Preantral Folliculogenesis

Question 1

Folliculogenesis- brief overview

Answer 1

Initiation of growth of primordial follicles
Will progress through the stages
Recruitment of antral follicles into the menstrual cycle
Selection of the dominant follicle from the cohort of smaller antral follicles
Maturation and ovulation of the dominant follicle
Conversion into the CL / degeneration

Question 2

Investigating folliculogenesis (techniques)

Answer 2

Animal models – mono-ovulatory/poly-ovulatory
Need to be careful as many animals are poly-ovulatory (produce litters) so the models may not reflect humans

Genotype/phenotype associations in naturally occurring mutations or from knock-out mice
You can have natural occurring mutations of genes in humans, you can then follow the phenotypic association of the mutation.
Or can create KO mice

Culture tissue of whole ovaries/slices/biopsies/large follicles/small follicles/cells
Very difficult in human because of limited supply of tissue
Primary cells are difficult to obtain; granulosa cell line but no suitable theca cell line

Question 3

Where do eggs come from?

Answer 3

Primordial germ cells are seen very early on: about 3-4 weeks in the human embryo.
Epiblast cells in yolk sac at base of allantois differentiate into PGC (primordial germ cells)
Then at 5-6 old human embryo
The PCGs will divide Mitotically to increase in number.
The PGCs then migrate along dorsal mesentery of hind gut to colonise genital ridge (which will become the gonads) → it is believed that a chemotactic substance secreted by ridge to attract PGCs towards it.
It maybe Kit ligand (KL) as the receptor cKit is present on surface of PGCs

Question 4

Formation of oocytes and follicles

Answer 4

As the mitotically dividing primordial germ cells colonise the genital ridge. If the embryo is destined to become female they will differentiate to form oocytes, and they will start to build nests.
They have cytoplasmic bridges around them which connects them together to form syncitia (or nests).
The role of the nests is thought to be to exchange organelles (like ER or mitochondria

Question 5

Role of retinoic acid in germ cell entry into meiosis

Answer 5

Retinoic Acid (the biologically active variant of Vitamin A) has been identified as key extrinsic regulator of germ cell entry into meiosis
So the cells have been dividing by mitosis to increase in number, they then stop and enter into meiosis. (which is regulated by retinoic acid)

Question 6

DAZL expression

Answer 6

Using KO mice it has been shown that there is another gene crucial in the process called DAZL.
DAZL expression ↑before meiosis at 9-14 weeks gestation. DAZL k/o mice germ cells do not develop past primordial germ cell stage, DAZL mutations in human assoc. sub-fertility. So see genotype/phenotype associations in humans and then confirm it using KO mice.

Question 7

Cyst breakdown and primordial follicle formation

Answer 7

The syncitia nests breakdown and the surrounding cells will infiltrate around the oogonia to start forming primordial follicles.
In humans primordial follicle formation occurs before birth. However in MICE this occurs after birth.

Question 8

Formation of primordial follicle

Answer 8

The primordial germ cells that have migrated and are mitotically dividing to increase in number, colonise the genital ridge which will then become an ovary. The germ cells will become oogonia, then the cytoplasmic bridges between the nests breakdown and the surrounding somatic cells will invade to surround the oogonia to form the primordial follicle.

Syncytia breakdown & somatic cells invade to surround oogonia to form primordial follicle (PF)

PF formation regulated through the following:
Numerous transcription factors identified in mice & human eg FIGLA, Nobox & Activin βA
FIGLA gene k/o female mice are sterile with no PF.
Activin βA expression ↓ just before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if k/o → loss of oocytes → “streak” ovaries, contrasts with male as can have testes with no sperm

You also need Co-ordination of several signalling pathways: KIT, Notch and TGFβ
Hormones : FSH promotes formation of primordial follicles and E2 and P oppose it

Question 9

Embryo-Menopause

Answer 9

There is germ cell migration, colonisation of the ovary and formation of the follicles occurring.
There are large numbers going up to 7m.

