Antral Folliculogenesis Flashcards
What is the theca layer and what does it differentiate into?
Theca = envelope of connective tissue
➝Differentiates into theca interna & theca externa
➝Contains steroidogenic cells, vascular tissue, immune cells and matrix factors
Theca is critical for …..
➝ Maintains structural integrity of follicle
➝ Delivers nutrients to the avascular granulosa cell layer
Theca brings blood-borne factors e.g. gonadotrophins(LH,FSH) to the follicle + also produces steroids (androgens, progesterone)
Studies injected radio-labelled LH/hCG (both bind to LHr) into adult female rats, the radiolabeled hormone specifically localised to theca layer of large preantral, small antral and small pre ovulatory follicles but NOT on primordial follicles
What conclusions can be drawn from this?
➝ Only theca cells have LH r
➝ No theca in primordial follicles but starts to develop at some point along the pre antral follicle pathway
What happens in GDF9 knockout mice?
➝ GDF9 knockout mice/humans w GDF9 mutns fail to develop theca layer = follicles arrest
➝ Oocyte-derived GDF9 regulates theca layer formation
Where is GDF9 secreted and what is its function?
➝ GDF9 is secreted from the oocyte, into the GCs and attracts the theca layer towards it
Why is the formation and differentiation of the theca layer important for preantral->antral progression?
➝ Neoangiogenesis brings all the systemic endocrine factors to interact w follicle
➝ Theca produces steroids = Acquisition of steroidogenic function (the substrate for aromatase activity is androgens coming from the theca layer – you cannot get oestrogen if there is no theca layer)
What is the theca layer made up of?
theca interna and theca externa
What are theca cells derived from?
Theca cells derived from 2 different sources in the embryonic gonad:
➝ Mesenchymal (from mesonephros) cells that diff into steroidogenic cells
➝ Stromal cells (indigenous to the medullary region) diff into fibroblasts, vascular smooth muscle cells
When does the follicle become subject to circulating influences?
➝ When highly vascularised theca develops; at 200-400 µm
How does antrum formation occur?
➝ Fluid-filled spaces form b/w the GCs which coalesce to form a single large fluid-filled cavity = ‘antrum’ = ↑ foll fluid , ↑ foll size = pushes oocyte to 1 side + forms mural GCs(around edge) + cumulus GCs(surround oocyte).
COC is ovulated
➝ Antral follicles size range: 0.4-25mm diameter
Antrum is filled w …..
How is this formed?
➝ Antrum is filled w follicular fluid
Filtration of theca layer blood vessels produces plasma fluid exudate which then flows through the GCs to form the antrum. Fluid collects the secretory products of GCs + theca cells + oocyte = follicular fluid
What does follicular fluid contain?
➝ FF = fluid formed as exudate of plasma from theca cell blood; Contains secretory products of oocyte + GCs + theca cells
Name the factors essential for antrum formation
Kit Ligand & Connexin37
-Genes important for FF – Connexin37 – connects GCs + oocytes
-Kit Ligand
Knock out these genes = no antral follicle
What is follicular fluid formed by?
➝ filtration of thecal blood
How is follicular fluid different from plasma?
➝ contains secretory products of oocyte and granulosa cells
What are the approximate dimensions of the dominant follicle at ovulation (diameter + follicular fluid vol)?
➝ 25mm diameter
➝ 7ml follicular fluid
Change in GC & FF volume
➝Small fluid spaces b/w GCs coalesce = fluid volume ↑ = foll grows in size = num of antral folls ↑
➝Large changes in num of GCs + fluid vol
What are the components of antral follicle?
Components of antral follicle:
➝Antrum
➝Theca externa - smooth muscle
➝Theca interna - LHr = LH binds, stimulates theca to produce androgens + prog (steroidogenic function)
➝Mural GCs - FSHr, p450Aromatase
➝Cumulus GCs
What is the theca externa made up of?
➝ Concentrically arranged smooth muscle cells
Theca externa features
➝ Smooth muscle cells
➝ Innervation from the autonomic nerves
➝ Lymphatic vessels
➝Important during ovulation bc brings in inflamm markers
Where is theca interna located?
Theca interna is adjacent to Basement Membrane
What cells does the theca interna consist of?
How is theca interna vascularization?
Steroidogenic cells; contains LHr + insulinR
Highly vascularized
What do granulosa cells differentiate into and where are these found?
