Preantral folliculogenesis

Question 1

what has to be achieved to reproduce?

Answer 1

· Differentiation into male or female.
· Sexual maturation from an infant to a sexually mature person.
· Production, storage of eggs and release of sufficient supply of eggs and sperm.
· Correct number of chromosomes in eggs and sperm - both must be haploids to produce a diploid when they come together.
· Eggs and sperm have to meet - need the right environment to aid transport.
· Creation of new individual with genes from both parents - genetic variability during meiosis (crossing over and random alignment).

Question 2

what are primordial germ cells (PGCs)?

Answer 2

· Cells that will become eggs or sperm are called PGCs.
· PGCs are first identifiable in the yolk sac of the developing foetus at around 3 weeks.
· PGCs undergo many cycles of mitosis:
o They migrate to the genital ridge in the foetus.
o The genital ridge becomes the gonads, therefore, PGCs are bipotential - they can become testes and ovaries.
o Further differentiation of the PGC depends on the development of the gonads e.g. ovaries or testes.

Question 3

what are oocytes?

Answer 3

· If PGCs migrate to the area which will become the ovaries, they become oocytes - these increase in number via mitosis. When it migrates to the ovaries and the area has received signals to produce ovaries, the PGCs become oogonia.
o Oogonia are egg-precursors, diploid and multiply by mitosis.

Question 4

what happens when oogonia enter the first stage of meiosis?

Answer 4

no more divisions occur and they become primary oocytes. The primary oocytes remain in the first phase of meiosis until it is ovulated or dies.

Question 5

give an overview on mitosis

REFER TO NOTES

Answer 5

1. Chromosomes are replicated during S-phase of the cell cycle–> 2 chromatids attached by a centromere.
2. Chromosomes align one behind the other–> spindle fibres form, attach to the chromatids and contract = pull apart the chromatids, breaking the centromere = 1 chromatid to each centriole–> forms a new cell with original number of chromosomes.

Question 6

give an overview on meiosis

REFER TO NOTES

Answer 6

1. Chromosomes replicate similar to mitosis, but instead they align up next to each other.
2. During meiosis 1) chromosomes pulled apart to the centrioles.
3. During meiosis 2) chromatids pulled apart.

The primary oocytes remain arrested in meiosis 1 until you have a surge in LH and ovulation occurs. Older eggs are more problematic as their chromosomes have been aligned for much longer on the spindles.

Question 7

where are primary oocytes localised?

Answer 7

Primary oocytes are packed into the outer layer of the ovary, in the cortex

Question 8

what are primary oocytes surrounded by?

Answer 8

· protective cells and protective layers, never on their own.

· In the foetal ovary, the surrounding cells condense around the oocyte and differentiate into the granulosa cells—> secrete an acellular layer called the basal lamina. The whole structure is known as the primordial follicle.

Question 9

what is folliculogenesis?

Answer 9

· Folliculogenesis is the growth and development of follicles from the earliest resting stages as laid in the foetus through to ovulation.
· Most of the follicles in the ovary are not growing - after puberty, only a few grow each day out of the resting phase, as a continuum.
· As the follicles start to grow, the granulosa cells multiply and the oocytes secretes another protective acellular layer called the zona pellucida, which stays attached after ovulation. Therefore, sperm have to get through the zona pellucida to fertilise the oocyte.
· Once growth of the follicles has started, a second layer of cells then differentiate around the granulosa cells, the theca cells.

Question 10

Describe follicle growth

Answer 10

· The factors controlling initiation of growth and the early stages are unknown - it’s not hormonal, mainly autocrine and paracrine. The follicles and surrounding follicles release factors - these can be inhibitory or stimulatory in nature.

EARLY GROWTH = FSH-independent, driven by these local factors.
LATE GROWTH = FSH-dependent.

· Therefore, in FSH-deficient patients (pregnant or on contraceptives) or those with mutations of FSH receptors will still get continual early growth of the follicles, but then they die - this is why contraceptives don’t preserve your fertility, you still get continual early growth.

· As follicles start to grow, they rapidly increase in diameter and granulosa cell divisions increase BUT gaps begin to form in these granulosa cell layers —>known as antrums - these fill with follicular fluid.

Question 11

what is one way we can we characterise follicle growth?

Answer 11

We can characterise follicle growth: 1) absence of antrum 2) presence of antrum.

Follicles with an antrum are known as antral/secondary follicles.

Question 12

Describe antral development

Answer 12

Stage 1) antrums form and get filled with follicular fluid.

Stage 2) fluid spaces fuse together and expand.

Stage 3) fluid separates the granulosa cells, into the mural and cumulus GCs.

Stage 4) can grow up to 20mm.

