Embryology I
OOGENESIS
autumn 2024
Folliculogenesis
Zuzana Holubcová
Department of Histology and Embryology
zholub@med.muni.cz
Folliculogenesis overview
- Follicle = female germ cell + somatic cells
- symbiotic syncitium, functional unit of the ovary
- oogenesis (female gamete development) a folliculogenesis (follicle development)
are interconnected processes
- endocrine, paracrine and autocrine regulation
- first germ cells colonize primordial ovaries in 5. week of gestation (wg)/ 3. week post
conception (pc) and divide completely producing fully separated oogonia
- second division wave is characterized by incomplete cytokinesis, daughter cells remain
connected by intercellular bridges
syncitium
germ cell nest/cluster
➢ Proliferation of oogonia
Primordial follicles formation
- surrounding somatic cells invade syncitium a enclose individual oocytes which
entered meiosis
- prenatally in humans (15-22. wg), perinatally in mouse
➢ Germ cell nest breakdown
Primordial follicles formation
primordial follicles (PF)
➢ Germ cell nest break down
- primordial follicles represent extremely long-living
symbiotic unit
- somatic cell supply germ cells (oocytes) with
nutritients and signalling factors
- oocytes dictate follicular cell function
= diplotene non-growing oocyte
surrounded by a single layer of flattened
pre-granulosa cells
Primordial follicles formation
- thin sheet-like structure that surrounds and protects
primodial follicle
- formed from extracellular matrix and mesnchymal
cells of indiferent gonad
- components: colagen IV + laminin
(+ fibronectin only later in antral stadium)
➢ Basement membrane
Primordial follicles formation
- Selective resorption/degeneration of germ cells/follicles during oogenesis
Germ cell
syncytium
Somatic cell
invasion
Primordial
follicle
Primary follicle Preantral follicle
ATRESIA ATRESIA ATRESIA
Granulosa
cells
Oocyte
Zona Pellucida
Oocyte
Pregranulosa
cells
Invading
somatic cellsApoptotic
oogonia
Oogonium
Somatic
cells
BEFORE BIRTH AFTER BIRTH
• 4-5. gestation month: 6-7 milions of germ cells
• at birth: ~ 1-2 milion of germ cells
• at puberty: ~ 400.000-500.000 of germ cells
Atresia
- Cell death mechanism?
- apoptosis – rarely observed (rapid
progression)?
- autophagy - increased lysosomal
activation detected
- Purpose?
- Required for germ cell nest breakdown
and individualization of primordial
follicles?
- Degenerated cells nurture survivors?
- Quality control mechanism
- elimination of defective germ cells
(e.g. gene mutations, aneuploidy, non-functional
mitochondria,...)
Atresia
Activation of primordial follicles
➢ Dormant follicles
=„quiescent“/„resting“ PFs
- localised in the cortical region of mammalian
ovary
- long survival due to local inhibitory signals
(weeks, months, yeas, decades in different species)
- dormant state sustained until recruited for
growth by activating signal or receive signal to
undergo atresia (>90% !)
- compact units, relatively resistant to
environmental factors a cryopreservation
- number of PFs defines fertility
span
- exhaustion of „ovarian reserve“
(<1.000 primordial follicles) →
menopauze
- proliferation and diferentiation of flat
pregranulosa cells into cubic granulosa cells
(GCs)
- emergence of zona pellucida
- only little portion of PFs is activated (~1.000 per month)
Primary follicle
= single layer of
cubic granulosa
cells enclosing
diplotene oocyte
with zona
pellucida
Activation of primordial follicles
- a selective process by which a dormant PFs are selected
for further development in preparation for fertilization
Activation of primordial follicles
❑ 2 categories of PFs
- Medulla
- Postnatal activation
- Fast growth
- Early specification of preganulosa cells
- Cortex
- Dormant until puberty
- Periodical activation
- Late specification of pregranulosa cells
- Activation progresses form medulla to
cortex
Follicular development
Follicular develoment
Follicle growth
- continual long-lasting irreverzible process of follicle enlargement
- disproportional growth of follicle and oocyte
- surrounding stroma cells align with basal membrane of secondary
follicle an differentiate to theca cells
- weakening of cell contacts between granulosa cells produces multiple
fluid-filled foci, they expand and coalesce giving rise to large central
cavity called antrum with follicular fluid
40 m 100 m 200 m 400 m
4 months 2-3 months
corpus luteum
preantral phase antral phase
17 - 26 mm (!)
