Preantral Folliculogenesis Flashcards
How can we study reproduction?
What are some challenges with each method
1.Using animal models
These can be :
Mono-ovulatory
Poly-ovulatory
(We need to always check how many offspring these animals will produce to make the model similar to humans
2.Genotype/phenotype associations in naturally occurring mutations or from knock-out mice.
(for e.g for early follicle growth)
3.Culturing tissue -whole ovaries,slices,biopsies,large follicles ,small follicles,cells
These can be difficult in human because of limited supply of tissue
Primary Cells are also difficult to obtain
granulosa cell lines are available but not theca cell lines available
Where do primordial germ cells originate from?
Early on during 3-4 weeks human embryo the epiblast (primitive ectoderm) is one of two distinct layers arising from the inner cell mass) cell in the yolk sac at the base of all allantois (an extension of the posterior wall of the yolk sac) differentiate into PGC
What will happen to the PGCS which in the 5-6 weeks human embryo?
The mitotically dividing (increase in numbers)PGCS will migrate along the dorsal mesentery of the hind gut and colonise the genital ridge.
This will become the reproductive gonads depending on sex differentiation
Chemotactic substance is secreted by the ridge to attract the PGCS.This maybe the Kit ligand because the receptor cKit has been found on the surface of PGCS
What will happen to the PGCS to form oocytes?
Mitotically dividing PGCS will reach the genital ridge :
If female=oocytes
This will let them form syncytia (nests).
They have cytoplasmic bridges which connect them together to form syncytia and this is thought to allow the exchange of organelles e.g. ER and mitochondria
What is the role of retinoic acid ?
This is the biological active variant of Vitamin A and has been found to be a key extrinsic regulator of germ cell entry into meiosis.
These cells have been dividing through mitosis in order to increase in number and stop to enter meiosis.
What gene has been identified to be important in the process of oocyte formation from PGCS ?
DAZL expression has been found to increase before meiosis at 9-14 weeks gestation.
We know this because in Knock out mice germ cells do not develop past primordial germ cell stage
and mutations in DAZL have been associated with sub fertility
What happens following meiosis of cells whjich are developing following 16 week gestation?
At this stage there is cyst breakdown /primordial follicle formation
This stage will happen before birth in humans whereas in mice it was found to happen following birth
Describe the nest breakdown to form primordial follicles
Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF) – these somatic cells go on to become the granulosa cells. This occurs postnatally in mice, in humans this always occurs before birth.
Numerous transcription factors identified in mice & human follicle formation e.g FIGLA, Nobox & Activin βA…
FIGLA k/o female mice sterile.
Activin βA expression ↓ just before nest breakdown.
Important signalling pathways: KIT signalling, Notch and TGFβ.
Hormones : FSH promotes and E2 /P oppose primordial follicle formation
Describe embryo menopause and some of the reasons this is caused
In utero prior to birth, there is a mass reduction in the oocyte reserve by apoptosis to form the primordial follicles which represent the entire pool of germ cells available during reproductive life of the female.
This is called the ovarian reserve
Predicted range: 35,000-2,500,000 primordial follicles.
Loss of PF prior to birth…
Aneuploidies
Errors in follicle formation – naked oocytes.
Chromosome damage – unrepaired DNA damage.
Dysfunctional spindle.
Germ cell selection – selecting good quality oocytes.
Where are primordial follicles initially located and how do the migrate as they grow towards ovulation?
These are located in the avascular ovarian cortex
Blood vessels are found in the central hilum of the ovary
As the follicles grow they will move towards the blood supply towards the vascular section of medulla.
Following selection of dominant follicule and ready to ovulate, it will move out towards periphery and outer cortex
What are the stages of follicle growth ?
1..Resting
This is when they are in meiotic arrest
A cohort of these will initiate growth
2.Pre-antral
Following initiation , the follicles will grow in slow and controlled manner to create pre antral follicles. This will last around 65 days and is gonadotrophin independent.
3.Recrutiment
At the early antral stage the follicles will no begin to require FSH so a cohort will be recruited.
-Antral
4.Selection
The selection of the dominant follicle
(ovulatory)
Describe each of the preantral follicle stages
Primordial Follicles 🡪 oocyte with layer of flattened granulosa cells.
The growth of the follicle will occour by expansion of the oocyte and proliferation of granulosa cells.
