pre-antral follciulogenesis Flashcards
How can we investigate folliculogenesis
Animal models – mono-ovulatory/poly-ovulatory
Genotype/phenotype associations in naturally occurring mutations or from knock-out mice
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
Where do eggs come from (embryology)
3-4 week human embryo
Epiblast cell in yolk sac at base of allantois differentiate into PGC (primordial germ cells)
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
Describe the formation of primordial follicles and oocytes
cytoplasmic bridges between mitotically-dividing oocytes to form syncitia or “nests”
» Role? Maybe the exchange of organelles eg. ER and mitochondria
Retinoic Acid (the biologically active variant of Vitamin A) has been identified as key extrinsic regulator of germ cell entry into meiosis
DAZL expression ↑before meiosis at 9-14 weeks gestation. DAZL k/o mice germ cells do not develop past PGC, DAZL mutations in human assoc. sub-fertility
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 k/o female mice 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, contrast with male as can have testes with no sperm
Co-ordination of signalling pathways: KIT, Notch and TGFβ
Hormones – FSH promotes and E2 and P oppose
What is ovarian reserve
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)
Describe germ cell selection
After meiotic prophase 1, the follicles go in meiotic arrest, a layer of granulosa cells form and they become primordial follicles
However if there is inadequate follicle formation and DNA damage, these follicles are not selected and die
What are the 3 stages of follicle growth
Initiation- gonadotrophin dependent (resting and pre-antral)
recruitment- gonadotrophin independent (antral stage)
selection-gonadotrophin independent (ovulatory)
Describe the pre-antral follicle stage
Primordial
Transitional (granulosa cells grow)
Primary (granulosa become cuboidal)
Secondary follicle (precursor theca cells form around cortex, zona pellucida and basement membrane form . Theca formation begins here and is gonadotrophin independent
ZP is around inner region, basement membrane separates theca and granulosa cells
What is the technique to isolate pre-antral follicles
Elective Caesarean section patients consented for ovarian cortical biopsy
Digested in Collagenase +DNAse for <1hr
Follicles dissected out of stroma & placed individually in drops of media
Describe the primoridal to primary transition stages
1) Change in granulosa cells (~15 cuboidal granulosa cells)
2) Massive increase in oocyte growth & activity
3) Controlled & very slow process
Describe zona pellucida formation
ZP is a thick extra-cellular coat separating the egg from surrounding gc
ZP formation is a marker of follicle/oocyte growth
Human follicles made up of four ZP proteins:
ZP1, ZP2, ZP3, ZP4
Permeable to large macromolecules
Follicle extensions continue through it
Pre-antral follicle structure
Nucleus, oocyte, zona pellucida, granulosa cells, basal lamina, theca, capillary (inner to outer structure)
Intracellular communication between oocyte and GC via gap junctions that penetrate ZP
Also communication via connexins i.e. Cx43 between GC and Cx37 between GC & oocyte
Describe the location of the primordial follicle
Primordial follicle arrested in dictyate stage of mieosis
Primordial follicles located in ovarian cortex & have no blood supply. Consequences?
Basal lamina around the follicle creates microenvironment for gc & oocyte i.e not in contact with other cells in the ovary
What are the 3 possible fates of the primordial follicle
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
What are the 2 theories that cause initiation of follicle growth
1)initiation is regulated by loss of an inhibitor
Resting follicles under constant inhibitory influence (local paracrine/autocrine factors) to remain dormant. Where from?
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 inhibitory and stimulatory factors
Production-line” hypothesis: those that enter meiotic arrest first in foetal ovary, will initiate growth first
Also dependent on size of PF pool and ratio at which enters the growing pool.
Describe the importance of the extracellular matrix
ECM consists of collagen, lamimin, fibronectin, proteoglycans & polysaccharides
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
Genes involved in primordial follicle activation and suppression
Endocrine disruptors BPA (Bisphenol A), Genistein, DES (diethylstilbestrol) – inhibits nest breakdown
Genes involved in nest breakdown & Primordial Follicle Assembly: FIGLA (human & mice) Zona Pellucida 1-4 (human & mice) Activin βA & BDNF (human) AMH? (mouse) Oestrogen? (baboons, human)
Genes involved in primordial follicle activation: KIT ligand & KIT receptor (cKIT) FOXL2 NOBOX (newborn ovary homeobox) SOHLH 1&2 (transcription factors)
Genes involved in primordial follicle maintenance and repression: PTEN FOXO3 (forkhead family) AMH (Anti-Mullerian Hormone) SDF-1 (stromal derived factor)
Describe the genes 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
Describe the genes involved in primordial 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
Newborn mice injected with antibody to cKIT, that blocks interaction with KL do not progress beyond primordial follicle stage.
Genes involved in primary to pre-antral growth and defects that can arise
FOXL2 (forkhead transcription factor) ⇒ granulosa cell proliferation
FOXL2 -/- have Type 1 BPES and POF (premature ovarian failure) – 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
Genes involved in 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
Do androgens play a role in early follicle growth
Do androgens play a part?
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?
Explain 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)
Is the ovary capable of forming new oocytes?
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
