Preantral Folliculargenesis

Question 1

Where do eggs come from? - 3-4wks

Answer 1

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

Question 2

Where do eggs come from? - 5-6wks (2)

Answer 2

1) Mitotically dividing PGCs migrate along dorsal mesentery of hind gut to colonise genital ridge

2) chemotactic substance secreted by ridge to attract PGCs. (maybe Kit ligand (KL) as cKit receptor present on PGCs surface)

Question 3

Formation of oocytes and follicles (4) - nest form + BD

Answer 3

1. During migration: PGC undergo epigenetic re-programming (genome-wide DNA de-methylation + erasure of genomic imprinting)
2. Cytoplasmic bridges b/w mitotically-dividing oocytes = form syncytia/“nests” (Role? Maybe the exchange of organelles eg. ER and mitochondria)
3. Retinoic Acid (biologically active variant of Vitamin A) derived from somatic cells drives germ cell entry into meiosis (from mitosis)→ induces Stra8 gene expression in oogonia
4. Germ cell nest breakdown -> form primordial follicle

Question 4

Formation of primordial follicle (4)

Answer 4

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

2) PF formation reg. through
complex network of molecular signals b/w oocytes + somatic cells.

» Numerous transcription factors identified in mice & human e.g. FIGLA, Nobox & Activin βA

» Co-ordination of signalling pathways i.e. KIT, Notch + TGFβ

Question 5

Ovarian reserve birth to Menopause (5)

Answer 5

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)

b/w birth + puberty - lose some follicles.

After puberty: some enter menstrual cycle, die off, never enter etc.

Menopause: works as a continuum whole cycle stops due to depletion of all follicles

Question 6

Why is there a massive loss of oocytes and follicles just before birth? (5)

Answer 6

faulty (checkpoints) - if the follicle hasn’t formed properly (eliminating ‘the bad eggs’)

because of:
- failure of mitosis/meiosis involving defective chromosome spindle function
- Unrepaired DNA damage during egg/follicle formation
-Insufficient pre- GC’s = naked oocytes which degrade
- Degeneration of oocytes during nest breakdown and follicle formation.

Question 7

Gross Anatomy of the Ovary- fallopian/uterine tube + ovary (2)

Answer 7

image
1)fallopian tubes w/ fimbriae (relatively free for picking up ovulated oocyte) = adhesions from surgery/ fibroids = cause of infertility (not having open/patent tubes)

2) ovary - blood vessels @ medulla (follicles start at cortex + migrate into medulla (middle) and then back out for ovulation + release)

Question 8

Stages of follicle growth named (6)

Answer 8

1) oocytes in mitotic arrest (gonad. indepen.)

2) Initiation of growth - toward preantral stage (gonad. indepen.)

3) Recruitment of foll- towards antral (gonad depe.)

4) Selection of dom foll (gonad depe.)

5) ovulatory stage (gonad depe.)

current follicle started this process 3 cycles ago.

Question 9

Preantral Follicle Stages - activation of resting follicle (Gougeon classification) (6)

Answer 9

1)Primordial/Resting follicle: flattened GC’s + oocyte= meiotic arrest

2) Transitional follicle: increase in cuboidal GC’s (mix of flat + cube) + oocyte= meiotic arrest

3) Primary follicle: all cube single layer + oocyte= meiotic arrest but cytoplasm starts to grow + sec. ZP proteins

4) Secondary follicle starts: starts to increase GC’S + formation of ZP(mark of growth) + precursor theca cells condense around follicle

5) Secondary follicle: 2 layers of GC’s = second. + formation of Theca, basement membrane sep. GC from Theca

6) Multilaminar follicle: then multiple layers build up

Question 10

Alternative Follicle Classifications (3)

Answer 10

others may say:
1) all preantral follicle stages = class 1/ pre-antral/ primary
2) two layers = secondary
2) anything w/ antrum = antral follicle/tertiary

Question 11

Primordial-Primary Transition Morphological changes (4)

Answer 11

1) Change in gc’s: no. + shape ~15 cuboidal GC’s

2) Massive increase in oocyte growth & activity- will grow until end of preantral stage (active but in meiotic arrest)

3) Controlled & very slow process

4) As follicle grow they move from collagen-rich, avascular cortex to perimedullary/medullary zone of ovary where ECM is of lower density
and rich blood supply

Question 12

What is the Zona Pellucida? (5)

Answer 12

• ZP is a thick, extra-cellular coat separating the egg from surrounding gc
• marker of oocyte/ follicle growth
• Human follicles made up of four ZP proteins:
  ‒ ZP1, ZP2, ZP3, ZP4

‒ Permeable to large macromolecules (b/w ZP + GC)

‒ Follicle extensions continue through it

Question 13

What is the role of the ZP in fertilisation? (2)

Answer 13

• it helps with the binding of the sperm to ZP
• • to avoid polyspermia

Question 14

Preantral Follicle Structure - allowing 2 types of communication (2)

Answer 14

• strong/Intracellular communication b/w oocyte and GC via gap junctions (made of proteins) that penetrate ZP

-Also communication via connexins i.e.Cx43 b/w GC and Cx37 b/w GC & oocyte

Question 15

Location of the Primordial Follicle - avascular cortex so need… w/o moving (3)

Answer 15

-Primordial follicles located in ovarian cortex & have no blood supply.

