Antral folliculogenesis

Question 1

Name the stages of follicle regulation

Answer 1

1. Primordial follicle formation
2. Primordial follicle activation
3. Preantral follicle growth – growth is under control of intra-ovarian regulators
4. Antral follicle growth – this lecture

Question 2

What does the theca form in response to?

Answer 2

The theca forms in response to gonadotropins as supported by studies in 1970s which found that radiolabelled LH/hCG localised specifically to the theca layer of preantral, antral and pre-ovulatory follicles.

Question 3

what is the theca?

Answer 3

The theca is an envelope of connective tissue → differentiates into theca interna & externa containing vascular tissue, immune cells and matrix factors.

Formation and differentiation of theca extremely important for preantral to antral progression.

Question 4

why is the theca important?

Answer 4

1. Acquisition of steroidogenic function.
2. Neo-angiogenesis, hence follicle interaction with systemic endocrine factors.
3. GDF9 K/O mice fail to develop theca layer and follicles arrest → so intra-ovarian factors still important as well as the systemic endocrine factors.

Question 5

Describe antrum formation

Answer 5

When the follicle reaches a diameter of 200-400µm, surrounded by a vascularized theca, thus, subject to circulating influences, fluid-filled spaces appear between the GCs. These spaces soon coalesce together to form a single, large, fluid-filled cavity or “antrum”.
•Contains fluid formed as exudate of plasma containing secretory products of oocyte & GC. Known as follicular fluid.
•KL and Connexin 37 essential for antrum formation in lab animals – as K/O of these genes result in no antral follicles at all.

As the fluid volume increases, the follicle continues to expand greatly in size.

Antrum formation begins with indentations, which coalesce together. As the fluid volume/antrum expands, the follicle expands greatly and the GCs get pushed to the edges, while the oocyte remains. The GCs which are surrounding the oocyte are known as cumulus GCs. While the other GCs are known as mural cells.

Question 6

describe the structure of the follicle

refer to PPT

Answer 6

From the outside, in:

1. Theca externa = concentrically arranged SMCs; innervated by autonomic nerves; lymphatic vessels; important during ovulation.
2. Theca interna = steroid-producing cells; contain LHR & InsulinR; richly vascularized – brings hormones in AND circulates hormones produced e.g. oestrogen, progesterone and androgens.
3. Mural granulosa cells = involved in endocrine feedback control; express both FSHR and LHR in the dominant follicle, and P450 aromatase.
4. Cumulus oophorus = remains in contact with oocyte & interact with oocyte via gap junctions; mitotically active; no LHR.

Question 7

what is the cumulus-oocyte-complex (COC)?

Answer 7

• This complex travels down the uterine tubes and the sperm needs to penetrate through the sticky proteins surrounding the oocyte.
• Although the cumulus GCs do not have LHR, it responds rapidly to the LH surge as the GCs produce EGF-like ligands that bind LH and allow for secretion of hyaluronan. A complex of hyaluronan cross-linking proteins then causes expansion of the COC.

Question 8

describe the 2-cell,2-gonadotrophin theory

Answer 8

• The HPG axis acts to control antral follicle growth at this stage.
• In response to LH, theca express key steroidogenic enzymes to make androgens from cholesterol.
• In response to FSH, GCs upregulate aromatase (CYP19A1) and 17β-HSD to make oestrogens.
• Androgens cross from theca cells to GCs, where they are converted into androgens via aromatase.

Question 9

what do we mean by “many follicles to one”?

Answer 9

• Once puberty has begun, everyday a cohort of primordial follicles will initiate growth.
• Early antral follicles at the right stage and time will be recruited into the MC, in response to FSH (gonadotrophin-dependent growth).
• However, majority of the follicles die through atresia.

Question 10

what is the role of FSH in antral follicles?

Answer 10

• increase granulosa cell proliferation
• increase aromatase expression
• induce and maintain FSHr
• induce and maintain LHr
• interact with paracrine factors

Question 11

describe the autocrine control of FSHR expression

Answer 11

FSH binds to FSHR–> downstream cascade which produces cAMP–>phosphorylates PKA which leads to the consequences of FSH.
Activin binds to ActivinR –>downstream cascade = drives activin and FSHR expression.