➝GC diffs into mural/cumulus
➝ Mural GCs = adjacent to BM, contain FSHr, produce aromatase(for androgen->oestrogen conversion), (GCs in DF contain LHr)
➝ Cumulus GCs = surround oocyte, mitotically active = can increase in num, no LHr, remain in contact with oocyte & interact with oocyte via gap junctions
What are mural granulosa cells involved in and what receptors do they express?
➝ Endocrine feedback control: Produce oestrogen - conversion of androgens(produced by theca) -> oestrogen via aromatase enzyme (P450aromatase)
➝ express FSHr, P450 aromatase (bc produce estrogen), + eventually LH receptor in DF
Where are the cumulus oophorus granulosa cells and how do they interact with the oocyte?
Cumulus GCs = surround oocyte
➝ in contact with the oocyte and interact with the oocyte via gap junctions(Cx37)
➝Mitotically active = can increase in num
➝No LHr - odd bc LH causes cumulus cell expansion, resumption of meiosis etc.
What layers does sperm have to fight through in order to fertilise the oocyte?
➝Thick ZP
➝Cumulus cells around oocyte – will expand + get more sticky at ovulation
➝Sperm fights its way through this (cumulus cells, ZP) once COC is ovulated
How does the cumulus-oocyte-complex respond so rapidly after the LH surge if there are no LH receptors on the cumulus granulosa cells?
➝LH surge = cumulus cells expand, become thick + mucified
➝Picked up by a pipette
Cumulus GCs produce EGF-like ligands. EGF-like ligands bind LH = triggers GCs to secrete hyaluronan + a complex of hyaluronan cross-linking proteins that triggers COC to expand, become thick + mucified
What happens to COC hyaluronan in IVF?
Hyaluronidase = enzyme breaks down hyaluronan.
Used in IVF to:
-Digest cumulus GCs to obtain only the oocyte to inject it
-Sperm for ICSI – to inject only 1 sperm
What happens at the intercycle rise in FSH?
➝ Recruitment of antral follicles (in the right stage at the right size) into the menstrual cycle
In response to LH what does the theca do?
➝ theca expresses key steroidogenic enzymes to make androgens from cholesterol
LH binds to LHr on theca = stimulates theca to produce androgens + progesterone (theca cells steroidogenic function)
In response to FSH what do granulosa cells do?
➝ upregulate aromatase (CYP19A1) and 17β-HSD to make estrogens
(need to find out 17β-HSD function/which reaction step it catalyses)
**2-Cell,2-Gonadotrophin theory - get clearer understanding of this + add in a fc on this*********
Describe how estradiol is produced in GCs
➝ LH binds to LHr on the theca cells. GPCR = activates PKA signalling cascade to express enzymes for conversion of cholesterol->progesterones + androgens.
➝Androgens diffuse from theca cells, across BM into GCs
➝FSH binds to FSHr on GC = stimulates aromatase activity(upregulates CYP19A1+17β-HSD) which converts androgens->estrone
➝17β HSD converts estrone into estradiol
**just confused ab last 2 bullet pts on 17β HSD and oestrone/oestradiol/testosterone/androstenedione*****
What is FSH crucial for?
FSH recruits early antral follicles into menstrual cycle + maintains their survival
How do most primordial follicles die?
Atresia
most PFs die through atresia, only few undergo initiation into menstrual cycle
Roles of FSH on GCs (antral follicles)
➝ ↑ GC proliferation
➝ ↑ Aromatase (upregulates CYP19A1+17βHSD)
➝ Induces + Maintains FSHr
➝ Induces + Maintains LHr
What does activin do to the granulosa cells and where does it come from?
➝ GCs produce activin
➝ Activin acts on GCs to induce + maintain FSHr (stimulates FSH release at pituitary)
What is needed for effective FSH action?
FSHr expression
What type of receptor does FSH have?
➝ GPCR
What do androgens do to granulosa cells?
➝ act on granulosa cells (paracrine interaction) to upregulate AR + FSHr
What do androgen knockout mice have?
➝ reduced FSHr mRNA
What does AMH do to FSH and how?
AMH ↓ FSH recruitment of antral follicles by :
➝ ↓ FSH sensitivity
➝ ↓ FSH-stimulated aromatase expression
Where is AMH produced?
➝AMH is produced by GCs of small antral follicles
What counterbalances the effect of AMH and how?
➝ Androgens + FSH counterbalance AMH
➝AMH = ↓ FSH recruitment by ↓ FSH sensitivity + ↓ FSH-stimulated aromatase
➝Androgens efect on GCs: Androgens upregulate AR + FSHr on GCs.
