Reproductive biology and Embryology
Brno, March 2019
Gametes
• Meiosis
• Structure and development
• Differences between oogenesis and spermatogenesis
• Regulation of gametogenesis
• Ovarian and menstrual cycles
• Ovulation
• Transport of gametes, sperm capacitation, acrosome reaction
Fertilization and Early Embryogenesis
• Cortical reaction
• Cleavage, morula, blastocyst
• Activation of embryonal genome
• Embryonic stem cells, nuclear transfer (cloning)
Lecture 3
Zygote
Multicellular
embryo
Dynamic
multicellular
organism
CELLDIFFERENTIATION
and
MORPHOGENESIS
STABLE GENOME
Genomic equivalence
(= equal amount of DNA and the
same nucletide sequence in all
cells of a organism – cloning)
VARIABLE
TRANSCRiPTOME
Transcription Regulators
X
Embryology: what does it cover?
Embryonal x Fetal Development
All the organs
are established
Embryo Fetus
Week 8Fertilization Week
39-40
Parturition
The
primitive
heart
starts
beating at
4 weeks. 7 weeks
Early embryo
before implantation
Any use of understanding principles
of reproduction and embryonal/fetal development?
• Infertility treatments
• Contraception
• Avoidance of developmental abnormalities
Genetic basis of gamete development
Examination of genetic status (amniotic fluid)
Understanding the effects of teratogenic compounds
Intrauterine examination - sonography
Intrauterine surgeries
Others to come
Instruments
Cell culture
Molecular biology
and genetics
Reproduction
• allows for continuity of a given species via propagation
of its individuals
• key element in reproduction is the transfer of DNA
duplicate from parents onto progeny
Indviduals of different sex produce different gametes
Key element in sexual reproduction
Sexual reproduction mediated by gametes
may seem to be too complicated and much less effective than asexual but
serves very significant adaptation role.
This adaptation role realizes via unique genetic processes,
which take place during development of gametes – eggs and sperm.
Although development of eggs and sperm differ in many morphogenetic details,
key genetic processes taking place in both types of gamates are principally the same.
Genetic processes that are crucial for gametogenesis
take place during meiotic cell division - MEIOSIS
These genetic processes include:
• „Crossing over“
• Independent segregation chromosomes
• Reduction of the number of chromosomes
Reduction of the number of chromosomes
Why?
Somatic cell Somatic cell
2n 2n
Progeny
4n
Gametes have to contain haploid number of chromosomes (n) in order to
prevent multiplication of chromosomes in progeny above a diploid number (2n)
In principle, the number of chromosomes could be reduced in one step by just separating
homologous chromosomes without preceding replication of DNA (DNA synthesis)
2n
1n
1n
Meiosis – two divisions
instead of one
DNA replication
establishment of bivalents
„crossing over“
1. meiotic division
Independent segregation
of chromosomes
2. meiotic division
Separation of chromatids
2n
2C
2n
4C
1n
2C
1n
2C
1n
1C
1n
1C
Fully functional
germ cells
MI oocyte - tetrades
Dr. Zuzana Holubcová, June 2018
• „Crossing over“
• Independent segregation of chromosomes
• Fertilization
are sources of genetical diversity, that
underlies adaptation of living organisms.
Sperm Eggs
Significance for
embryogenesis
(reproduction)
Morphological
and physiological
properties
Development and
underlying regulatory
mechanisms
Genetic function
D
I
F
F
E
R
E
N
T
S
A
M
E
X
• numbers of ocytes (follicles)
in ovary is given at the time
of birth and do not increase
(in woman ~500 000)
• only small number of oocytes
develop into fertilizable eggs
(in woman ~400)
• at the time of menopause,
ovary contains only small
number of remaining oocytes
(in woman ~100-1000)
• after reaching puberty, sperm
cells are produced in testes
continuosly until high age (two
testes of man produce about 1000
spermatozoa every second)
Primordial germ cells – PGC
• stem cells, which are common to both sperm cells and oocytes
• originate in yolk sac (extraembryonally)
• divide mitotically while migrating into gonad anlagen (genital ridges)
(due to signals from surrounding environment – laminin, kit-ligand, TGF-beta1, …)
• in man PGCs are sexually indifferent until the 6th week
of embryonic development
+
DEVELOPMENTAL
PROCESSES
Ooocyte
~0,12 mm
One of the biggest and most „precious“
(by both number and significance) cells.
Paradoxical cell
Highly specialized cell.
The only cell in the female body that
can undergo meiosis and fertilization,
and thus give rise to a new individual.
„Totipotent“ cell
It can generate all the cellular
diversity that si typical for
multicellular organism.
&
Even the era of cloning did not replace the functions of egg !
