pre-antral follciulogenesis

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

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

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

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)

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

Initiation- gonadotrophin dependent (resting and pre-antral) recruitment- gonadotrophin independent (antral stage) selection-gonadotrophin independent (ovulatory)

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

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

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

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

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

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

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

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.

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

Endocrine disruptors BPA (Bisphenol A), Genistein, DES (diethylstilbestrol) – inhibits nest breakdown Genes involved in nest breakdown &amp; Primordial Follicle Assembly: FIGLA (human &amp; mice) Zona Pellucida 1-4 (human &amp; mice) Activin βA &amp; BDNF (human) AMH? (mouse) Oestrogen? (baboons, human) Genes involved in primordial follicle activation: KIT ligand &amp; KIT receptor (cKIT) FOXL2 NOBOX (newborn ovary homeobox) SOHLH 1&amp;2 (transcription factors) Genes involved in primordial follicle maintenance and repression: PTEN FOXO3 (forkhead family) AMH (Anti-Mullerian Hormone) SDF-1 (stromal derived factor)

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

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.

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

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 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?

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)

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