Embryology I
OOGENESIS
autumn 2024
Development
of reproductive system
Zuzana Holubcová
Department of Histology and Embryology
zholub@med.muni.cz
Development of reproductive system
• 0-7 week - indiferent stage
• from week 7 sexual differentiation
• mammalian gonads develop as
integral part of mesoderm-derived
urogenital system
• formation of gonads due to
interaction of germ cells with
mesonefric mesenchyme
• genital portion of reproductive
system develops 1-2 weeks later
XX - Müller (paramesonefric) duct
XY - Wolff (mesonefric) duct
XY/XX
Primordial germ cells timeline
week 3
week 4
week 5
week 8
week 7
week 6
week 9
week 10
➢ Specification
➢ Migration
➢ Colonization of genital ridges
➢ Sexual diferentiation
post conception (pc)
P
R
O
L
I
F
E
R
A
T
I
O
N
- sex cells progenitors
= primordial germ cells (PGCs)
oogonia
(ovary)
gonocytes
(testis)
PGCs specification
• In lower species germ line specified based on
inheritance of specific area of cytoplasm
(germplasm) containing maternally encoded
proteins and RNAs (maternal factors)
• In mammals, specification occurs within
posterior epiblast of postimplantation embryos
before gastrulation onset based on inductive
cell-cell signalling
/ WNT
WNT
BMP
FGF?
BLIMP1
Fragilis
BLIMP1
Fragilis
Stella
„PGCs-competent“
epiblast
Posterior epiblast Move out trough
developing
primitive streak
=PRMD1
PGCs specification
priming → commitment → licencing → specification
Waddington´s landscape
Kobayashi and Surani, 2018.
cylindr bilaminární disk
PRDM1=BLIMP1
TFAP2C = AP2gamma
Azim Surani
T=TBXT=brachyury
Pregastrulation embryo
PGCs specification
• Human PGCs first detected in endodermal epithelium of yolk sack wall close to allantois
~21-22 days pc
• ~50 cells expressing BLIMP1, Fragilis, Stella and tissue non-specific alkalic phosphatase
(TNAP)
• Transient extraembryonic deposition allows PGCs to escape from molecular signals
inducing somatic differentiation into 3 germ layers (ectoderm, mesoderm, endoderm) and
thus prevent their pluripotency marked by expression of Oct3/4 and Nanog (in mice also Klf4
and Sox2)
Primitive streak Allantois
Visceral endoderm
PGCs specification
PGCs morphology
- large round/oval cells (10-20 m)
- large excentric nucleus with prominent
membrane
- dense a granular cytoplasm
- cytoplasmic deposits of glycogen a lipid droplets,
round pale mitochondria, abundant ribosomes,
ER and GA underdeveloped
- glycogen and lipids consumed during PGCs
development and the number of mitochondria
increases
- „nuage“ material – electron-dense granules
lokalized on cytoplasmatic side of nuclear
membrane
- histologically detectable alkalic phosphatase and
PAS staining (periodic-acid-Schifft)
- imunofluorescence detection of transcriptional
factors Vasa (DDX4), Nanos, Oct3/4, Stella,
Fragilis, BLIMP1 a glycoprotein surface antigens
SSEA-1/SSEA-3/SSEA-4
PGCs migration
• passive – ~week 4 pc
- embryonic disc is bending and a portion of
the yolk sack is incorporated into the
embryo body
- PGCs are translocated to endodermal
epithelium of the hindgut
• active – 5-6th week pc
- PGCs penetrate the mesenchyme of the
hindgut in the 16th somite region and
migrate through dorsal mesentery in the
lateral direction towards nesonefros
- Here, PCGS colonize mesenchymal ground
of urogenital ridge in L1-L3 area → genital
ridge → basis of paired gonads
12.týden
mezonefros
ovarium
➢ Active migration towards urogenital ridge
➢ Ligand-receptor chemotaxis
- atraction/repulsion signals from coelomic epithelium of gonadal ridge
SCF/c-Kit ligand --- c-Kit receptor PGCs
SDF1---- CXR4 receptor of PGCs pseudopodia
➢ Migration along
autonomous nerve
fibres a Schwan cells
➢ Interaction with ECM
(1 integrins)
➢ ameboid movement