Just before birth there is mass apoptosis of the oocytes and follicles
- At birth a woman is born with her entire stock of primordial follicles that she will have for the rest of her reproductive life.
- This loss is due to:

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.

Question 10

Once puberty is established, you get growth of

Answer 10

follicles occurring continuously until all the follicles are depleted and the woman enters into menopause.

Once formed primordial follicles represent the entire pool of germ cells available during reproductive life of the female – known as “ovarian reserve”. Predicted range: 35,000-2,500,000 primordial follicles (mathematical modelling & histological counting)

Question 11

Loss of primordial follicles is related to…

Answer 11

The loss it thought to be related to the “Germ Cell Selection theory” – to select oocytes of highest quality
Allows the ovary to select the oocytes of the highest quality to establish the true ovarian reserve

Question 12

Briefly describe the anatomy of the ovary

Answer 12

All of the resting primordial follicles which are in meiotic arrest are located in the avascular ovarian cortex.
Blood vessels are in the central hilum of the ovary
As follicles grow, they will move inwards towards the blood supply and towards the vascular central medulla
Once there is selection of the dominant follicle and it is ready to ovulate, it then moves out again toward the outer cortex and surface ready for ovulation.

Question 13

Stages of follicle growth

Answer 13

Have resting follicles that are in meiotic arrest.
A cohort of these follicles will initiate growth every day once puberty is established. What causes this initiation is still unknown.
Once the follicles have initiated growth, they grow in a very slow and controlled manor to form preantral follicles.
This process lasts over 65 days
This process is gonadotrophin independent

Once the follicles have reached the early antral stage, and have stated to form an antrum, they need FSH to continue growth.
So a cohort of these follicles are recruited into the menstrual cycle.
These will grown, then from this cohort, the dominant follicle is selected.
This is gonadotrophin dependant

The follicle that is ovulated would have initiated its growth nearly 3 cycles beforehand.

Question 14

Preantral Follicle (Stage 1)

Answer 14

In the primordial follicle there is the oocyte, surrounded by a single layer of flattened granulosa cells.
Growth of the follicle will occur by expansion of the oocyte and proliferation of the granulosa cells.

Once the follicle initiates growth, the granulosa cells start to change in appearance and number. This is called a transitional cell – initiation of growth and formation of 1 or 2 cuboidal granulosa cells.
They go from being flattened to more cuboidal.
There is also growth of the oocyte.

As it continues to grow you will get a single layer of cuboidal granulosa cells, it is now known as a primary follicle.

It then acquires a second layer of granulosa cells.
You start to get theca formation too, which comes from precursor cells which are condensing around the follicle.

You will also see formation of the zona pellucida.

SO… When you have a full secondary follicle (with two layers of granulosa cells) you then have theca formation, you have a definitive basement membrane which separates the theca from the granulosa cells.

Question 15

Preantral Follicle (Stage 2)

Answer 15

Primordial follicle
Can see the oocyte with a single later of flattened granulosa cell

Transitional follicle
Can still see flattened granulosa cells, but some are starting to become cuboidal

Primary follicle
More expanded oocyte. Have a single later of expanded granulosa cells which are all cuboidal.

Secondary follicle
Has two layers of granulosa cells
Can see the zona pellucida and the basement membrane.

Multilaminar follicle
Has several layers of granulosa cells forming

Different classification systems – need to be aware when reading papers. Some call all follicles Primary follicles if they have a primary oocyte i.e. whilst still in mieotic arrest and once completed mieosis 1 and ejected 1st polar body, known as Secondary follicles because they have a secondary oocyte. (some call them secondary follicles when you get more than 1 layer of granulosa cells forming)

Question 16

Technique to isolate pre-antral follicles

Answer 16

Elective Caesarean section patients consented for ovarian cortical biopsy
The piece is dissected out into many smaller pieces in the lab
It is then Digested in Collagenase +DNAse for under 1hr
As the ovarian stroma is very thick

The follicles are then dissected out of the stroma and placed individually in drops of media in a dish
They are then looked at under high power magnification.