Graafian follicle
mural
cells
Follicle growth
➢ Theca (lat. a case)
- vascularized somatic layer which provides nutrition, mechanical support and protection to the follicle,
and plays the role in follicle ruture during ovulatory process
- recruited from surrounding stromal tissue by factors secrted by activated primary follicle
❑ Theca interna
- inner layer in contact with basal membrane
- endocrine cells responsible for synthesizing
androgens
❑ Theca externa
- external part made up of connective tissue
- smooth muscle-like cells and fibroblast-like cells
producing ECM
Mural granulosa cells
„2 hormones – 2 cells concept“
Amstrong 1979
- under control of LH,
thecal cells synthesize
androgens
- stimulation of FSH
receptors in granulosa
cells leads to activation
of aromatase, which
converts androgens to
estrogens
Follicle growth
Follicle selection
Initial recruitment Cyclic recruitment
Primordial
follicles
Primary
follicles
Secondary
follicles
Antral
follicles
Preovulatory
follicle
ATRESIA
depletion
Continuing initial recruitment
time
- Cyclic rise of
circulating FSH
allows group of
follicle to escape
atresia
- dominant follicle selection based of abundance of FSH receptors (FSHR) at granulosa
cells
- rollicle with the highest number of FSHR gows faster
- dominant follicle secrete estrogens and inhibin → lower pituitary FSH release → the
growth of other follicles slowed → apoptosis
Follicle selection
Dominant follicle faces LH peak
→ resumption of meiosis
→ oocyte maturation
→ ovulation
→ luiteinisation of GCs
Follicle selection
Regulation of folliculogenesis
➢ Regulation of primordial follicles formation
Ge et al 2019
SDF(KL) -cKit
- coordinated expression of genes in both somatic and germ cells
- interplay of secreted factors
- depletion of
primary
follicles
- oocytes at
birth not
surrounded ny
preGCs
- sterility
-/-
decrease of
cells adhesion
selective
atresia
follicle formation
↓ E-catherinproliferation
Oocyte-specific
genes
➢ Regulation of primordial follicles activation
- not completely understood
- ratio of local activation and inhibition signals
- (1) oocyte-secreted factors
- (2) granulosa cells-secreted factors
- (3) paracrine inhibition signals from other growing follicles
- (4) endocrine signalling? Insulin?
Primordial
follicle
Primary
follicleTransition
Stromal cells
Regulation of folliculogenesis
➢ Regulation of primordial follicles activation
❖ PI3K signalling
- growth factors stimulate receptor tyrosine kinase which
phophorylates-activates PI3K
- PIP3 formed from PIP2 binds and activates PDK1
- PDK1 phosphorylates Akt
- Akt inactivates transcription factor Fox (translocation
from nucleus)
PTEN
FOXO3
P27
TSC1/2
PIP3
Akt
PDK1
mTORC
Pten-/-
Foxo3-/-
Pdk1-/-
premature activation
- depletion of primordial
follicles
- premature ovarian failure
(inferility)
Regulation of folliculogenesis
➢ Regulation of primordial follicles activation
❖ TGF signalling
- binding of hetero-/homo-dimeric ligand on outer
domain of receptor kinase I and II
- formation of heterotetrameric complex
- receptor´s subunit II phophorylates subunit I which
activates SMAD pathway regulating transcription
▪ AMH – Antimüllerian hormone
- produced by GCs of preantral follicles
- negative regulator of folliculogenesis
- prevents activation and growth of PFs
- prevents proliferation and diferentiation of GCs
- inhibits aromatase
- suppresses FSH-stimulated growth of antral follicles
Amh-/-
loss of dormant PFs
- untimely activation
Activin
BMPs
GDFs
TGFs
Regulation of folliculogenesis
▪ AMH as a clinical marker
- indicates presence of dormant PFs
cohort during reproductive lifespan
- the level decreases with reproductive
aging in correlation with the exhausion
of ovarian reserve
- circulating level used a predictor of
response to hormonal stimulation in
ART
- extremely high levels imply risk of PCOS
and OHSS
- inappropriately advertised as a marker
of female fertility (!)
- contraception potential?