(AR k/o mice = ↓ FSHr mRNA)
To prevent:
➝ Premature depletion of follicles in the pool of resting primordial follicles
➝ Premature selection of follicles by FSH
As antral follicles grow, …… prod ↑
➝ estradiol production
What does estradiol production by antral follicles do?
➝ exerts negative feedback at the hypothalamus and pituitary which results in a drop in FSH levels
Describe how the dominant follicle is selected
➝ Antral folls grow = ↑ E2 prod = neg fb = FSH ↓
➝ ↓ FSH = DF survives fall in FSH, other antral follicles die off (foll atresia)
➝ Foll w the most FSHr is selected to become DF = ↑ sensitivity to FSH; survives w the little FSH
What is the threshold?
➝ Threshold : amount of FSH required to recruit one follicle
The foll w the lowest threshold will be recruited = e.g. the foll w the most FSHr
What does the dominant follicle have?
➝ the most FSHr
➝ ↑ GC prolif
➝ ↑ Aromatase
➝ ↑ Androgens + Oestrogens
➝ ↑ intrafollicular cAMP
➝ ↑ area of theca vasculature
What does ↑ theca vasculature of the DF mean?
➝ more susceptible to circulating influences
➝e.g. insulin, IGF, EGF and gonadotrophins
How does FSH activate LHr exp in the GCs of the DF?
➝ FSH binds to the FSHr + sets up downstream signaling (↑ cAMP, ↑ PKA = ↑ LHr exp) = ↑ LHr exp in the DF
↑ GC prolif
↑ aromatase exp + activity
How does the dominant follicle survive the fall in FSH?
➝ the most FSHr = ↑ sensitivity to FSH
➝ ↑ GCs
➝ DF GCs acquire LHr (FSH activates LHr gene transcription)
➝IGF1 + IGF2 possible involvement
What does IGF do?
IGF1, IGF-2
➝ IGF enhances FSH effects, it stimulates androgen output and hence estrogen
IGF binds to both theca + GCs
-Stimulates androgen prod from theca + oestrogen production in GCs
What is IGF activity suppressed by?
➝ IGFBP (IGF-binding protein)
How is IGF made?
➝ PAPP-A cleaves IGF from IGFBP
(pregnancy-associated plasma protein A)
➝IGFBP binds IGF = prevents it from acting
➝IGF activity depends on IGF/IGFBP ratio
Where is PAPP-A expression high?
➝ in the dominant follicle
↑ PAPP-A exp in DF = ↑ IGF in DF = aids FSH
PAPP-A cleaves IGF from IGFBP
Unselected antral folls have ↓PAPP-A/↑IGFBP
Why isn’t there a co-stimulatory effect of IGF & FSH?
➝ Unselected antral follicles = ↑ IGFBP = prevents co-stim effect of IGF + FSH
What happens to the rest of the growing follicles in the ovary?
Die off by atresia
What happens if a woman has an inactivating mutation of the LH receptor?
➝ Normal early follicular phase estradiol level
➝ don’t ovulate ; accumulate multiple cysts (unruptured follicles)
➝ morphologically normal antral follicles
What is estradiol like in hypogonadotropic women?
➝ significantly reduced but detectable
↓ LH/FSH but androgen prod from adrenal cortex.
androgen->oestrogen aromatisation in GCs
What is the treatment for hypogonadotropic women?
FSH treatment is effective as long as some LH is present:
➝need some LH - theca androgen production
➝FSH - androgen->oestrogen
What happens in LH knockout mice?
➝ antral stage growth is blocked
What are the functions of LH once the DF has been selected?
➝ FSH activates LHr exp in GCs of DF = LHr on theca + GCs of DF
➝ LH = ↑ theca function of steroidogenic enzymes (CYP11a, CYP17) = ↑ steroidogenesis + growth of DF
➝ Gap junctions close b/w GCs+oocyte + resumption of meiosis
➝ COC expansion
➝ Ovulation + Luteinization
What does LH do when luteinization of the GCs occurs?
➝ induces Prog Rs in the GCs
➝ Still inhibin A prod = fb
Describe LH signalling in the theca interna
Theca interna: LH-LHr binding (GPCR) = cAMP + PKA prod = downstream signalling, steroidogenesis: cholesterol->androgens
Insulin = co-conadotrophin - binds to theca Rs; enhances LH action
What happens in women with PCOS & insulin resistance?