As the follicle grows, the oocyte is displaced to one side.

Question 13

how can follicles be visualised?

Answer 13

Follicles can be visualised using ultrasound, but it’s only the follicles which are larger than 1-2mm which can be visualised. People think contraceptives preserves fertility but actually what happens is, the follicles are arrested in the early growth phase which is FSH-independent, therefore they’re too small to be picked up by ultrasound but they’re still there.

Question 14

describe follicle initiation and follicle recruitment

Answer 14

· A cohort of early follicles leaving the resting pool and grow continuously–> follicle initiation.

· They will NOT continue to grow unless they reach the size at which they respond to changes in FSH that occur in the menstrual cycle–> follicle recruitment.

· The human pelvis is only designed to carry a single foetus, therefore, from the group that is recruited, only one will be selected for ovulation - this is the dominant follicle.

Question 15

what do we mean when we say “many follicles to one”?

Answer 15

· Majority of the eggs will die during reproductive life, even before puberty, but majority will die during puberty.
· There are thousands of primordial follicles most die through atresia, and very few make it into the menstrual cycle.
· But only one follicle is ovulated (dominant follicle) which has more FSHr so it grows even during low levels of FSH and the FSHr has a more efficient downstream IC cascade.

Question 16

describe the antral follicle

Answer 16

· The follicle is avascular until when the theca is formed–> forms vasculature which allows blood borne influences to act on the follicles e.g. hormones, cortisol, insulin, etc. This suggests a lot of stuff comes into play during the later stages of antrum growth, in the presence of theca cells.

Question 17

follicles produce steroids. Describe where the receptors are and which cells produce which steroid

Answer 17

· Theca cells have LHr–> produces progesterone and androgens which goes into circulation or into granulosa cells.
· Granulosa cells have FSHr–>stimulates aromatase which converts the androgens into oestrogen (allows granulosa cells to continue proliferating).
Known as the 2-cell, 2-gonadotrophin theory.

· The dominant follicle develops LHr on the granulosa cells so it starts to produce progesterone.
· Oestrogen and progesterone feedbacks to the hypothalamus and anterior pituitary–> decreases levels of FSH and LH.

Question 18

give an overview on preantral follicle stages

Answer 18

A single layer of flattened granulosa cell surrounds the oocyte. Growth is determined by changing shape of granulosa cell and increased proliferation of granulosa and oocyte.

Flattened granulosa form cuboidal (transitional) so a single layer of cuboidal granulosa cells around the oocyte, at this point it is considered primary.

Multiplication of layers, ZP forms around oocyte and other cells in the ovarian cortex, which condenses around the follicle and form the theca (secondary), a defined basement membrane separating it from granulosa. It is metabolically active even though it is in meiotic arrest, stops growing at the end of the preantral stage.

Question 19

how can we investigate folliculogenesis?

Answer 19

1. Animal models - mono-ovulatory (e.g. sheep and monkeys) and poly-ovulatory (e.g. rabbits and mice, however produce litter which is not an accurate representation of reproduction).
2. Introduce defined mutations into embryonic stem cell by injecting into blastocysts, breed them to be homozygous for the mutations and then examine the phenotype.
3. Genotype/phenotype associations in naturally occurring mutations or from knock-out mice.
4. Culture of whole ovaries/slices/biopsies/large follicles/small follicles/cells.
   - Very difficult in human because of limited supply of tissue
   - Primary cells difficult to obtain; granulosa cell line but no suitable theca cell line (inherent or induced mutations to keep growing them)

Question 20

describe the formation of oocytes

Answer 20

1. As the PGCs divide, they form nests (cytoplasmic bridges between dividing oocytes). The purpose of this is to exchange organelles between the oocytes e.g. mitochondria and ER.
2. Eventually, you get breakdown of the nests to allow the individual oogonia to form primordial follicles.
3. DAZL + Retinoic acid are thought to be involved in nest breakdown.
   - Retinoic acid is thought to regulate entry into meiosis (from mitosis).
   - DAZL expression increases before meiosis at 9 - 14 weeks of gestation. DAZL K/O mice germ cells don’t develop past the PGC, they don’t go onto form primordial follicles. In humans, DAZL mutations associated with sub-fertility.

Therefore, this highlights the importance of both of these genes for entry into meiosis.

Question 21

what is the difference between humans and mices (regarding the formation of cytoplasmic bridges)?

Answer 21

In humans: PGCs divide along the ridge and colonise the ridge. Cytoplasmic bridges form between the dividing oocytes to form nests of germ cells. They then enter into meiosis + nests are broken down = primordial follicles are formed. Therefore, we are born with all the primordial follicles.