These begin to change in shape and number
At this stage the oocyte is still in meiotic arrest but becomes metabolically active and begins to make RNA /protein for future stages
Transitional 🡪 oocyte surrounded by growing granulosa cells, visible mixture of flattened and cuboidal granulosa cells.
Primary 🡪 oocyte surrounded by a single layer of cuboidal granulosa cells.
Secondary 🡪oocyte develops second layer of granulosa cells and zona pellucida forms between the oocyte and granulosa cells (thick glycoprotein coat). Surrounding precursor theca cells condense around the follicle. A basement membrane forms, separating the granulosa and theca and remaining stromal cells.
Why can we not see theca cells in the real images of the preantral follicles?
This is because when digesting the tissue , the theca cells also get digested
Why are the follicles called pre-antral?
They do not contain an antrum which is the portion of an ovarian follicle filled with follicular fluid.
What are the classifications of the pre antral follicle stages?
Class 1 Primordial follicle Transitional follicle Primary follicle Secondary follicle Multi-laminar follicle
(the last two are sometimes both referred to as secondary follicles)
How can we isolate and classify pre-antral follicles?
Elective caesarean section patients consented for ovarian cortical biopsy
( outer portion of the ovary)
This is then digested in collagenase +DNAse for 1 hour
Collagenases are enzymes that break the peptide bonds in collagen
Follicles are the digested out of the stroma and placed individually in drops of media
What are the morphological changes which take place from primordial to primary transition changes ?
- There is a change in the granulosa cells
- Massive increase in oocyte growth and activity
- controlled and very slow process
Describe the process of Zona pellucida formation
Define the zona pellucida
This is a thick extra-cellular coat separating the egg from surrounding gc.
Zona pellucida formation is a marker of follicle/oocyte growth.
Human follicles made up of four zona pellucida proteins:
ZP1, ZP2,Zp3,ZP4
They will form a thick glycoprotein coat but it is still permeable to large macromolecules
Follicle extensions continue through it.
Describe what the cross section of a preantral follicle structure would be
Outside in: Capillaries Theca layer Basal lamina Granulosa cell layer Zona pellucida Oocyte (in meiotic arrest) Nucleus
What are the communicatiosn which exist between oocytes and granulosa cells ?
There is intracellular communication between oocyte and GC via gap junctions that penetrate ZP as well as communication via connexions i.e. Cx43 between GC and Cx37 between GC & oocyte. The gap junctions are withdrawn when the follicle restarts meiosis
Where can primordial follicles be found ?
These are found along the cortical region very close to the surface.
Avascular - no blood supply
What stage are primordial follicles arrested in ?
They are arrested in the dictytate stage of meiosis
the resting phase in the oogenesis in certain mammals
What are the consequences of primordial follicles not having a blood supply?
This means the follicles are not subject to blood-borne influences such as hormones
What does the basal lamina do to the primordial follicle ?
Basal lamina around the follicle will create a microenvironment for the granulosa cell and oocyte.
Not in contact with other cells in the ovary
therefore the important factors required for initiation are thought to come from the follicle itself
What are the three possible fates of a primordial follicle ?
1.Quiescent and die out directly at dormant stage
2.Begin development but arrest and later undergo atresia
(the breakdown of the ovarian follicles,)
This can happen at any stage
3.To develop mature and ovulate
What will cause initiation of follicle growth ?
- Initiation is regulated by loss of an inhibitor
- Resting follicles under constant inhibitory influence (local paracrine/autocrine factors) to remain dormant. - Initiation is regulated by stimulatory factors :
From the microenvironment (other follicles, stromal cells , blood)
Gradient of diffusion from centre to periphery
blood borne influence will come in and diffuse
Combination of both =Inhibition/stimulation
-Production line hypothesis :those that enter meiotic arrest first in foetal ovary will initiate growth first.
Also dependent on size of PF pool/ration at which they enter the growing pool
What is the importance of the extracellular matrix ( ECM)
ECM consists of collagen,lamimin, fibronectin,proteoglycans and polysaccarides
It is a 3D matrix around the follicles.
Mixture of proteins which exert force on the follicle to keep its integrity also keep it quiescent (dormant)
ECM turns over and remodelled during folliculogenesis to allow for growing follicle -very dynamic layer
May regulate follicle growth especially the interactions between gc and oocyte.
Can regulate chemical diffusion between different layers.