Women with PCOS develop insulin resistance/hyperinsulinemia
Impacts LH-mediated steroidogenic activity
➝ Insulin = co-gonadotrophin w LH = insulin enhances LH effects = ↑ androgen prod in theca cells
What is the second messenger of LH and FSH?
➝ cAMP
How does the cell distinguish between LH & FSH if they have the same second messenger?
➝ FSH produces ↓ cAMP levels
➝ LH produces ↑ cAMP levels
Both LHr + FSHr = GPCRs, same 2nd messenger system: cAMP, PKA (Gs, adenylate cyclase)
Why is there a difference in cAMP levels between FSH & LH?
➝ Diff density FSHr vs LHr
➝ ↑ LHr, ↓ FSH receptor once induced, OR LHr are more effectively coupled to generate high cAMP levels
What are angiogenic factors stimulated by?
Androgens
+ Oestrogens
-theca, GCs, stroma involved
What are the two angiogenic factors and what do they do?
➝ bFGF (basic Fibroblast Growth Factor)
=Endothelial cell mitogen
-most potent angiogenic factor
➝ VEGF (Vascular Endothelial Growth Factor)
=Endothelial cell mitogen
-↑ vascular permeability
What is the effect of VEGF-R3?
➝ Recruits ovarian lymphatic vessels to theca and stroma layers around the growing follicle
Why do we need angiogenesis?
➝ Constant re-modeling to allow for growth of follicle (2-20mm) through the ovarian tissue,angiogenesis of CL, tissue repair etc.
Androgen action on Endothelial cells
➝ Androgen-AR complex translocates into nucleus, binds to AREs + induces HIF-1 expression (HIF-1=TF for VEGF)
➝ HIF-1 binds to HIF-1 response elements on VEGF gene + induces VEGF transcription
➝ VEGF binds to VEGFR on endothelial cells + induces endothelial cell proliferation
When is AMH secretion maximal and what happens after this?
➝ during small antral follicle stage <4mm; decreases to undetectable levels later
What does serum AMH reflect and why?
➝ serum AMH reflects AFC(small antral follicles)
➝growing antral follicles produce AMH (secrete AMH into serum)
-serum AMH = reflects AFC; good indicator of functional ovarian reserve(num of small antral follicles)
-Functional ovarian reserve = Antral Follicle Count (u/s)
-True ovarian reserve = pool of resting primordial follicles
What does the number of antral follicles in the early follicular phase correlate with?
➝ numbers of growing antral follicles only
What is a sign of ovarian ageing?
↓ AFC = indicates ovarian ageing
What are FSH levels controlled by and what happens as women age?
➝ growing antral folls produce ↑ oestrogen = neg fb = ↓ FSH
➝ ↑ reproductive age = number of folls ↓ = estradiol ↓ = ↓ neg fb = FSH ↑
What is used as a marker for menopause?
↑ FSH
What is used to determine functional ovarian reserve?
➝ AFC + serum AMH (+other markers - FSH, E2 and Inhibin)
Used to assess menopause: AFC + serum AMH
What are FSH levels like in menopause?
High FSH bc no neg fb from oestrogen
folls depleted(↓) = ↓ oestrogen = ↓ neg fb = ↑ FSH
When does a womans fertility decline?
➝ at 35
What is POI and how many women does it affect?
➝ premature ovarian failure
➝ 1%
What is POI defined as?
POI: Ovarian dysfunction <40 years → oligomenorrhoea/amenorrhoea
What is the overarching feature of POI?
↓ foll reserve (accelerated depletion) = infertility
What are the 5 possible reasons for POI?
➝ Hormonal imbalance
➝ Environmental genotoxins induce DNA damage e.g chemo/radio-therapy for cancer treatment
➝ Gene mutns - e.g BRCA1 and BRCA2 that repair DNA double strand breaks = diminished ovarian reserve
➝ Chrs defects (Turner’s - XO, streak ovaries)
➝ Autoimmune diseases - thyroiditis, Addison’s
What are 2 types of 3D culture systems?
➝ Collagen
➝ Alginate (product of seaweed)
Why is a hydrogel matrix needed in 3D cultures?
➝ to support foll - maintain contact b/w oocyte + GC
➝ Also to be permeable to media
How does a 3D printed ovary work?
➝ 3D printer makes ovary from microporous hydrogel scaffolds
➝ Follicles seeded through out to create mouse bio-prosthetic ovary
➝ Scaffold provides 3D support to follicles – allowing for vascularization and ovulation
➝ Ovarian function restored when implanted in surgically sterilized mice
➝ Pups born through natural mating