PGCs migration
PGCs proliferation
- occurrs during migration and
intensifies after arrival to genital ridges
- mitogen signalling from microenvironment
- selective mechanisms → survival vs. apoptosis
weeks of gestation
PGCnumberperembryo
Cotticchio et al. Oogenesis. Springer 2013
0
50000
100000
150000
200000
250000
300000
350000
3 4 5 6 7 8 9
FEMALE MALE
Ectopic localisation of PGCs
- defects of migration/colonisation/apoptic
process can lead to germ cells tumors
PGCs colonisation of gonads
- (uro)genital ridge
- in Th6-S3 area
- derived from mesoderm
- formed by mesenchyme and covered by coelomic
epithelium
- gonads develop in L1-L3 region
- epithelial cells from degenerating structures of
mesonefros undergo epithelial-mesenchymal transition
(EMT) and contributes to formation of sexually
indiferent gonads
- Gata4 expresion in somatic tissue of gonadal ridge is
critical for PGCs „ licencing“ for sexual differentiation
Hu et al 2015
- in Gata4 KO mice PGCs arrive to genital
ridge but do not differentiate
- somatic portion of gonads is derived from
➢ mezonefric mesenchyme
➢ mezonefric epithelium
➢ coelomic epithelium mesonefros
+ nerves, vessels, blood elements,...
XY XX
- first PGCs arrive to sexually indiferrent
primary sex cords ~30 day pc and
colonisation of gonadal mesenchyme
continues in following weeks (6-7th
week pc)
- PGCs expression of pluripotency markers
decreases and so does PGCs´capability to
generate pluripotent cells in vitro
PGCs colonisation of gonads
Sexual differentiation
PGCs
gonocytes
spermatogonia
(quiescence)
PGCs
oogonia
oocytes
(entry to meiosis)
„Ovary-determining
genes“
„Testis-determining
genes“
„Battle of sexes“
(antagonism)
- genetically encoded by Sry and Sry-box (Sox9) genes localized on chromosome Y
- expression of testis-/ovary-determing genes in somatic compartment of future gonads is
critical for PGCs entry to either male or female gametogensis, sex of germ cells and
phenotype of the whole organism
Anne McLaren
- PGCs have a potential to enter
either spermatogenesis or
oogenesis
- „the phenotypic sex is not
determined by chromosomal
constitution of germ cells (XX/XY)
but cellular environment PGCs are
exposed to during embryonic
development“
Sexual differentiation
McLaren 1988
The sex determining switch
- germ cells are dispensable for
organogesis of reproductive
system!
Testis development
• Sry-Sox9-FGF9 expresion in somatic cells of indiferent gonadal region induces growth and
sprouting of primitive sex cords in medullary region and their differentiation in seminiferous
tubules which are colonized by PGCs (gonocytes)
• connecting primitive seminiferous tubules to mesonefros forms anastomotic network (rete testis)
• Sertoli cells arise from sex cord endothelium and start to produce antimüllerian hormone (AMH)
• Leydig cells originate from intermediate mesenchyme and produce testosteron
• PGCs immigrated into a developing testis (gonocyty) intensively proliferate, and during 2nd
trimestr differentiate to mitotically-inactive prospermatogonia
Ovary development
• No Sry-Sox9-FGF9 expression
• primary sex cords in medullar region undergo fragmentation and are replaced by
vascular fibrous tissue
• in cortical part of ovary, primary sex cord cells undego secondary diferentiation and
surround immigrated PGCs
Ovary development
• Immigrated PGCs diferentiate to oogonia, which first divide by mitosis and later enter to
meiosis but arrest in prophase
• clusters of oogonia are surrounded by somatic cells which later invade syncytium
• individual meiotically arrested oocytes enclosed by single layer of follicular (pre-granulosa)
cells form primordial follicle
Asociation with somatic cells is
critical for oocyte survival!
