Prenatal follicugenisis Flashcards
Why would animal models not always be accurate for humans?
many animals are poly-ovulatory so may not be accurate for humans
How may folliculogenesis be studied?
- Animal models
- Genotype/phenotype associations in naturally occurring mutations or from knock-out mice
- Culture of whole ovaries/slices/biopsies/follicles/cells
Why are ovary cultures hard to create?
o Very difficult in human because of limited supply of tissue
o Primary cells difficult to obtain, granulosa cell line but no suitable theca cell line
Where are the eggs in a 3/4 week human embryo?
Epiblast cell in yolk sac at base of allantois differentiate into PGC (primordial germ cells)
Where are the eggs in a 5/6 week human embryo?
Mitotically dividing PGCs migrate along dorsal mesentery of hind gut to colonise genital ridge → chemotactic substance secreted by ridge to attract PGCs. It maybe Kit ligand (KL) as the receptor cKit is present on surface of PGCs
What are “nests”?
cytoplasmic bridges between mitotically dividing oocytes form syncitia/nests
What is the role of nests?
- To exchange organelles
- Not sure
Why is retinoic acid important?
It’s a key extrinsic regulator of germ cell entry into meiosis
What gene’s expression increases before meiosis?
- DAZL expression increases before meiosis at 9-14 weeks gestation.
- DAZL mutations in humans are associated with sub-fertility
Name the steps before [primordial follicle formation
- Germ cell cyst formation
- Meiotic onset
- Cyst breakdown/primordial follicle formation
What happens during the cyst breakdown?
the nests breakdown and surrounding cells will infiltrate around the oogonia to form the primordial follicles – this happens before birth in humans
How do primordial follicles form?
Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF) – this all happens on the genital ridge
What regulates PF formation?
o FIGLA k/o female mice sterile with no PF
o Activin βA expression ↓ just before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if k/o → loss of oocytes → “streak” ovaries, contrast with male as can have testes with no sperm
o Co-ordination of signalling pathways: KIT, Notch and TGFβ
o Hormones – FSH promotes formation of primordial follicles and E2 and P oppose
Summarise formation of follicles?
Germ cell migration»_space; colonisation of ovaries»_space; formation of the follicles
What happens to oocytes and follicles right before birth?
Massive loss/apoptosis of oocytes and follicles right before birth
Why does that massive loss/apoptosis of oocytes and follicles happen right before birth?
- Failure of mitosis/meiosis involving defective chromosome spindle function
- Unrepaired DNA damage during egg/follicle formation
- Insufficient pre-granulosa cells resulting in naked oocytes which degenerate
- Degeneration of oocytes during nest breakdown and follicle formation.
What is the ovarian reserve?
Once formed primordial follicles represent the entire pool of germ cells available during reproductive life of the female - this is the ovarian reserve
What are the different stages of follicle growth?
- Resting
- Preantral
- Antral
- Ovulatory
What is germ cell selection?
to select oocytes of the highest quality for the ovarian reserve
Describe what happens as the primordial follicles grow
- All the resting primordial follicles are in the avascular ovarian cortex
- blood vessels are only on the hilum of the ovary
as follicles grow they move in towards the blood supply - after dominant follicle selection and it’s ready to ovulate it moves out towards the periphery and outer cortex
What happens in the resting stage?
Resting follicles are in meiotic arrest
What happens in the preantral stage
Resting follciles grow slowly to become preantral follicles - no gonadotrophins needed
What happens in antral stage?
At early antral stage gonadotrophins are needed – cohorts of follicles are recruited in the menstrual cycle, grow and a dominant follicle is selected
What are the preantral follicle stages?
- primordial
- transitional
- primary
- secondary
- multilaminar follicles
Describe the primordial stage
- oocyte is surrounded by a single layer of flattened granulocytes
- growth occurs by expansion of oocyte and proliferation of granulosa cells
describe the transitional stage
- A few granulosa cells become cuboidal
- oocyte becomes metaboliccaly active - starts to make RNA and proteins
describe the primary stage
- when all cells become cuboidal
- still have a single layer
describe the secondary stage
- Another granulosa cell layer acquired (2 layers)
- start to get theca formation from precursor cells condensing around the follicle
- Formation of the zona pellucida
- A definitive basement membrane - separate theca from granulosa
describe the multilaminar follicle
has several layers of granulosa cells
Can theca cells be seen in real life pictures?
No - they get digested
How are preantral follicles isolated?
- elective caesarean section patients consented for ovarian cortical biopsy
- the biopsy is taken to lab and dissected into small pieces
- digested in collagenase and DNAase for one hour because ovarian stroma is thick
What are the morphological changes seen in the primordial to primary follicle transition???
- changes in granulosa cells
- massive increase in oocyte growth + activity
- controlled and very slow process
- as they grow they move away from collagen rich ovarian cortex and towards perimedullar zone of ovary, ECM is of lower density there
What is the zona pellucida?
A thick extracellular coat separating the egg from the surrounding granulosa cells
what is ZP a marker of?
follicle/oocyte growth
How many ZP proteins make up the human follicles
4 - ZP1, ZP2, ZP3, ZP4
What’s a property of ZP?
It’s permeable to large macromolecules - allows nutrients, follicle extensions and cGMP to come in
cGMP function in oocytes?
helps the egg stay in meiotic arrest
What is the fully formed preantral follicle structure?
- Thick ZP layer
- Has layers of granulosa cells
- A basal lamina/membrane between granulosa cells and theca cells
- intracellular communication between oocyte and granulosa cells via gap junctions (they penetrate ZP)
- communication between granulosa cells via connexins
Where is the location of primordial cells
in the ovarian cortex with no blood supply - follicles will not have any blood borne influences
any factors that initiate growth come from within the ovaries
basal lamina around the follicle creates microenvironment for granulosa cells and oocyte
What are the 3 possible fates of primordial follicles?