Regulation of folliculogenesis
➢ Regulation of primordial follicles activation and growth
▪ GDF9 (Growth differentiation factor 9)
▪ BMP15 (Bone morphogenic protein 15)
- oocyte-secreted factors from TGF family of growth factors
- synergic activity (formation of homo-/hetero-dimers)
- major positive regulators of GCs growth, proliferation and
diferentiation
- inhibition of apoptosis, stimulation of secretion of
paracrine growth factors in GCs
- regulatory feedback loop promoting follicule growth
- Oocyte-secreted
factors stimulate
GCs proliferation
and secretion of KL
(Kit ligand/SDF)
- KL activates
oocyte´s c-Kit kinase
promoting oocyte´s
growth
Oocyte
growth
Granulosa cell
proliferation
BMP15/GDF9
KL/cKit
Regulation of folliculogenesis
➢ Regulation of primordial follicles activation
and growth
• mutace GDF9/BMP15 spojené s předčasným
ovariálním selhání a neplodností
• GDF9 mutace spojené s familiárním výskytem
dyzigotických dvojčat
• Abnormlání exprese BM15/GDF9 u PCOS
• Polymorfismus BMP15 u OHSS
• Gdf9-/-
development arrested in primary follicle stage
- infertility
• Bmp15-/-
decreased ovulation frequency
- subfertility
• Gdf9+/-Bmp15-/-
low numebr of growing follicles
- infertility
Differences between
mono- a poly-ovulatory
species?• BM15 overexpression – depletion of
folllciles
- infertility
• Bmp15-/- i Gdf9-/•
Bmp15+/- i Gdf9+/•
mutation fo BMP15 receptor
• immunisation against BMP15
High
fecundity
Regulation of folliculogenesis
➢ Regulation of primordial follicles activation and growth
Robert Gilchrist
Poly-ovulatory species have higher GDF9:BMP15 ratio than mono-ovulatory species
Non-covalent
homo/heterodimer
Regulation of folliculogenesis
Single point mutation
causes latency of
human GDF9
Robert Gilchrist
Supplementation
of IVM media
with recombinant
cumulin
➢ Regulation of primordial follicles activation and growth
Regulation of folliculogenesis
Robert Gilchrist
Development of low-molecular
drug promoting short-term
BMP15+GDF9 pairing would
allow natural selection of
multiple best quality oocyte
from the cohort for IVF
purpose...
Availability of BMP15 and altered ratio of GDF9:BMP15
heterodimer changes the timing of LH receptor expression
Reduced level of BMP15
• Smaller and more follicles acquire LH receptors
• More follicles „see“ the LH surge and are selected for ovulation
cumulin
➢ Regulation of primordial follicles activation and growth
Regulation of folliculogenesis
Normal level of BMP15
Increased fertility
➢ Role of gonadotropins
- stimulation of antral follicles
- preantral phase of folliculogenesis is
regarded as gonadotropin-independent
BUT
some data indicate that
in prenatral stage
FSH effect is transmitted
via local factors and acts
in synergy with
paracrine factors
produced by ovarian
tissue
Regulation of folliculogenesis
Insuline?
Estrogen?
Progesterone?
Hyppo signalling pathway
microRNA
Regulation of folliculogenesis
❖ Hyppo signalling
- Tumor supresor signalling pathway
regulating organ size
- limits proliferation and induced apoptosis
during organogenesis
- dysregulation leads to tissue overgrowth
- Hpg gene (MST in mammals) named after
mutant Drosophyla phenotype which
head resembles a hippo
- normal follicle growth requires
suppression of Hyppo pathway
Regulation of folliculogenesis
❖ microRNA
- small non-coding RNAs involved in regulation of
gene expression in ovarian cells
- influence development of gonads,
folliculogenesis, ovulation, steroidogenesis,
luteinisation and apoptosis
- stage-specific oocyte/GCs miRNA profiles
- pathophysiological miRNA profiles
Reviewed in Nouri et al., Cell Commucation and signaling, 2022
Regulation of folliculogenesis
Bidirectional communication between
oocyte a follicular cells
- coordination of
(1) oocyte growth and maturation
(2) granulosa cells proliferation and
differentiation
(3) steroidogenesis
(4) metabolism
(5) apoptosis
Paracrine signalling
Communication via:
(A) Paracrine signals (growth factors, microRNA)
(B) Extracellular vesicles
(C) Transzonal projections
Oocyte and granulosa cells interaction
❖Extracelular vesicles
= mebrane vesicles
- exosomes (40-100 nm)
← fusion of endosomes with plasma membrane
- microvesicles (>100nm)
← released from cell membrane
Chen et al, 2023
- transfer of membrane and cytosolic
proteins, lipids, nc-RNAs, microRNA,..
- present in follicular fluid
- potential biomarkers of
oocyte quality?