Mechanical stimuli are communicated throughout the follicle as various cell types are physically connected e.g via connexins
What genes are implicated in primordial follicle activation?
Genes for nest breakdown and primordial follicle assembly:
FIGLA (human & mice) Zona Pellucida 1-4 (human & mice) Activin βA & BDNF (human) AMH? (mouse) Oestrogen? (baboons, human)
Genes for Primordial Follicle Maintenance/Repression: PTEN FOXO3 (forkhead family) AMH (Anti-Mullerian Hormone) SDF-1 (stromal derived factor)
Genes for Primordial activation
KIT ligand & KIT receptor (cKIT)
FOXL2
NOBOX (new-born ovary homeobox)
SOHLH 1&2 (transcription factors)
Describe and outline the FOXO3 and AKT pathway
This is important to go from primordial to primary follicle
FOXO3 (transcription factor) binds to Cyclin D2 (cell cycle gene) in the nucleus and maintains the cell in meiotic arrest.
KIT ligand is produced from the granulosa cells, acts on the cKIT receptor in the oocyte which activates the PI3 Kinase pathway. PI3 Kinase stimulates the formation of PIP3 from PIP2 and activates (phosphorylates)AKT. AKT phosphorylates FOXO3, allowing it to move out of the nucleus, releasing Cyclin D2 and allowing it to be activated.
This will then activate the cell cycle
What is PTENs role?
This is a factor which is also present in the oocyte and it prevents the conversion of PIP2 to PIP3 , it maintains PIP2 in the oocyte and keeps the primordial follicle in the repressed state
What are some controls and inhibitors of the AKT pathway ?
AMH
and SDF-1 from the surrounding follicles and stroma
What are some factors involved in primordial follicle repression
Oocyte-derived factors (inhibitory):
PTEN (tumour suppressor gene) inhibits signalling by Akt/PI3K signalling pathway.
loss of PTEN global activation of primordials
FOXO3a (transcription factor) also part of PI3K and restrains follicle activation
FOXO3 k/o have global activation of primordials
SDF-1 (stromal-derived factor) chemokine inhibits follicle activation in autocrine/paracrine fashion
Granulosa-derived factors (inhibitory):
AMH (anti-Müllerian hormone) acts in paracrine fashion to inhibit primordial follicle initiation
k/o have less stock of primordial follicles & more growing follicles
What are some factors involved in follicle activation?
Granulosa-derived factors (stimulatory):
KL (KIT ligand aka stem cell factor SCF) secreted from granulosa cells evidence that KL may inactivate Foxo3a
Oocyte-derived factors (stimulatory):
cKIT (KIT ligand tyrosine kinase receptor) in oocytes necessary for follicle activation
What is FOXL2 involved in ?
This is in the primary to pre antral growth stage .
It is a forkhead transcription factor and is important for granulosa cell activation.
What is a consequence of FOXL2 mutation?
Women without FOXL2 have type 1 BPES and POF (premature ovarian failure) no progression of follicles to secondary stage
What is BPES?
BPES: affects development of eyelids
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
What are the factors which are involved in the progression of primary follicle growth ?
Oocyte-derived factors:
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
Granulosa-derived factor:
Cx43 (connexin 43) gap junction protein (between gc & gc)
k/o deficient in germ cells and no progression beyond 1°/2° stage
Extra-follicular factors:
Insulin & IGF-1 & IGF-II
increase primary stage follicles in cultured human ovarian cortex
NGF (Nerve Growth Factor)
k/o have ↓no. growing follicles → no correlation in domestic animals/humans
What are the roles of androgens in early follicle growth ?
Testosterone rapidly increases intra-oocyte PI3K/Akt/FOXO3 pathway in mouse follicles → increasing >2-fold ratio of primary:primordial follicles (Yang et al, 2010 Endocrinology)
monkeys treated with androgens have more primary follicles and increased FSH receptors
Inhibiting AR in bovine ovaries prevents primary to secondary follicle transition (Yang & Fortune, 2006, BoR)
Is there a human equivalent? (Weber et al 2003 Lancet)
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
(Aittomaki K, 1996; Tapanainen, 1998; Touraine, 1999)
FSHR have been found on primary stage follicles
may not be coupled to 2nd messenger system
(Otkay et al, 1997; Rice et al, 2007)
Whilst FSH is not essential for preantral growth, low tonic levels keep the follicle healthy and in a better state to respond i.e. Healthy pool of selectable follicles.
