antral folliculogenesis

Studies in 1970s showed that when radio-labelled LH/hCG injected into adult female rats localized specifically to the theca layer of small preantral, antral and pre-ovulatory follicles but not primordial follicles Theca of follicle is envelope of connective tissue → differentiates into theca interna & externa containing vascular tissue, immune cells and matrix factors Theca is critical for maintaining structural integrity of follicle and delivering nutrient to avascular GC layer Formation and differentiation of theca extremely important for preantral to antral progression. Why? GDF9 k/o mice (& GDF9 mutations in human & sheep) fail to develop theca layer and follicles arrest → oocyte-derived GDF9 regulating formation of theca cell layer. Neo-angiogenesis, hence follicle interaction with systemic endocrine factors Acquisition of steroidogenic function

Theca cells derived from 2 different sources in the embryonic gonad: Mesenchymal (from mesonephros) cells become steroidogenic cell Stromal cells (indigenous to medullary region) become fibroblasts, perivascular smooth muscle cells and interstitial ovarian tissue

Formation of antrum occurs when the follicle reaches a diameter of 200-400µm, surrounded by a vascularized theca, hence subject to circulating influences. Fluid-filled spaces appear between the granulosa cells which soon coalesce together to form a single, large, fluid-filled cavity or “antrum”. Contains fluid formed as exudate of plasma containing secretory products of oocyte & GC Known as follicular fluid KL and Cx37 essential for antrum formation in lab animals – as k/o of these genes result in no antral follicles at all As the fluid volume increases the follicle continues to expand greatly in size.

theca externa-Concentrically arranged smooth muscle cells; innervated by autonomic nerves; lymphatic vessels; important during ovulation theca interna-Steroid-producing cells; contain LH-r & Insulin-r; richly vascularized GC differentiate into 2 mature cell lineages: mural and cumulus cells. Mural granulosa cells-Mural Granulosa – involved in endocrine feedback control; express FSHr, P450arom, LHr Cumulus oophorus cells-Remain in contact with oocyte & interact with oocyte via gap junctions; mitotically active; no LHr

GC produce EGF-like ligands that bind LH and allow for secretion of hyaluronan and a complex of hyaluronan cross-linking proteins that cause expansion of COC

The HPG axis acts to control antral follicle growth at this stage In response to LH, theca expresses key steroidogenic enzymes to make androgens from cholesterol. Likewise granulosa cells respond to FSH by up-regulating aromatase (CYP19A1) and 17β-HSD to make oestrogens Inter-cycle rise in FSH crucial for recruitment of AF into the menstrual cycle Progression of antral follicles Selection of dominant follicle Fate of remaining AF

↑ granulosa cell proliferation ↑ aromatase ↑ induce and maintain FSHr ↑ induce and maintain LHr Interact with paracrine factors

Androgens act on granulosa cells (paracrine interaction) to upregulate AR & FSHR AR k/o mice have reduced FSHR mRNA AMH (produced by GC of small antral follicles) acts as a brake on FSH recruitment of antral follicles by: decreasing FSH sensitivity decreasing FSH-stimulated aromatase expression Counter-balancing effect of AMH, Androgens and FSH to ensure against premature depletion of PF pool and/or premature selection of follicles by FSH

amount of FSH required to recruit one follicle follicle with the lowest threshold will be recruited size? Serial USS has revealed that largest follicle is not always selected FSH receptors Increased numbers Coupled more effectively to down-stream signalling Growth and Oestradiol production increased cell division (2-5 million GC in EFP and 50-100 million at ovulation) Size ~5.5-8.2mm in EFP and 18-20mm in LFP increased aromatase (200x more E2 than other follicles) Androgens & Oestrogens Intra-follicular cAMP Increased area of theca vasculature  consequence?