Pietro et al., JARG 2016
Oocyte and granulosa cells interaction
- thick extracellular coat surrounding mammalian oocytes
- composed of 4 glycoproteins ZP1-4*
- ZP-2 and ZP 3 form long chains
- ZP-1 and ZP-4 (its paralog) crosslinks the chains
- sialylation and sulfation → acidic character
- FUNCTION:
- protection of oocyte (and early embryo)
- storage of biactive molecules
- receptivity and sperm attachment during fertilization
- prevention of polyspermy
❖Zona pellucida (ZP)
Oocyte and granulosa cells interaction
*u myší jen ZP1-3
❖Transzonal projections (TZP)
- projections of cumulus GCs enabling their communiation with oocyte through ZP
- FUNCTION:
(1) mechanical contact
(2) intercellulra signalling
(3) supply of nutrients
- presence of cytoskeletal
structures (actin/acetylated
microtubulin) stabilizes TZP
and facilitates intercellular
transport of molecules
Oocyte and granulosa cells interaction
❖Transzonal projections (TZP)
Beana and Terasaki 2018
Oocyte and granulosa cells interaction
❖Transzonal projections (TZP)
prenantrallargeantral
- prominent during
preantral stage
(oocyte growth)
- retracted in late antral
stage
- maintain meiotic
arrest
- mechanical removal of
cGCs → spontaneous
resumption of meiosis
in denuded oocytes
Oocyte and granulosa cells interaction
❑ Gap junctions
- transmembrane channels
- made of two „connexomes“ (hemichannels)
located on both apposed membranes
- each connexom composed of six connexins with
4 transmembrane domains
- connection:
(A) between oocyte and GC (B) between two GCs
functinal syncitium
Cx 43/Gja1
Cx 43/Gja4Cx 37/Gja4
cGC
Cx 43/Gja1
cGCcGC
Oocyte
Oocyte and granulosa cells interaction
❑ Adherent junctions
- bridge neighboring plasma membranes via
transmembrane glycoprotein cadherin
- cytoplasmic face linked to actin
- E-cadherin and N-cadherin involved in physical
contact of GCs with oocyte´s ZP
Oocyte and granulosa cells interaction
❑ Cilia of granulosa cells
- primary cilia (9+0 pairs of microtubules)
- communication between oocyte and GCs
- senzoring cumulus microenvironment
Shu et al, 2023
Daugther
centriole
Oocyte and granulosa cells interaction
Folliculogenesis disorders
➢MOF – multiple oocyte follicle syndrome
Gaytan et al 2014.
- presence of multiple preovulatory follicles within one
follicle
- failure of follicle individualisation
- two oocytes of different maturation stage enclosed by
one ZP commonly observed in cohorts of oocytes
retrieved for IVF
➢EFS – empty follicle syndrome
- no oocytes obtained from preovulatory follicles during IVF
procedure despite normal hormonal response and UZK
monitoring
- usually technical problem during COCs aspiration and/or hCG
trigger administration
- genuine absence of oocyte very rare ((„genuine EFS“ - 0.0016%)
- Possible cause?
- COC stick to follicle wall due to insufficient LH/hCG trigger
which causes COC expansion and detachment
- Oocyte degeneration
- genetic predisposition for proapoptotic genes
expression in GCs
- defect ZP leading to impaired GCs-oocyte
communication
Chen et al., 2017.
Folliculogenesis disorders
➢Premature ovarian insuficiency (POI)
= premature ovarian failure - POF
Cause?
- genetic mutations (e.g. Figla, Nobox, Sohlh)
→ failure of oocyte individualisation
and apoptosis of oogonia
non-surrounded by follicular cells
→ deregulated activation of
primordial follicles and depletion of
ovarian reserve
- poor prognosis,
gonadotropin stimulation
inefficient, usually necessary
to become a recipient of
donor egg
Folliculogenesis disorders
→ premature depletion of ovarian reserve (<40
years), early menopause and infertility
- 1% women
- amenorrhea, hypoestogenism, low AMH,
elevated FSH
➢Polycystic ovary syndrome (PCOS)
- enlarged ovaries with thickened sclerotic capsules
(ultrasound visible cysts)
- high number of small antral follicles (2-9 mm), but
no preovulatory
- oligo-/a-menorrhea, dysmenorrhea, anovulation,
subfertility
- ↓FSH, ↑LH, ↑ androgens (hirsutismu, acne),
extremely high AMH
- inzuline resistance, obesity, hypertensis
- 5-10 % women in reproductive age (12-45 years)
- Difficult hormonal stimulation, risk of OHSS
Cause?
- defect of growth and/or selection
of dominant follicle
- genetic predisposition
- altered miRNA profile
Lean vs. obese
PCOS
Folliculogenesis disorders
Folliculogenesis disorders
Possible to „unleash“
development of small
follicles?