Question 17

Alternative Follicle Classification

Answer 17

Some papers will class them all as pre-antral or class 1 follicles
At antral formation they are called antral follicles or tertiary follicles
When they are ready to be ovulated they are fall graafian or preovulatory follicle

Question 18

Primordial -> Primary Transition Morphological changes

Answer 18

There are distinct morphological changes as follicles initiate grown from primordial follicles to becoming primary follicles:

1) Change in granulosa cells (~15 cuboidal granulosa cells)
2) Massive increase in oocyte growth & activity
3) Controlled & very slow process

Also as they start to grow they move away from the collagen-rich ovarian cortex towards the perimedullar zone of ovary, where the ECM is of lower density.

Question 19

Zona Pellucida formation

Answer 19

As the follicle and oocytes start to grow you get formation of the Zone Pellucida:
ZP is a thick extra-cellular coat separating the egg from surrounding granulosa cells
ZP formation is a marker of follicle/oocyte growth

Human follicles made up of four ZP proteins:
ZP1, ZP2, ZP3, ZP4
Even though they form a thick glycoprotein coat, they are permeable to large macromolecules. (gap junctions)
Follicle extensions continue through it

Question 20

Preantral Follicle Structure

Answer 20

What you would expect to see in a multilaminar or pre antral follicle:
Oocyte with nucleus still in meiotic arrest
Thick glycoprotein ZP
Several layers of granulosa cells
Basal laminar – which separates the granulosa cells from the theca
The theca has started to form around the secondary follicle stage, secondary follicle = 2 layers of granulosa cell.
The theca is very well vascularised unlike the granulosa

Even though there is a thick ZP coat, there is a lot of Intracellular communication between oocyte and granulosa cells via gap junctions that penetrate the zona pellucida.

But there is also communication between granulosa cells. This communication is formed by connexin proteins:
Also communication via connexins i.e. Cx43 between GC and Cx37 between GC & oocyte

Question 21

Location of the Primordial Follicle

Answer 21

The primordial follicles are found along the cortical region, close to the surface which is quire avascular. They group together in this region.

Primordial follicles are arrested in dictyate stage of meiosis

Primordial follicles located in ovarian cortex & have no blood supply. Consequences?
This means thee follicles are not subject to blood borne influences

In addition the basal lamina around the follicle creates microenvironment for granulosa cells & oocyte i.e not in contact with other cells in the ovary

This shows that any factors that are involved in initiation of growth of the follicles must be coming from within the ovary itself.

Question 22

Fate of the Primordial Follicle

Answer 22

Primordial Follicle has 3 possible fates:
To remain quiescent and die out directly at dormant stage
To begin development but arrest and later undergo atresia
To develop, mature & ovulate

Question 23

What causes initiation of follicle growth?

Answer 23

Initiation is regulated by loss of an inhibitor
Resting follicles under constant inhibitory influence (local paracrine(adjacent)/autocrine factors(within follicle) to remain dormant. Where from?

Initiation is regulated by stimulatory factor/s
Need something to stimulate the follicles to grow
Can come From the microenvironment (other follicles, stromal cells) and/or blood
Could be Gradient of diffusion from centre to periphery

Question 24

Changes to what may initiate follicle growth?

Answer 24

However we now think that it is a combination of both inhibition and stimulation:

So as you get a decrease in the inhibitor, it will allow the stimulating factors to dominate and activate the follicles.

Is also a “Production-line” hypothesis: those that enter meiotic arrest first in foetal ovary, will initiate growth first
Initiation of follicle growth is Also dependent on size of primordial follicle pool and ratio at which enters the growing pool.
As women come into the perimenopausal period, as the PF pool has decreased, you get more follicles activating and entering the growing pool at a Faster rate.

Question 25

Importance of Extracellular Matrix (ECM)

Answer 25

Need to take into account the ECM or surrounding stromal cells.
The structural architecture around the follicle plays a crucial role in regulating follicle growth.

ECM consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides
This will exert force onto the follicle to keep its integrity, but also to keep it quiescent.

