preantral folliculogenesis

Question 1

Folliculogenesis - overview

Answer 1

Question 2

Where do eggs come from?

Answer 2

At 3-4 week human embryo

Epiblast cell in yolk sac at base of allantois differentiate into PGC (primordial germ cells)

Question 3

What happens at 5-6 week human embryo?

Answer 3

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

Question 4

cytoplasmic bridges between mitotically-dividing oocytes to form..

its role?

Answer 4

syncitia or “nests”

Role: the exchange of organelles eg. ER and mitochondria

Question 5

Retinoic Acid (the biologically active variant of Vitamin A) has been identified as

Answer 5

key extrinsic regulator of germ cell entry into meiosis

Question 6

DAZL expression increases (when?)

Answer 6

before meiosis at 9-14 weeks gestation.

DAZL expression in mice - mice germ cells do not develop past PGC

Question 7

Primordial follicle formation occurs (in humans - before or after birth?)

Answer 7

before birth in humans.

Mice: Primordial follicle formation occurs after birth

Question 8

How are Primordial Follicles formed?

Answer 8

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

Question 9

Primordial follicle formation is regulated through:

Answer 9

Numerous transcription factors identified in mice & human:
FIGLA (no figla no PF)
Nobox
Activin βA
Co-ordination of signalling pathways: KIT, Notch and TGFβ
Hormones – FSH promotes E2 and Progesterone oppose

Question 10

Which receptor is downstream of Activin βA?

Answer 10

TRKβ receptor

Question 11

(Activin βA)
If TRKβ receptor is knocked out this can lead to..?

Answer 11

-> loss of oocytes
->“streak” ovaries

In males, you can get testes with no sperm

Question 12

Activin βA expression decreases just before..

Answer 12

Nest breakdown

Question 13

ovarian reserve

Answer 13

Once formed primordial follicles represent the entire pool of germ cells available during reproductive life of the female

Question 14

“Germ Cell Selection” theory is the reason as to why not all primordial follicles formation survive

Answer 14

Ovaries are to select oocytes of highest quality

Question 15

Answer 15

Question 16

Answer 16

One layer of Cuboidal granulosa cells - primary follicle

Secondary follicle - two layers of granulosa cells, theca cell formation and basement membrane formation

Question 17

Primordial-Primary Transition Morphological changes

Answer 17

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

Question 18

Zona Pellucida (ZP) Formation is a marker of..

Answer 18

ZP formation is a marker of follicle/oocyte growth

Question 19

What is the. Zona Pellucida (ZP)

Answer 19

ZP is a thick extra-cellular coat separating the egg from surrounding gc

Human follicles made up of four ZP proteins:
ZP1, ZP2, ZP3, ZP4

Permeable to large macromolecules

Follicle extensions continue through it

Question 20

How does Intracellular communication between oocyte and GC take place?

Answer 20

Via gap junctions that penetrate ZP

and Connexins (another card)

Question 21

Which specifc connexin between Granulosa cells and which between GC and oocyte?

Answer 21

Cx43 between GC and Cx37 between GC & oocyte

Question 22

Primordial Follicle has 3 possible fates:

Answer 22

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

2 main ideas:
1) initiation is regulated by loss of an inhibitor:
Resting follicles under constant inhibitory influence (local paracrine/autocrine factors) to remain dormant.

2) initiation is regulated by stimulatory factor/s:
From the microenvironment (other follicles, stromal cells) and/or blood
Gradient of diffusion from centre to periphery

3) Combination of both – inhibition & stimulation:
As inhibitory factors decline then you get an increase in the effects of stimulatory ones. It is also dependent on the size of the PF pool (ovarian reserve) and the ratio at which it enters the growing pool.

Question 24

“Production-line” hypothesis:

those that enter meiotic arrest first..

Answer 24

..those that enter meiotic arrest first in foetal ovary, will initiate growth first

Question 25

Extracellular Matrix (ECM) consists of:

Answer 25

consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides

Question 26

Importance of Extracellular Matrix (ECM)

Answer 26

ECM turns over and remodelled during folliculogenesis to allow for growing follicle

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 27

Genes & Primordial Follicles

Nest breakdown & Primordial Follicle Assembly

Answer 27

FIGLA (human & mice)
Zona Pellucida 1-4 (human & mice)
Activin βA & BDNF (human)
AMH? (mouse)
Oestrogen? (baboons, human)

Question 28

Genes & Primordial Follicles

Primordial Follicle Activation

Answer 28

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

Question 29

Primordial Follicle Maintenance/Repression

Answer 29

Oocyte-derived factors (inhibitory):
- PTEN (tumour suppressor gene) –> inhibits signalling by Akt/PI3K signalling pathway.

• FOXO3a (transcription factor) -> also part of PI3K and restrains follicle activation
• 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

Question 30

Primary → Preantral Growth

FOXL2

Answer 30

FOXL2 (forkhead transcription factor) ⇒ granulosa cell proliferation

NO FOXL2
–> have Type 1 BPES and POF (premature ovarian failure) – no progression of follicles to secondary stage

Question 31

Factors relevant in progression of Primary Follicle Growth

Three groups: oocyte derived, granulosa derived, Extra follicular

Answer 31

Oocyte-derived factors:
- GDF-9 (growth differentiation factor-9)
- BMP-15 (bone morphogenetic protein-15)
- Cx37 (connexin 37) gap junction protein (between oocyte & gc)

Granulosa-derived factor:
- Cx43 (connexin 43) gap junction protein (between gc & gc)

Extra-follicular factors:
- Insulin & IGF-1 & IGF-II –>
increase primary stage follicles in cultured human ovarian cortex
NGF (Nerve Growth Factor)

Question 32

Androgens & Early follicle growth: Do androgens play a part?

Answer 32

Testosterone rapidly increases intra-oocyte PI3K/Akt/FOXO3 pathway in mouse follicles → increasing >2-fold ratio of primary:primordial follicles

Inhibiting AR in bovine ovaries prevents primary to secondary follicle transition

Question 33

Importance of gonadotrophins during basal follicular growth?

Answer 33

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

• 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

Question 34

Summary of Preantral folliculogenesis

Answer 34