➢Polycystic ovary syndrome (PCOS)
❖Ovarian drilling
Therapeutical strategies to improve folliculogenesis
- historical empiric technique for fertility
treatment of PCOS patients
- mechanical disruption of ovariant tissue
integrity
- surgical multiperforation or laparoskopic
diathermy
- risk of adhesions, vaskular changes,
bleeding, tissue damage
(1) Mechanical fragmentation of
surgically removed ovarian
tissue
(2) Cryopreservation of ovarian
strips
(3) In vitro treatment with
activating substances
(4) Autologous transplantation of
ovarian tissue
❖In vitro activation (IVA) of primordial follicles
Mature oocytes for IVF
- method of last choice for
POI patients Kawamura et al. PNAS 2013
Kazuhiro Kawamura
Therapeutical strategies to improve folliculogenesis
- experimental intervention
technique
❖In vitro activation (IVA) of primordial follicles
- „drug-free“ IVA
Vo KCT, Kawamura K. Int J Mol Sci. 2021
Kawamura K. Et al, RMBO. 2020
Therapeutical strategies to improve folliculogenesis
- overactivation of Hyppo
signalling pathway blocks
growth of follicles in PCOS
patients
- tissue irritation induces
reparation and actin
polymerization which
inactivates Hyppo
signalling
Therapeutical strategies to improve folliculogenesis
❖In vitro activation (IVA) of primordial follicles
❖In vitro maturation (of follicles)
- clinical strategy involving collection of immature
oocytes surrounded by follicular cells from small
or mid-sized follicles (2-10 mm) and their in vitro
culture obtain fertilizable eggs
- follicle in vitro culture for 24-48 hours
in maturation media suplementaed
with gonadotropins (FSH, LH/hCG)
- mature (MII) oocytes fertilized by ICSI
(IVF not recommended du to ZP hardening)
- relatively low efficiency
- method of choice
- in patients with risk/history of OHSS (e.g. PCOS)
- oncological patients (fertility preservation)
- in „poor-responders“
Hormones**
nutritients
antioxidants
serum/HSA
growth factors*
* např. GDF9, BMP15, KL, cumulin,...
Therapeutical strategies to improve folliculogenesis
❖In vitro maturation (IVM)
MII ~ 50%
MII ~70%
hCG „priming“ in vivo – oocyte maturation in vitro
- Poor/delayed response to stimulation, maturation
incomplete in 36hours after hCG
„Rescue“ in vitro maturation
, EGF
Therapeutical strategies to improve folliculogenesis
- in cancer patients (including children!) undergoing
radiotherapy and/or gonadotoxic treatment
- cryopreservation of ovarian tissue containing
primordial follicles followed by autologous
transplantation or in vitro culture
❖ Fertility preservation
- hormonal replacement therapy?
- reimplantation of thawed tissue graft might
constitute risk of reintroducing malignant
cells and cancer remission!
Therapeutical strategies to improve folliculogenesis
❖Egg development in vitro
option for prepubertal
cancer survivors with
high risk of cancer cell
reintroduction from
frozen-thawed tissue
Telfer et al 2023
Therapeutical strategies to improve folliculogenesis
❖In vitro foliculogenesis
Evelyn Telfer
Optimalisation of in vitro
folliculogenesis
(A) isolation of follicles from stroma
(B) cultivation in media containing grow
factors, low-molecular inhibitors,
hormones, nutrients,
antioxidants,...
(C) supporting 3D biomatrix, nonadheren
culture conditions
Suboptimal
quality of in vitro
grown oocytes
Therapeutical strategies to improve folliculogenesis
❖In vitro gametogenesis (IVG)
Future prospects
- experimental technique of
making female/male gametes
outside of body
- full recapitulation of
gametogenesis in vitro
- use of reprogrammed
somatic/embryonic cells
- co-culture of in vitro-produced
gametes and gonadal somaderived
cells
- chance of having biological
child for cancer survivals
- possibility to rescue
endangered and/or revive
extinct animal species
- efficiency and safety?
- ethical and legal aspects
❖In vitro gametogenesis (IVG)
Saitou and Hayashi, Science 2021
Future prospects
❖In vitro gametogenesis (IVG)
Yamashiro et al.,Science 362, 356–360 (2018) Hwang et al., Nature Communication, 2167–2179, 2021
Mitinori Saitou Kotaro Sasaki
Future prospects
❖In vitro gametogenesis (IVG)
Future prospects
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