- to remain quiescent and die out directly at dormant stage (atresia)
- to begin development but arrest and later undergo atresia
- to develop, mature and ovulate
What causes the initiation of follicle growth?
- Loss of an inhibitor: resting follicles are under the constant influence of local/autocrine factors to remain dormant
- Stimulatory factors from the microenvironment and blood (they diffuse in from centre to periphery)
- As inhibitor decreases it allows stimulatory factors to predominate and activate
- Initiation is also dependent on the size of the primordial follicle pool and the ratio at which stimulatory factors enter the growing pool
What is the production-line hypothesis?
Those that enter meiotic arrest first in foetal ovary will initiate growth first
What is the importance of the extracellular matrix?
- Consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides – exert force on follicle to keep its integrity and to keep it quiescent
- ECM turns over and remodelled during folliculogenesis to allow for growing follicle
- May regulate follicle growth especially interactions between gc & oocyte – through a combination of mechanical signals compressing it and chemical diffusions
- Mechanical stimuli are communicated rapidly throughout follicle as various cell types are physically connected eg via connexins
What inhibits nest breakdown?
Endocrine disrupters that mimic oestrogen
- occupy oestrogen receptor and prevent it from binding
Which transcription factor keeps the primordial factor in arrest?
FOXO3 - binds to cycline D2 and keeps the follicle in arrest
Which pathway is important in the primordial follicle to primary follicle transition?
AKT pathway
How does the AKT pathway play a role in the activation of the cell cycle?
- As follicle activates K ligand is produced in the GC
- it binds to cKIT in the oocyte
- This activates PI3 kinase which mediates the conversion of PIP2 into PIP3.
- PIP3 phosphorylates AKT which phosphorylates FOXO3.
- causes the FOXO3 to leave the nucleus, releases cyclin D2, allows activation of cell cycle
What does PTEN do?
In the oocyte it prevents the conversion of PIP2 to PIP3 - keeps the primordial follicle in repressed state
What factors promote the primary to primordial follicle transition?
- K ligand: granulosa derived, KL (KIT ligand aka stem cell factor) secreted from granulosa cells, evidence that KL may inactivate Foxo3a
- cKIT: oocyte derived, cKIT (KIT ligand tyrosine kinase receptor) in oocytes, necessary for follicle activation
What oocyte derived factors inhibit the primary to primordial follicle transition?
- PTEN (tumour suppression gene): inhibits signalling by AKT/PI3K signalling pathway, loss of PTEN leads to global activation of primordial
- FOXO3a (transcription factor): Part of PI3K signalling pathway, restrains follicle activation, knockout leads to global activation of primordial
- SDF-1 (stromal derived factor) chemokine: inhibits follicle activation in autocrine/paracrine fashion
What granulosa derived factors inhibit the primary to primordial follicle transition?
- AMH (anti-Müllerian hormone): acts in paracrine fashion to inhibit primordial follicle initiation
o k/o have less stock of primordial follicles & more growing follicles
What is needed for primary to preantral growth?
- FOXL2 (forkhead transcription factor): granulosa cell proliferation
- FOXL2 mutations lead to Type 1 BPES and POF (premature ovarian failure) – no progression of follicles to secondary stage
What is BPES?
BPES: Blepharophimosis, Ptosis and Epicanthus Inversus Syndrome
- Blepharophimosis = narrowing of eye opening
- Ptosis = droopy eyelids
- Epicanthus Inversus Syndrome = upward fold of the skin of the lower eyelid near the inner corner of the eye
Which oocyte derived factors are needed for the progression of primary follicle growth?
- GDF-9 (growth differentiation factor-9) - k/o no progression beyond primary
- BMP-15 (bone morphogenetic protein-15) - k/o mice sub-fertile ≡ equivalent mutation in sheep (Inverdale sheep FecXi) profound infertility
- Cx37 (connexin 37) gap junction protein (between oocyte & gc) - k/o failed folliculogenesis
Which granulosa derived factors are needed for the progression of primary follicle growth?
• Cx43 (connexin 43) gap junction protein (between gc & gc) - k/o deficient in germ cells and no progression beyond 1°/2° stage
Which extra-follicular factors are needed for the progression of primary follicle growth?
- Insulin & IGF-1 & IGF-II - increase primary stage follicles in cultured human ovarian cortex
- NGF (Nerve Growth Factor) - k/o mice have ↓no. growing follicles → no correlation in domestic animals/humans
What do androgens do in early follicular growth?
- Androgen receptors seen on preantral follicles from early stages
- In women with PCOS there’s more androgens and increase no. of primary and growing follicles
- In animals androgens increase/stimulate follicle growth
What is the importance of gonadotrophins during basal follicular growth?
Physiological & pathological states where circulation gonadotrophin levels are low still see follicular growth:
• e.g. physiological= infancy, pregnancy
• e.g. pathological= Kallman’s syndrome, anov. PCOS
- FSHß & FSHR k/o mice have normal preantral growth
- inactivating mutations of the FSH receptor - follicle growth to antral stage, but less follicles
- FSHR have been found on primary stage follicles, may not be coupled to 2nd messenger system – protein is not fully functional
- Low tonic FSH levels = healthier follicles
Can the ovary form new oocytes?
New research suggests no answer yet
- mouse germline stem cells (GSCs) in ovarian surface epithelium that capable of regenerating ovary depleted of follicles – under normal physiological conditions they do NOT contribute to fertility of mice.
- Found similar structures in human, cows and other species – but very controversial and no consistent markers
- Could be left over oocytes from ovary formation