Question 12

Describe the role of androgens and AMH on the antral follicle

Answer 12

Androgens act on GC (paracrine interaction) to upregulate AndrogenR & FSHR.
•AR K/O mice have reduced FSHR Mrna.

AMH (produced by GC of small antral follicles) acts as a brake on FSH recruitment of antral follicles by:
•Decreasing FSH sensitivity.
•Decreasing FSH-stimulated aromatase expression.

Counter-balancing effect of AMH, Androgens and FSH to ensure against:
•premature depletion of PF pool and/or,
•Premature selection of follicles by FSH.

Question 13

describe the role of AMH secretion

Answer 13

AMH secretion is maximal during small antral follicle stage and decreases to undetectable levels later, hence serum AMH reflect small antral follicles.

The number of antral follicles in the early follicular phase correlates with numbers of growing follicles only.
-Low numbers of antral follicles are a sign of ovarian ageing.

Question 14

How can we get an indication of a functional ovarian reserve?

Answer 14

We can use serum AMH and antral follicle count (ultrasound to count the number of 2 – 8mm follicles at the start of the cycle) to get an indication of a functional ovarian reserve.

But women with PCOS produce an abnormal amount of AMH, therefore, this cannot be done in these women.

Serum markers FSH, AMH, E2 and Inhibin B used to determine “functional ovarian reserve” – but does not indicate true PF reserve, instead, growing follicles.

Question 15

How quickly does fertility decline?

Answer 15

Fertility declines fairly slowly, until you reach 34/36, then it declines rapidly.

Poorly stocked initial reserve –> infertility/early menopause.

Question 16

define premature ovarian failure (POF)/ Primary ovarian insufficiency (POI)

Answer 16

Affects 1% of women worldwide.

Defined as ovarian dysfunction < 40yrs → oligomenorrhoea or amenorrhoea (different from premature menopause).

•Overarching feature is infertility resulting from accelerated depletion or reduced follicle reserve.

Question 17

describe the aetiology of premature ovarian failure/primary ovarian insufficiency

Answer 17

Aetiology is poorly understood:
• Environmental genotoxins induce DNA damage e.g. chemo/radio-therapy for cancer treatment.
• Mutations in genes e.g. BRCA1 and BRCA2 that repair dsDNA breaks, resulting in diminished ovarian reserve.
• Altered hormonal signalling.
• Chromosomal defects e.g. Turner’s syndrome (XO) → have streak ovaries.
• Autoimmune diseases including thyroiditis & Addison disease.

Question 18

Explain how the dominant follicle is selected

Answer 18

Inter-cycle rise in FSH allows for recruitment of a cohort of early antral follicles into the MC and subsequent selection of a dominant follicle.

The raised FSH presents a “window” of opportunity. The most sensitive follicles respond first to the FSH rise – FSH threshold hypothesis.
o They produce oestrogen + Inhibins –> negative feedback to FSH, therefore we get a fall in FSH – this prevents further follicle growth.
o Consequently, we get selection of a dominant follicle.

Clomiphene citrate is an anti-oestrogen. It binds to the OestrogenR and prevents Oestrogen binding + acting–> this maintains the FSH rise (no negative feedback) –> recruits more follicles – this is useful for women who are not growing up their follicles properly (infertility treatment).

Question 19

what allows the dominant follicle to be selected?

Answer 19

The one follicle which is selected as the dominant follicle has a lower FSH recruitment threshold to keep it growing + alive, within its microenvironment.
(Size has no effect as a study revealed that the largest follicle is not always the one selected)

Question 20

why does the DF have a lower FSH recruitment threshold?

Answer 20

Because of the FSHRs:

• Increased number – responds to FSH even at low concentrations.
• Coupled more efficiently to downstream signalling.

Growth and oestradial production – also a reason:
- Increased cell division–> more GCs –> more oestrogen production.

• Androgens + Oestrogens – increased steroidogenesis environment.
• Increased theca vasculature – more open to other factors (beyond what’s being produced in the follicle).
• Intra-follicular cAMP–> phosphorylation of PKA –> upregulation of FSHR…

Question 21

How do the granulosa cells of the DF acquire LH receptors?