-Primordial follicle (arrested in dictyate stage of meiosis) = Meiotic arrest

Basal lamina around the follicle creates microenvironment for gc & oocyte (in local environment) i.e not in contact with other cells in the ovary

Question 16

What are the 3 Fates of the Primordial follicle? (3)

Answer 16

»To remain quiescent and die out directly at dormant stage

»To begin development - but arrest and later undergo atresia

»To develop, mature & ovulate

Question 17

What causes initiation of follicle growth? - 2 main ideas (+mix + others) (3)

Answer 17

1) initiation is reg. by loss of an inhibitor:
‒ Resting follicles under constant inhibitory influence (local paracrine/autocrine factors) to remain dormant from adjacent follicles or the ovary

2) initiation is reg. 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
- default = inhibition (something is keeping it dormant)
- loss of inhib. + stim factors overcome inhib.
- as you age and lose more follicles = loss becomes accelerated

4) - “Production-line” hypothesis: those that enter meiotic arrest first in foetal ovary, will initiate growth first
- Also dep. on size of PF pool and ratio at which enters the growing pool

Question 18

What’s the importance of Extracellular Matrix (ECM)? (4)

Answer 18

*ECM consists of collagen, lamimin, fibronectin, proteoglycans &
polysaccharides

*ECM turns over(changes conc.) + remodelled during folliculogenesis = grow + movement of follicle

*important communicator w/ follicles = May reg. follicle growth esp. interactions between gc & oocyte (DIRECT)

*Mechanical stimuli are communicated rapidly throughout follicle as various cell types are physically connected e.g. via connexins (INDIRECTLY)

Question 19

Genes & Primordial Follicles growth reg. stages (4)

Answer 19

1)Nest breakdown = Primordial Follicle form.:
FIGLA (human & mice)
Zona Pellucida 1-4 (human & mice), Activin βA & BDNF (human)
AMH?(mouse)
Oestrogen? (baboons, human)

2)Endocrine disruptors (substances from plastic):
BPA (Bisphenol A),
Genistein, DES(diethylstilbestrol) –
inhibits nest breakdown

3)Primordial Follicle Maintenance/Repression:
PTEN, FOXO3 (forkhead family), AMH (Anti-Mullerian Hormone), SDF-1 (stromal derived factor)

4)Primordial Follicle Activation:
KIT ligand & KIT receptor (cKIT), FOXL2, NOBOX (newborn ovary homeobox), SOHLH 1&2 (transcription factors)

Question 20

Primordial → (x) → Primary Follicle Activation - pharm (2)

Answer 20

1)primordial follicle:
- @ growth rest = not prog. w/ cell cycle (because of FOXO3)
- GC increase their Ccnd2 protein = bind + repress growth cells = cell cycle arrest
- Hippo signal. keeps YAP protein Phosphorylated

2)Transitional follicle:
- (TGFbeta sig.) activate mTOR = phosp. SMAD3 = removal of SMAD3 = tF’s bind + cell cycle inhib. removed
= growth begins by activation of Myc
- inactiv. of hippo sign. = YAP dephos. , moves into nucleus GC’s + aids process
-KITL binds to cKit (r on oocyte) = activates PI3K pathway (PIP2->PIP3 (w/PTEN) = activ. Akt = Phos. FOXO3
- FOXO3 out of nucleus = Cell cycle activated + growth occurs

Question 21

Primordial Follicle Repression evidence (7)

Answer 21

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 = acts in paracrine fashion to inhibit primordial follicle initiation
» k/o have less stock of primordial follicles & more growing follicles

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 22

Primary → Preantral Growth: FOXL2 (2)

Answer 22

-FOXL2 (forkhead tf) ⇒ granulosa cell proliferation (necessary in prog. from primary to preantral growth)

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

Question 23

BPES: (4)

Answer 23

-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 24

Preantral Follicle growth + progression: GDF9 + BMP-15, connexins, GF’s, Neutrophins - species differences (7)

Answer 24

models:
-in GDF9 k/o mice = no prog. beyond primary
-BMP-15 k/o mice = sub-fertile
-BMP-15 mutated sheep = profound infertility

-some women w/POF have mutations of BMP-15 + GDF9 = rare = menopause early (20/30s)
-mutation in Cx37 gene = infertility (disrupts GC + oocyte)
-Insulin, IGF1.II, androgens = early follicle growth
_Neurotrophins (NTF5 +BDNF) = r on oocytes

Question 25

Progression of Primary Follicle Growth (6)

Answer 25

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

Question 26

Do androgens play a part in early follicle growth? - animals (3)

Answer 26

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

» monkeys treated with androgens have more primary follicles + increased FSHr

» Inhibiting AR in bovine ovaries prevents primary to secondary follicle transition

Question 27

Is there importance of gonadotrophins during basal follicular growth?

Answer 27

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 pre-antral 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

Question 28

Can the ovary form new oocytes? (3)

Answer 28

*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

*Recent single-cell RNA-seq could not detect any oogonial stem cells