In mice: This process doesn’t occur until after birth - made it problematic to research this area.

Question 22

Describe the formation of primordial follicles

Answer 22

The germ cell nests are broken down and the surrounding somatic cells (pre-granulosa cells) invade to surround oogonia to form primordial follicles. This process is highly regulated through transcription factors e.g. FIGLA, Nobox and Activin βA.
o FIGLA is important for primordial follicle formation as female FIGLA K/O mice develop no primordial follicles
o Activin βA expression decreases just before nest breakdown. Downstream of Activin βA is TRKβ receptor, which if K/O → loss of oocytes = “streak” ovaries.

· Co-ordination of signalling pathways: KIT, Notch and TGFβ, and hormones is also required (FSH promotes, while oestrogen and progesterone oppose formation of primordial follicles).

NOTE: As these primordial follicles have entered meiosis, they are now under arrest.

Question 23

what is the follicle reserve?

Answer 23

· Once all the primordial follicles are formed, the entire pool of germ cells available during reproductive life of the female = ovarian reserve (varies from 35,000 - 2.5million follicles).

Question 24

Just before birth, there is a massive loss of follicles. Why does this occur?

Answer 24

1. Failure of mitosis/meiosis (forming defective chromosome spindles).
2. Unrepaired DNA damage.
3. Insufficient pre-granulosa cells (naked oocytes).
4. Degeneration of oocytes during nest breakdown + follicle formation.

= “Germ cell selection”, allows us to select oocytes of the highest quality.

Question 25

how can we predict the ovarian reserve?

Answer 25

Histological counting: take ovarian sections, stain, count the follicles, then estimate

mathematical modelling

Primordial follicles are located in the avascular ovarian cortex (move to the vascular medulla for growth).

Question 26

Describe zona pellucida formation

Answer 26

· ZP is a thick extra-cellular coat which separates the oocyte from the surrounding GCs.
· ZP formation is a marker of follicle/oocyte growth.
· Human follicles made up of four ZP proteins: ZP1, ZP2, ZP3, ZP4
o Permeable to large macromolecules.

Question 27

Describe the primordial to primary preantral follicle transition

Answer 27

Controlled and very slow process.

1. Change in GC size and morphology.
2. Massive increase in oocyte growth.
3. As the follicles begin to grow, they migrate from the collagen-rich ovarian cortex towards the ovarian medulla where the ECM is of lower density and there is a rich blood supply.

Question 28

describe preantral follicle structure

refer to notes

Answer 28

Oocyte is in meiotic arrest, but actively synthesising mRNA and proteins as the egg contributes most of the proteins needed for the early stages of pre-implantation development to occur.
· Theca layer gets differentiated into Theca Interna (highly vascularised) + Theca externa.
· Intracellular communication exists between the oocyte and GCs via gap junctions that penetrate through the ZP layer. Connexin 37 allows communication between the oocyte and GCs, while Connexin 43 allows communication between the GCs.

Question 29

what is the importance of the ECM?

Answer 29

The ECM is important to regulate growth of the oocyte, through substances moving in and out of the oocyte.

· ECM consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides.
· ECM turns over and remodelled during folliculogenesis to allow for growing follicle.
· The ECM regulates follicle growth, through interactions between the GCs and oocyte.
· Mechanical stimuli are communicated rapidly throughout follicle as various cell types are physically connected e.g. via connexins.

Question 30

why have 3D-culture systems been developed?

Answer 30

The important message is, we need the surrounding ECM and environment to be able to successfully grow follicles in vitro e.g. hydrogel matrix needed to support follicle, to maintain the contact between the oocyte and surrounding GCs.

· All the components of the mice media that were needed to grow a mice follicle all the way to pre-ovulatory, fertilise it, then continue growing it until it forms an embryo, were identified.
· While in humans + monkeys, they’ve identified two key components - collagen + alginate (product of seaweed).
o Alginate gels were able to produce antral follicles from secondary follicles in humans and monkeys.

Question 31

What are the 3 possible fates of primordial follicles?

Answer 31

1. To remain quiescent and die out directly at dormant stage.
2. To begin development but arrest and later undergo atresia.
3. To develop, mature & ovulate.

Question 32

what are the 2 main ideas regarding primordial follicle growth initation?

Answer 32

1. Initiation is regulated by loss of an inhibitor:
   o Resting follicles under constant inhibitory influence to remain dormant. Removal of inhibitory factor = growth. Thought this inhibition was coming from the surrounding stroma.
2. Initiation is regulated by stimulatory factors:
   o Thought these stimulatory factors are coming from the microenvironment (other follicles, stromal cells) and/or blood.
   o Gradient of diffusion from centre to periphery.