Key periods of oocyte development
G2/M block
mitotic
division
growth
(months)
meiotic maturation
(hours)
PGC & oogonia
MI MII
Resumption of
meiosis
Block of meiosis in diplotene
of the 1. meiotic division
Fertilizable egg,
capable of supporting
early embryonal
development
Primary oocytes
Secondary oocyte
Where and how the oocyte development is achieved ? (1)
DiploteneoftheMeiosisI
Inside the ovary
Preantral follicle
(multilaminar)
Recruited follicle
(unilaminar)
Secondary
(antral) follicle
(medium size)
Tertiary
(Graafian) follicle
Primordial follicle30 mm
20 mm
Selection of the Dominant follicle
(the one most sensitive to FSH)
~14 days
Growth
Primary
follicles
Massive hormone
production
Androstendione
Theca folliculi interna
Estradiole
Granulosa cell
Takes place in ovary (along with the growth of follicle)
It is fully dependent on the contact of oocyte with granulosa cells
of the follicle (mediated for example by the gap junction protein connexin-37)
Signal that initiates growth is not known
(it is not FSH - hypophysectomy does not prevent growth)
Communication between oocyte and granulosa cells is bidirectional
&
&
&
kit-
ligand
GDF-9
Where and how the oocyte development is achieved ? (2)
Ooocyte growth
100x increase in volume – accumulation of organelles a molecules providing
egg with the ability to support early embryogenesis until reaching autonomy
(about 105 mitochondria accumulated in oocyte supports embryogenesis until blastocyst stage)
Slow process (several months in woman)
Intensive transcription –
accumulation of mRNA in dormant
state (regulated by polyadenylation and ?)
oocyte growth
transcription
Fully grown oocyte – ~2,5 ng total mRNA
Intensive translation – many proteins (very limited knowledge)
Example: ZP1, ZP2, ZP3 – proteins of zona pellucida
Fully grown oocyte – ~120 ng total protein
Transkriptome
and proteome –
underlie unique
properties of
oocyte
Where and how the oocyte development is achieved ? (3)
Ooocyte growth
Reactivation of X chromosome
• somatic cells – one X chromosome is inactivated by hypermethylation
of cytosine residues in molecule of DNA
• growing oocyte – both X chromosomes are active (crucial for oocyte
development – karyotype 45, X0 results in an abnormal development of ovaries)
Genomic imprinting
• epigenetic modification of autosomal chromosomes that leads to
monoallelic expression of genes – due to activity of enzyme DNA
methyltransferase
• PGCs are globally demethylated
• imprinting is newly established during oocyte growth (about 70-80 genes)
&
Abnormalities in imprinting may result in
spontanneus abortions in assisted reproduction !!!
(in vitro manipulation with gametes and embryos may produce
abnormalities in imprinting)
Epigenetic changes occuring during oocyte growth
Where and how the oocyte development is achieved ? (4)
MPFactivity
fully grown
oocyte (G2/MI) oocyte at MI
oocyte
in interphase egg at MII zygoteSperm
penetration
GVBD
LH
25 hours
Transcription inhibited Translation activatedX
drop of
cAMP
Where and how the oocyte development is achieved ? (5)
The last hours before ovulation – meiotic maturation
The final look into the ovary
Where and how the oocyte development is achieved ? (6)
Hormonal regulation
Ovarial cycle
Hypothalamus
Pituitary
gland
Gonadotropin-releasing
Hormone
(pulses)
Follicullar growth
Meiotic maturation
+ Ovulation
Corpus luteum
Gonadotropins
FSH LH
Menstrual ph. Proliferative phase Secretory (lutal) phase Ischemic Menstrual ph.
0 5 14 28 527
Estrogen Progesteron
+ Estrogen
Sperm cell development - Spermatogenesis (1)
Minimal ejaculate (WHO)
• Volume - 1.5 ml
• Sperm number - 15.1 millions/ml
• Motility - 40%
Sperms on the oocyte
~4 mm ~60 mm
~0.5 mm
Sperm cell development (2)
Before
puberty
Slowly mitotically dividing
spermatogonia in seminiferous tubuli
After
puberty
~0.25 mm
~0.5 km
Lumen
Spermatocytogenesis (mitotic) Meiotic phase Spermiogenesis
Sperm cell development (3)
A0 Spermatogonia – Stem cells
A1 Spermatogonia
A2 Spermatogonia
A3 Spermatogonia
B Spermatogonia
• Mitotic divisions
• Connected to basal membrane
• 2N, 4C – primary spermatocytes
Primary Spermatocytes
Secondary Spermatocytes - 1N, 2C
1. Meiotic division
2. Meiotic division
Spermatides – 1N, 1C
Spermiogenesis
• No division
• Differentiation
Sperm cell development (4) - Spermiogenesis
Histones to Protamines
Genome inactivation
Loss of cytoplasm
Sperm production
• 1 million sperms every hour
• Spermatogenesis takes ~70 days
• Transport through epididimis ~8-17 days
• Cyclic character
(Cycle of the seminiferous epithelium – 16 days
– the same developmental stage at the same place)
Sperm cell development (5) - Regulation
Sertoli cells
•Support , protect, and nourish
•Phagocyte
•Blood-testis barrier (zon. occlud.)