Dominant follicle survives fall in FSH by: increased sensitivity to FSH  increased FSH receptors increased numbers of granulosa cells acquisition of LH receptors the LHR gene is switched on by FSH possible involvement of insulin-like growth factors 1&2 (IGF-2 particularly important in humans)

IGF-2 enhances FSH effects; stimulates androgen output and hence oestrogen IGF activity suppressed by IGFBP (IGF-binding protein) IGF cleaved from IGFBP by PAPP-A (pregnancy-associated plasma protein A) PAPP-A expression high in DF Thought that other AF in cohort may have higher levels of IGFBP hence preventing co-stimulatory effect of IGF & FSH

inactivating mutations of LH receptor normal *EFP E2 , anovulatory, multiple cysts, morphologically normal antral follicles (Toledo, 1996) hypogonadotrophic women FSH treatment effective as long as some LH present E2 is significantly reduced but detectable, why? can some A be accessed from adrenal? LH k/o mice Antral stage growth blocked

↑ theca function of CYP11a, CYP17 ↑ growth & steroidogenesis in dominant follicle Withdrawal of gap junctions between gc & oocyte and resumption of meiosis Expansion of COC Ovulation & luteinization

LH and FSH have same 2nd messenger - cAMP How does the cell distinguish between them? FSH produces low cAMP levels LH produces high cAMP levels Yong, Baird, Hillier (1992) Clin.Endocrinol.(Oxf) provides energy for biosynthetic activity mediates effects of FSH and LH on protein production eg. aromatase, SCC, LHr, proteolytic enzymes

Angiogenic factors stimulated by primarily by androgens but also oestrogens – theca, gc, stroma all involved Basic fibroblast growth factor (bFGF) endothelial cell mitogen, most potent angiogenic factor Vascular endothelial growth factor (VEGF) endothelial cell mitogen, enhances vascular permeability Ovarian lymphatic vessels recruited to theca and stroma layers around growing follicle, under control of VEGF-R3 Why do we need angiogenesis? Constant re-modelling to allow for growth of follicle (2-20mm) through the ovarian tissue, angiogenesis of CL, tissue repair etc.

AMH secretion is maximal during small antral follicle stage (≤4mm) and decreases to undetectable levels later Hence serum AMH reflect small AFs The number of antral follicles (AFC) in the early follicular phase correlates with numbers of growing follicles only Use ultrasound to count number of 2-8mm follicles at start of cycle & correlate ≈ AMH serum levels Low numbers of antral follicles are a sign of ovarian ageing Observable earlier than a rise in FSH serum level Intriguingly, right ovary has been shown to be larger and have higher AFC than left ovary → thought to be due to larger PF pool in right ovary formed in fetal life So before selection (early part of menstrual cycle) will have a cohort (~10 is normal) of follicles about 2-10mm diameter visible on ultrasound. So if scan ovary in early folliclular phase can count them – known as AFC and it has been shown that this correlated well with what is known as “functional” ovarian reserve. Preantral follicles and those <2mm diameter cannot be visualised by current u/s technology. All serum markers are the function of the GC of growing follicles and NONE are products of the primordial follicle. Hence only reflect number and size distribution of AFs. AMH production is highest in AF≤4mm & AF of 5-8mm contribute most to serum AMH levels.

POI is clinical term for premature ovarian failure Affects 1% of women worldwide Defined as ovarian dysfunction <40yrs → oligomenorrhoea or amenorrhoea Overarching feature is infertility resulting from accelerated depletion or reduced follicle reserve. Aetiology is poorly understood: Environmental genotoxins induce DNA damage eg chemo/radio-therapy for cancer treatment Mutations in genes e.g. BRCA1 and BRCA2that repair DNA double-stand breaks, resulting in diminished ovarian reserve Altered hormonal signalling Chromosomal defects e.g. Turner’s syndrome (XO) → have streak ovaries Autoimmune diseases including thyroiditis & Addison disease

Hydrogel matrix to support follicle so that contact maintained between oocyte and gc Also to be permeable to media Modifiable in terms of rigidity 2 types: Collagen Alginate (product of seaweed) Alginate gels able to produce antral follicles from 2° in monkey & human 3D-printer makes ovary from microporous hydrogel scaffolds Follicles seeded through out to create mouse bio-prosthetic ovary Scaffold provide 3D support to follicles – allowing for vascularisation and ovulation Ovarian function restored when implanted in surgically sterilized mice Pups born through natural mating!