Combination of both - inhibition & stimulation”Production-line” hypothesis: those that enter meiotic arrest first in the fetal ovary will initiate growth first.

Question 33

what are the genes thought to be implicated in primordial to primary antral follicles?

Answer 33

· FOXO3 (TF) is thought to maintain the primordial follicle in arrest. It is bound to cyclin D2 in the nucleus of the GCs and keeps it in arrest.

· This arrest is overcome by KIT ligand being secreted from the GCs which binds to cKIT on the oocyte —> activates PI3 pathway:

• PI3 kinase is activated and converts PIP2–>PIP3, which activates AKT, which then phosphorylates FOXO3.
• Phosphorylated FOXO3 moves out of the nucleus, releasing cyclin D2 = cell cycle progression.

· Upstream of FOXO3 is PTEN which facilitates conversion of PIP3 to PIP2 (opposes action of AKT).
· AMH + Stroma-derived factor (SDF-1) from other follicles can also keep the primordial follicles arrested.

THUS, WE HAVE AUTOCRINE + PARACRINE WHICH PLAYS A ROLE.

Question 34

what are the factors that activate primordial follicles?

Answer 34

Granulosa-derived factors (stimulatory):
· Kit ligand secreted from GCs –> activates PI3 pathway –> inactivates FOXO3 through phosphorylation (unbinds Cyclin D2)

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 KIT ligand do not progress beyond primordial follicle stage.

Question 35

what represses primordial follicle activation?

Answer 35

Oocyte-derived factors (inhibitory):

1. PTEN (TSG)–> inhibits AKT/PI3K signalling. Loss of PTEN = global activation of primordial follicles.
2. FOXO3 (TF)–> inhibits follicle activation. FOXO3 K/O = global activation of primordial follicles.
3. SDF-1 (stromal-derived factor) chemokine–> inhibits follicle activation in autocrine/paracrine fashion.

Granulosa-derived factors (inhibitory):
AMH–> acts in paracrine fashion to inhibit primordial follicle initiation. AMH K/O have less stock of primordial follicles & more growing follicles.

Question 36

what factors are involved in preantral follicle progression?

Answer 36

· GDF9, BMP-15 + AMH - all part of the TGFb family.

· Insulin, IGF-1/2 + Androgens drive preantral follicle growth.

Question 37

what factors are involve in the progression of primary follicle growth?

Answer 37

Oocyte-derived factors:
1. GDF9 (growth differentiation factor-9). K/O = no progression beyond primary follicles.
2. BMP-15 (bone morphogenetic protein-15). K/O mice = sub-fertile. But K/O sheep = profoundly infertile.
3. Connexin 37 gap junction protein. K/O = failed Folliculogenesis.
Granulosa-derived factor:
· Connexin 43 gap junction protein. K/O = deficient in germ cells and no progression beyond 1°/2° stage.
· FOXL2 (TF) which is responsible for GC multiplication.
Extra-follicular factors:
1. Insulin & IGF-1 & IGF-II –> increase primary stage follicles in cultured human ovarian cortex.
2. NGF (Nerve Growth Factor). K/O ↓number of growing follicles → no correlation in domestic animals/humans.

Androgens - required for follicle progression (from primary to secondary).

Question 38

How do androgens play a role in early follicle growth?

Answer 38

· Testosterone rapidly increases intra-oocyte PI3K/Akt/FOXO3 pathway in mouse follicles → increasing >2-fold ratio of primary : primordial follicles (Yang et al., 2010).
· Monkeys treated with androgens have more primary follicles and increased FSH receptors.
· Inhibiting androgen receptors in bovine ovaries prevents primary to secondary follicle transition (Yang & Fortune, 2006).

Androgens are required for follicle progression; increase in androgen receptors

Question 39

what is the importance of gonadotrophins during basal follicular growth?

Answer 39

While FSH is not essential for preantral growth, low tonic levels keep the follicle healthy and in a better state to respond e.g. healthy pool of selectable follicles.

· Physiological & pathological states where circulation gonadotrophin levels are low still see follicular growth:

• e.g. physiological= pre-pubertal + pregnancy.
• e.g. pathological= Kalman’s syndrome + anovulatory PCOS.

· FSHb & FSHR K/O mice have normal preantral growth.
· Inactivating mutations of the FSH receptor = follicle growth to antral stage, but less follicles. Therefore, this suggests that gonadotropins are not needed for preantral growth.
o (Aittomaki K, 1996; Tapanainen, 1998; Touraine, 1999).

· FSHR have been found on primary stage follicles… may not be coupled to 2nd messenger system.
o (Otkay et al, 1997; Rice et al, 2007).