ECM turns over and remodelled during folliculogenesis to allow for growing follicle to pass through
It may regulate follicle growth especially interactions between gc & oocyte.
Mechanical stimuli are communicated rapidly throughout follicle as various cell types are physically connected eg via connexins

Question 26

When does primordial follicle formation occur?

Answer 26

when the nests breakdown, allowing for surrounding cells to invade, surround the oogonia and form flattened granulosa cells -> primordial follicle.

Question 27

Genes implicated in nest breakdown and primordial follicle assembly

Answer 27

FIGLA (human & mice)
Zona Pellucida 1-4 (human & mice)
Activin βA & BDNF (human)
AMH? (mouse) -Don’t think it acts the same in humans
Oestrogen? (baboons, human) – In the human estrogen levels will reach its peak in the third trimester, but the nest breakdown occurs much earlier. The majority of estrogen from the mother will not reach the foetus as it is aromatised by the placenta.
Baboons were given aromatase inhibitors, preventing the conversion of androgens to estrogens, they saw a decrease in PF formation, more nests were visible.

Question 28

Endocrine Disruptors

Answer 28

They mimic oestrogen by occupying the estrogen receptors, and stop estrogen from binding. Such as
BPA (Bisphenol A), Genistein, DES (diethylstilbestrol)
This inhibits nest breakdown

Question 29

Genes involved in primordial follicle maintenance/repression (inhibition)

Answer 29

Inhibitory factors keep the primordial follicles as PF
PTEN
FOXO3 (forkhead family)
AMH (Anti-Mullerian Hormone)
SDF-1 (stromal derived factor)

Question 30

Genes involved in primordial follicle activation

Answer 30

KIT ligand & KIT receptor (cKIT)
FOXL2
NOBOX (newborn ovary homeobox)
SOHLH 1&2 (transcription factors)

Question 31

AKT pathway

Answer 31

important in primordial follicle activation:

In the resting primordial follicle
In the nucleus of the oocyte is a transcription factor called: FOXO3.
FOXO3 will bind to cyclin D2 and keep the follicle in arrest.
It prevents it from entering into the cell cycle

As the follicle activates KIT Ligand is produce in the granulosa cells
This will bind to its receptor cKIT which is present in the oocyte and activate PI3K
This will mediate the conversion of PIP2 to PIP3
PIP3 will phosphorylate AKT
Which leads to the phosphorylation of FOXO3
When FOXO3 is phosphorylated it comes out of the nucleus releasing cyclin D2, allowing for activation of the cell cycle.

There is another factor in the oocyte called PTEN, PTEN will prevent the conversion of PIP2 - > PIP3. Hence it will prevent the activation of AKT and the whole signalling pathway.
So it will keep the primordial follicles in the repressed state.

AMH from surrounding follicles and SDF-1 from the surrounding stroma. Add to this

Question 32

Genes SUMMARY

Answer 32

PTEN and FOXO3 are oocyte derived factors as is cKIT (the receptor for KIT ligand). KL is granulosa derived.
FOXO3 (forkhead transcription factor) is found in nucleus bound to Cyclin D2, which keeps the follicle in arrest and prevents it entering cell cycle. How is this brake overcome? KL produced from gc, binds to it’s receptor (cKIT) and activates PI3 kinase. This then results in conversion of PIP2 to PIP3, which activates AKT which then phosphorylates FOXO3. Phosphrylated FOXO3 moves out of the nucleus releasing Cyclin D2 and allowing the cell cycle to progress. Upstream of FOXO3 is PTEN which facilitates conversion of PIP3 to PIP2, which the opposes actions of Akt to keep follicle in arrest. Aided by other factors like AMH and SDF-1 produced from other follicles and stroma.

