Antral Folliculogenesis Flashcards
Studies in 1970s showed that when radio-labelled LH/hCG injected into adult female rats localized specifically to the theca layer of small preantral, antral and pre-ovulatory follicles but not primordial follicles
Theca of follicle is envelope of connective tissue, what does it differentiate into?
theca interna & externa containing vascular tissue, immune cells and matrix factors
What is the theca critical for?
maintaining structural integrity of follicle and delivering nutrient to avascular GC layer
Formation and differentiation of theca extremely important for preantral to antral progression. Why?
GDF9 k/o mice (& GDF9 mutations in human & sheep) fail to develop theca layer and follicles arrest → oocyte-derived GDF9 regulating formation of theca cell layer.
Neo-angiogenesis, hence follicle interaction with systemic endocrine factors
Acquisition of steroidogenic function
Basically oocyte derived GDF-9 is regulating formation of theca cell layer but not sure if it’s directly/indirectly via other signalling pathways.
How is a theca formed?
Theca cells within the theca layer of growing follicles are derived from two different sources in the embryonic gonad; mesenchymal cells migrating into the ovary from the mesonephros region become the steroidogenic cells, and WT1+ stromal cells indigenous to the embryonic ovarian medullary region become fibroblasts, perivascular smooth muscle cells, and interstitial ovarian tissue, respectively, in the adult ovary
When does the antrum formation switch from preantral to antral follicle transition?
When the follicle reaches a diameter of 200-400µm, surrounded by a vascularized theca, hence subject to circulating influences
How is an antrum formed?
Fluid-filled spaces appear between the granulosa cells which soon coalesce together to form a single, large, fluid-filled cavity or “antrum”
What does the antrum contain?
Contains fluid formed as exudate of plasma containing secretory products of oocyte & GC
Known as follicular fluid
KL and Cx37 essential for antrum formation in lab animals – as k/o of these genes result in no antral follicles at all
What does exudate mean?
Filtered out plasma
As the fluid volume increases the follicle…
continues to expand greatly in size.
Antral follicle range from what sizes?
ranging in size from 0.4-25mm diameter
What is follicular fluid formed of?
Follicular fluid formed by filtration of thecal blood, composition different from plasma as contains secretory products of oocyte and gc
What is the theca externa?
Concentrically arranged smooth muscle cells; innervated by autonomic nerves; lymphatic vessels; important during ovulation
What is the theca interna?
Steroid-producing cells; contain LH-r & Insulin-r; richly vascularized
What does the granulosa cell layer differentiate into?
mural and cumulus cells.
Function of mural granulosa
Mural Granulosa – involved in endocrine feedback control; express FSHr, P450arom, LHr
Function of Cumulus cells
Remain in contact with oocyte & interact with oocyte via gap junctions; mitotically active; no LHr
How do they respond so rapidly after LH surge if no LH receptors on cumulus gc?
GC produce EGF-like ligands that bind LH and allow for secretion of hyaluronan and a complex of hyaluronan cross-linking proteins that cause expansion of COC
In IVF, how is the cumulus layer discarded and oocyte extracted?
Through an enzyme: hyaluronidase
Progression of antral follicles
Selection of dominant follicle
Fate of remaining AF
Inter-cycle rise in FSH crucial for recruitment of AF into the menstrual cycle
**Describe the 2 Cell, 2 Gonadotrophin Concept
In response to LH, theca expresses key steroidogenic enzymes to make androgens from cholesterol.
Likewise granulosa cells respond to FSH by up-regulating aromatase (CYP19A1) and 17β-HSD to make oestrogens
Thousands of primordial follicles…
most die through atresia, a few make it into the menstrual cycle. What determines the survival?
Astresia (cell death)
Effect of FSH on granulosa cells
↑ granulosa cell proliferation
↑ aromatase
↑ induce and maintain FSHr
↑ induce and maintain LHr
Interact with paracrine factors
Why is too much FSH not good?
Factors feeding on granulosa cells
- FSH
- AMH
- Inhibin B–> pituitary feedback
- Activin
- theca cells which release androgens (androgens act on granulosa cells
Effect of Androgens on GC’s?
Androgens act on granulosa cells (paracrine interaction) to upregulate AR & FSHR
AR k/o mice have reduced FSHR mRNA
Effect of AMH on GC’s
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 ensures against
premature depletion of PF pool and/or
premature selection of follicles by FSH
What triggers the selection of a dominant follicle?