Answer 21

FSH binds to FSHR –> cAMP production–> phosphorylates PKA–> switches on LHR.

LHR is important as in the mid-follicular phase, FSH decreases, while LH increases, therefore, the dominant follicle doesn’t lose its stimulant, even in low levels of FSH, and continues to produce oestrogen (now in response to LH).

Question 22

how does the dominant follicle survive the fall in FSH?

Answer 22

1. Increases sensitivity to FSH –> increased FSHR + more efficient downstream signalling.
2. Increased numbers of granulosa cells –> more oestrogen production.
3. Acquisition of LHRs (switched on by FSH).
4. Possible involvement of insulin-like growth factors (particularly IGF-2).

Question 23

what do IGFs (insulin-like growth factors) do?

Answer 23

• IGF-2 enhances FSH effects; stimulates androgen output and hence oestrogen.
• IGF activity suppressed by IGFBP (IGF-binding protein), which binds to IGFBP and stops it acting.
• PAPP-A (pregnancy-associated plasma protein A) is needed to release IGF from IGFBP (cleaves IGF from IGFBP).
(PAPP-A is expressed in high levels in the dominant follicle + other antral follicles have higher levels of IGFBP)

Question 24

what happens to the rest of the growing antral follicles?

Answer 24

They die off via atresia.
This is contrast to polycystic ovaries, where the antral follicles do not die, irrespective of dominant follicle selection.

Question 25

How do we know that LH has a role in antral follicles?

Answer 25

•Inactivating mutations of LHR = normal EFP E2 , anovulatory, multiple cysts, morphologically normal antral follicles (Toledo, 1996).

•Hypogonadotrophic women:
o FSH treatment effective as long as some LH present.
o E2 is significantly reduced but detectable, why?

•LH K/O mice = antral growth blocked.

Question 26

describe the role of lH in antral follicles

Answer 26

• increases theca function of CYP11a, CYP17
• increases the growth and steroidogenesis in dominant follicle
• Withdrawal of gap junctions between gc and oocyte and resumption of meiosis
• Ovulation and luteinization

Question 27

describe LH signalling in the theca interna

Answer 27

LH signaling, binds to LHR –> ↑cAMP –> phosphorylation of PKA–> production of progesterone + androgens.
InsulinRs also on the Theca –> acts as a co-gonadotrophin – enhances LH + drives androgen production via the same pathway.

Question 28

what do we mean by “one message, 2 signals”?

Answer 28

• LH and FSH have same secondary messenger, cAMP. But how do the cells distinguish between them?
o FSH produces low cAMP levels
o LH produces high cAMP levels
o Yong, Baird, Hillier (1992).
• cAMP provides energy for biosynthetic activity and mediates effects of FSH and LH on protein production e.g. aromatase, SCC, LHR and proteolytic enzymes.

Question 29

what else is needed to support follicle growth?

Answer 29

BLOOD SUPPLY!

• Angiogenesis is important to bring in hormones e.g. LH and FSH, but also to take hormones away.
• Angiogenic factors stimulated primarily by androgens but also oestrogens – theca, GC, stroma all involved.
• Basic fibroblast growth factor (bFGF) is an endothelial cell mitogen, most potent angiogenic factor.
• Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which enhances vascular permeability.
• Ovarian lymphatic vessels are recruited to the theca and the stroma layers around growing follicle, under control of VEGF-R3.

Question 30

describe the role of androgens and VEGF, regarding ovarian angiogenesis

Answer 30

• Androgens bind to cytoplasmic receptors. The complex then translocates into the nucleus and binds to the ARE region, where they act as TFs.
o Induces activation of the target genes e.g. HIF-1.
o HIF-1 binds to HRE –> increases transcription and translation of VEGF.
o VEGF binds to VEGFR on endothelial cells –>proliferation + migration to form the vascular network of the Theca.
• In short, androgens induce HIF-1 expression which is a TF for VEGF.

Question 31

Describe the interaction between androgens, AMH and FSH in follicles

Answer 31

• Androgens amplify their local effects by increasing AR expression and activity.
• Androgens induce FSHR expression and prime follicle for FSH-driven growth.
• AMH inhibits FSH-stimulated aromatase expression – the growing follicle is protected from premature selection into the MC by FSH.