•Produce anti-mullerian hormone
•Produce fructose
•Produce inhibin (inh. FSH prod.)
Leydig cells
•In interstitium
•10 % of testis
•Produce testosteron
Fertilization (1)
= the process that culminates in the union of one sperm nucleus
with the egg nucleus within the activated egg cytoplasm
Where do the gametes meet ?
Ovary
Uterus
Oviduct
Ampula
Fertilization (2) Oocyte makes itself ready for being penetrated
LH
surge
Graafian follicle
35-40 hrs
Preovulatory stage
Ovulation
Fertilization
Fimbriae
Infundibulum
Oocyte is fertilizable
for only 12 to 16 hours
Fertilization (3) Travel of sperm to the site of fertilization
Testis
Seminal gland
(vesicles)
Ductus
deferrens
Epididymis
Bulbourethral
gland
Prostate
Urin. bladder
Urethra
EJACULATE
(2-6 ml)
Sperms
200 – 600 million
Products of accessory
sex glands
• Cholesterol (in prostasomes)
• Prostaglandin
• Fructose
• Vesiculase (coagulates semen)
Fertilization (4) Travel of sperm to the site of fertilization
Vagina
200-600 millions of sperms
Oviduct
100-1000 sperms
Cervix Uterus
2 – 7 hours
• Cleans sperm
• Cervical mucus
stabilizes sperms
• Initiates capacitation
• Acid environment
• Sperms move actively
• Removal of glycoproteins from the head
• Change to composition of cell membrane
• Increase of motility
Fertilization (5) Entry of sperm into the oocyte
Acrosome reaction
Initiated by the contact with ZP proteins
Release of acrosin, neuraminidase, esterases
Zona pellucida
Perivitelline space
Entry into ooplasm
Fusion with oolemma
Release of hyaluronidase
Penetration of ZP
Release of lysosomal enzymes
from cortical granules
Modification
of ZP
Block of
polyspermy
Completion of meiosis II
Fertilization (6)
Zygote formation and the first cleavages
Gap junctionsZygote
pronuclei fused
(Syngamy)
DNA
synthesis
Sperm
• DNA
• Cenriol
• Oocyte-activating
factor
Ooocyte
• DNA
• Mitochondria
• Cytoplasm
~6-8 hrs ~12-14 hrs ~3-4 days~8-12 hrs
O v i d u c t U t e r u s
Blastocyst formation
Compacted
morula
Hatched
blastocyst
Hatching
blastocyst
Expanded
blastocyst
220-280 mm
Trophoblast
Blastocoel
Embryoblast
Day 5 – 6
(60 to 8O cells)
PB PN 20 44
G1 S G2/M G1 S G2/M
Translation of maternal mRNA
Translation of zygotic mRNA
Zygotic transcription
Repression of transcription
Significance of „enhancers“
Activation of embryonal
genome
A potency of oocyte cytoplasm
It is not a single discrete event
(first signs occur in zygote, in man it reaches its
maximum in 4- to 8-cell embryo)
Transcrips that replace
degraded maternal
mRNAs
Novel transcripts that
underlie new pattern of
gene expression
Two types of transcripts
It is „responsible“ for establishment of totipotency of blastomeres
It represents phenomenon known as genome REPROGRAMMING
&
Activation of embryonal genome
Effectivity of cloning is very low (1-3%)
Reprogramming is slow and most likely
incomplete (as the result, gene expression is
often abnormal)
Effectivity of reprogamming depends
on many factors (type of somatic cells,
position in cell cycle phase, …)
Nuclear transfer (cloning) - principle
Egg
Somatic cell
Reprogramming „Normal“ development
Activation
Human Embryonic Stem (hES) Cells
(Thompson et al, 1998)
Early embryo at blastocyst stage
Isolated embryoblast (ICM - Inner Cell Mass)
Isolated embryoblast after placing to
in vitro conditions (+ feeder cells + FGF2)
Propagation in culture by enzymatic
disaggregation (repeated passaging)
Derivation of postmeiotic germ cells from hESC
Prof. Harry Moore, University of Sheffield, 2009
B) C-KIT
C) I-97 antigen
D) Cells with condensed chromatin
and signs of flagellum
Structures that are highly
reminiscent to oocyte-granulosa
complexes (zona pellucida is not
developed)
Thank you for your attention !
Questions and comments at:
ahampl@med.muni.cz