Within the micro-environment: amount of FSH required to recruit one follicle
follicle with the lowest threshold will be recruited
Does size affect the selection of the dominant follicle?
size? Serial Ultrasound Scan has revealed that largest follicle is not always selected
What are the FSH receptors like in the dominant follicle?
Increased numbers
Coupled more effectively to down-stream signalling
Growth and Oestrodiol production in dominant follicles?
increased cell division (2-5 million GC in EFP and 50-100 million at ovulation)
Size ~5.5-8.2mm in EFP and 18-20mm in LFP
increased aromatase (200x more E2 than other follicles)
Other factors that may affect the dominant follicle selection?
Androgens & Oestrogens
Intra-follicular cAMP
Increased area of theca vasculature- increased growth factor hormones
How does the dominant follicle survive a fall in FSH?
increased sensitivity to FSH increased FSH receptors
increased numbers of granulosa cells
acquisition of LH receptors
the LHR gene is switched on by FSH
possible involvement of insulin-like growth factors 1&2 (IGF-2 particularly important in humans)
DF & IGFs (IN BOVINE FOLLICLES)
IGF-2 enhances FSH effects; stimulates androgen output and hence oestrogen
IGF activity suppressed by IGFBP (IGF-binding protein)
IGF cleaved from IGFBP by PAPP-A (pregnancy-associated plasma protein A)
PAPP-A expression high in DF
Thought that other AF in cohort may have higher levels of IGFBP hence preventing co-stimulatory effect of IGF & FSH
Change in GC & FF volume
INCREASES exponentiallu
Importance of LH in folliculogenesis
inactivating mutations of LH receptor
normal *EFP E2 , anovulatory, multiple cysts, morphologically normal antral follicles (Toledo, 1996)
hypogonadotrophic women
FSH treatment effective as long as some LH present
E2 is significantly reduced but detectable, why?
can some A be accessed from adrenal?
LH k/o mice
Antral stage growth blocked
FSH treatment effective as long as some LH present
LH needs to bind to the LHr and produce androgens which aid growth?*
Role of LH in ANTRAL follicles:
↑ theca function of CYP11a, CYP17
↑ growth & steroidogenesis in dominant follicle
Withdrawal of gap junctions between gc & oocyte and resumption of meiosis
Expansion of COC
Ovulation & luteinization
one message, 2 signals
LH and FSH have same 2nd messenger - cAMP
How does the cell distinguish between them?
FSH produces low cAMP levels
LH produces high cAMP levels (Difference in density of FSHr & LHr (LHr>FSHr or LHr more effectively coupled to cAMP generation))
provides energy for biosynthetic activity
mediates effects of FSH and LH on protein production eg. aromatase, SCC, LHr, proteolytic enzymes
How does ovarian angiogenesis help follicle growth?
Angiogenic factors stimulated by primarily by androgens but also oestrogens – theca, gc, stroma all involved
Basic fibroblast growth factor (bFGF)
endothelial cell mitogen, most potent angiogenic factor
Vascular endothelial growth factor (VEGF)
endothelial cell mitogen, enhances vascular permeability
Ovarian lymphatic vessels recruited to theca and stroma layers around growing follicle, under control of VEGF-R3
Why is angiogenesis needed?
Constant re-modelling to allow for growth of follicle (2-20mm) through the ovarian tissue, angiogenesis of CL, tissue repair etc.
What is a true ovarian reserve?
What you’re born with
Why does serum AMH reflect small AFs?
AMH secretion is maximal during small antral follicle stage (≤4mm) and decreases to undetectable levels later
The number of antral follicles (AFC) in the early follicular phase correlates with only what?
numbers of growing follicles
Use ultrasound to count number of 2-8mm follicles at start of cycle & correlate ≈ AMH serum levels
Low numbers of antral follicles are a sign of
ovarian ageing
Observable earlier than a rise in FSH serum level
Intriguingly, right ovary has been shown to be larger and have higher AFC than left ovary → thought to be due to…
larger PF pool in right ovary formed in fetal life
why does fsh rise in early menopause?
Decreased follicles–> less oestrogen–> less negative feedback
AFC & serum markers FSH, AMH, E2 and Inhibin B used to determine “functional ovarian reserve”, but does not indicate true Preantral Follicle reserve ie what you are born with
clinical term for premature ovarian failure?
Primary Ovarian Insufficiency (POI)
POI prevelance
Affects 1% of women worldwide
POI
Defined as ovarian dysfunction <40yrs → oligomenorrhoea or amenorrhoea
Overarching feature is infertility resulting from accelerated depletion or reduced follicle reserve.
