a negative feedback system that is designed to result in a steady state on the number and amount of follicles on the ovary. It is synergistic because Inhibin and oestrodiol act together to control FSH. This is a very intricate and highly controlled system in order to ensure the ovulation of only one egg per cycle.

Question 31

What system ensures that only one egg is released per cycle.

Answer 31

The synergistic negative feedback system whereby oestrogen and Inhibin A released by the follicles acts at the level of the pituitary to inhibit FSH production.

Question 32

Describe LH feedback.

Answer 32

The LH feedback mechanism is a synergistic negative feedback system mediated by oestrodiol and progesterone. Oestrodiol works in a negative fashion in terms of the control of pulsatile LH release but in a positive fashion in terms of surge release. If we have high levels of oestrodiol in the peripheral circulation we have a negative effect on LH pulsatile release initially and then this will switch to a positive surge effect when oestrodiol levels reach a certain threshold on the peripheral circulation. In contrast, progesterone can be regarded as an antagonist to oestrodiol because it has a negative effect on both the pulsatile and surge release of LH. When there is progesterone on the system we get a negative effect on both LH pulse frequency and the ability of oestrodiol to induce and LH surge.

Question 33

Give a summary of the control of hormone levels in the follicular phase.

Answer 33

During the follicular phase we have a situation where the ovary contains some small follicles but mainly a large preovulatory sized follicle which is secreting large amounts of oestrodiol and Inhibin (but no corpus luteum secreting progesterone). This means that it is mainly the secretions of this one large oestrogenic follicle which is controlling the release of FSH and LH from the pituitary.

Question 34

Give a summary of the control of hormone levels in the luteal phase.

Answer 34

In the luteal phase we have a large corpus luteum secreting large amounts of progesterone, oestrodiol, and Inhibin. Due to these high levels of reproductive hormones we have high levels of negative feedback controlling FSH. FSH and LH secretion during this stage of the cycle is low and as a result we don't see follicle development past the small antral follicle stage. We have complete suppression of follicle development during the luteal phase.

Question 35

Give a complete overview of the hypo-pituitary ovarian axis and its functions at the various stages of the cycle. How does it all work together?

Answer 35

If we look at the ovarian menstrual cycle and go through the secretion patterns stage by stage we can see how it all works together. In the late luteal phase we have a situation where the women is not pregnant, we have had luteal regression, she's just starting to menstruate. Progesterone, oestrodiol and Inhibin levels are falling. FSH levels are increasing. This means that during the late luteal phase we have very little negative feedback on FSH or LH from the pituitary. This results in an increase in FSH in the late luteal/early follicular phase. The consequence of this increase in FSH is the recruitment of small antral follicles to the gonadotrophin-dependent phase. We have the recruitment of 10 or so small antral follicles which grow from 3-4mm to 5-6mm quite quickly. These small antral follicles start producing increasing amounts of Inhibin B. This is the hormone secreted by the granulosa cells of these small antral follicles. This Inhibin B will start to feedback at the level of the pituitary and start to suppress FSH. One of the recruited follicles is able to withstand this depression in FSH and continue to grow. This follicle will begin to excrete more and more oestrodiol and Inhibin A. This accelerates the rate at which the FSH declines which will start to limit the growth of other recruited follicles from within its cohort. As this follicle continues to grow and becomes more oestrogenic, it develops LH receptors on its granulosa cells and is able to use LH as a surrogate for FSH and will therefore continue to grow and continue to secrete more and more oestrodiol and Inhibin A, driving FSH down further and becoming a dominant follicle. The eventual fate of this dominant follicle is that it will continue to secrete more and more oestrodiol until the concentration reaches a critical threshold in the peripheral circulation and the mode of LH secretion will turn from pulsatile to a massive surge-like release. This will induce ovulation of this dominant follicle and final maturation of the oocyte so we have ovulation of a developmentally competent oocyte around 24-48 hours later. The ovulated follicle will then transform into a corpus luteum, start secreting increasing amounts of progesterone, oestrodiol and Inhibin and drive down gonadotrophins to very low levels during the luteal phase, until the individual starts the cycle again where the whole process will repeat.

Question 36

What pituitary hormones have reproductive effects?

Answer 36

1) . The gonadotrophins 2) . Growth Hromone 3) . Prolactin