• Retinoic acid (RA)
= meiosis inducing factor
- derived from retinaldehyde
oxidation by dehydrogenase
produced in mesonefric tissue
surrounding the gonads
- causes activation of nuclear
receptors RAR/RXR which
modulate transcription
of RA-responsive elements
- in the ovary RA stimulates
oogonia to enter meiosis
- in fetal testis RA actively
degraded by cytP450 (CYP26B1)
Germ cell entry to meiosis
Elimination
of poor quality
follicles
From
medulla to
cortex
Meiosis in the ovary
Female meiosis timeline
Ovarian stem cells
Jonathan Tilly
- Report about presence of mitotically
active ovarian stem cells (OSC) in
adult mouse and human ovary
- de novo oogenesis in adults?
Johnson et al, Nature 2004.
White et al, Nature Medicine 2012.
v
v
v
Development of reproductive tract
MALE FEMALE
Development of reproductive tract
Leydig cells
testosterone
development
of Wolffian duct
Sertoli cells
AMH
regression
of Müllerian duct
8-9th week pc
Leydig cells
testosterone
regression
of Wolffian duct
Sertoli cells
AMH
development
of Müllerian duct
MALE FEMALE
gubernaculumLGR8
INSL3
= cranial
suspensory
ligament
gubernaculum
▪ testicular hormone
INSL3 activate LGR8
receptor in
mucofibrosus
genito-inguinal
ligament
(gubernaculum)
which pulls the testes
through abdomen and
the inguinal canal
down to scrotum
▪ cranial suspensory
ligament (CSL)
regresses
▪ in females, absence
of INSL3 prevents
gubernaculum grown
and shrinkage
▪ CSL persists and
keeps gonads in
lumbar region
AMH, Testosteron
Development of reproductive tract
UTERUS
← caudal part of Müllerian ducts
← cranial part of uterovaginal canal
VAGINA
← caudal part of uterovaginal canal
← cranial part of urogenital sinus (← cloaka ←endoderm)
*
* Gartner´s canal – remnant of mesonefric duct
Hymen
Development of female reproductive tract
Evolution of female reproductive tract
Major et al, 2022
Molecular regulation of PGCs development
o PGCs specification
o PGCs migration o Genital ridge colonization o Sexual diferentiation
Sry Wnt4
Sox9 Rspo1
FGF9 FoxL2
-catenin
o Meiosis entry
RARLDH (RA)
Stra8
Cyt26B1
BMP4/BMP8
WNT
FGF?
BLIMP1/PRDM1
Sox17
Fragilis
Stella
TNAP
Oct3/4
Nanog
SCF/c-Kit ligand - c-Kit receptor
SDF1- CXR4 recptor
ADAM
PECAM1
o PGCs proliferation
PTEN
WNT
Nanos3
Pog
Dazl Gata4
Ddx4
Wt1
Gcna1
Reproductive system development overview
XY XX
INDIFERENT PRIMITIVE
GONADS
TUBULI COLLECTORII
DUCTUS MESONEFRICUS
(DUCTUS WOLFFI)
DUCTUS PARAMESONEFRICUS
(DUCTUS MÜLLERI)
PHALLUS
PLICAE GENITALES
TORI GENITALES
testis ovary
Fallopian tubes, uterus,
cranial part of vagina
clitoris, labia majora et minora
no SrySry
low testosterone
regresion
low AMH
regresion
testosterone
Sertoli cells Leydig cells
AMH
(Antimüllerian hormone)
DHT
(dihydrotestosterone)
low DHT
penis, scrotum
ductus efferentes,
ductus epididymidis
ductus deferens
ductus ejaculatorius
5-alpha reduktase
Germ cells
meiosis entrypremeitic stage