Question 33

Oocyte derived factors (inhibitory): genes involved in primordial follicle repression

Answer 33

PTEN (tumour suppressor gene) ⇒ inhibits signalling by Akt/PI3K signalling pathway.
loss of PTEN (ko in mice) ⇒ 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

Question 34

Granulosa derived factors (inhibitory): genes involved in primordial follicle repression

Answer 34

AMH (anti-Müllerian hormone) ⇒ acts in paracrine fashion (comes from growing follicles) to inhibit primordial follicle initiation
k/o have less stock of primordial follicles & more growing follicles

Question 35

Primordial Follicle Activation

Answer 35

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
Newborn mice injected with antibody to cKIT, that blocks interaction with KL do not progress beyond primordial follicle stage.

Question 36

Primary–> Preantral growth

Answer 36

FOXL2 is another transcription factor (forkhead transcription factor family) ⇒ is involved in granulosa cell proliferation
Woman with mutations of the gene FOXL2 -/- have Type 1 BPES and POF (premature ovarian failure). So they get primary follicles but no progression of follicles to secondary stage
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

Question 37

Genes involved in Preantral Follicle Progression

Answer 37

GDF9 and BMP-15 both belong to the TGFb superfamily. (also includes AMH inhibins and activins).
These genes are species different:
GDF9 k/o will not have progression beyond primary stage
BMP-15 k/o are sub-fertile, but BMP-15 mutated sheep profound infertility
Some women with POF have mutations of BMP-15 & GDF-9 ⇒ but israre

Connexin gene
Cx37 gene is responsible for encoding the connexin proteins that connect the granulosa cells to the oocyte. Cx43 connects the granulosa cells to each other.
Mutations in gene encoding Cx37 results in infertility (disrupts both GC and oocyte)

Insulin (IGF-1,-II ) and androgens also have profound affects on follicles – can stimulate granulosa cell proliferation and follicle growth. BUT too much can be detrimental.

Neurotrophins – nerve growth factor. NTF5 and BDNF
Know that there is a neurotrophin receptor on the oocyte.

Question 38

SUMMARY (up to primordial growth genes)

Answer 38

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 → but no correlation in domestic animals/humans

Question 39

Androgens and Early Follicle growth

Answer 39

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)
So in increases the activation and initiation of follicle growth.

Monkeys treated with androgens have more primary follicles and increased FSH receptors on the follicles

Inhibiting AR in bovine ovaries prevents primary to secondary follicle transition (Yang & Fortune, 2006, BoR)

Is there a human equivalent? Seen in PCOS
Know that androgen receptor is present on pre antral follicles.
No AR on primordial follicles, but once they start to grow the amount of AR is increasing.
Women with PCOS have high levels of androgens, they have an increased number of primary and growing follicles.

Question 40

The pre antral stage is gonadotrophin independent. We know this because:

Answer 40

You have Physiological & pathological states where circulating gonadotrophin levels are low but you still see follicular growth:
e.g. physiological= infancy, pregnancy
FSH levels will be low but you can see pre antral follicles at all stages
e.g. pathological= Kallman’s syndrome, anov. PCOS
Deficit in FSH, but yet will sill see pre antral follicles at all stages in ovaries
FSHß & FSHR k/o mice have normal preantral growth
inactivating mutations of the FSH receptor
still see follicle growth to antral stage, but less follicles
FSHR have been found on primary stage follicles
may not be coupled to 2nd messenger system

Whilst FSH is not essential for preantral growth, but low tonic levels keep the follicle healthy and in a better state to reproduce i.e. Healthy pool of selectable follicles.

Question 41

Can the ovary form new oocyes?

Answer 41

Women are born with their complete stock of follicles, their ovarian reserve.
Once this is depleted women menopause.

Paper in 2004, indicated that under the ovarian surface epithelium, there was a germ line stem cell niche which was capable of regenerating the ovary with follicles if it got depleted. BUT there are no consistent markers

Question 42

SUMMARY

Answer 42

Primordial follicles are resting in meiotic arrest, they have flattened granulosa cells. It is thought that this arrest is being maintained by certain inhibitory factors. And once these factors are overcome it can activate various stimulatory factors causing growth in size, and the flattened granulosa cells become cuboidal.
After progression is determined by various genes, when they enter into the menstrual cycle there are also activins, inhibins, gonadotrophins, AMH.