Reproduksjonsbiologi

Reproduksjonsbiologi
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Hormoner og Reproduksjon
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Sperm
Spermatogenesis
Oogensis
Meiosis
Fertilisation
The first week
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The Testis
Tunica albuginea
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Seminiferous tubuli
Epidiymis
Vas Deference
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The Testis
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Spermatozoa
Seminiferous tubuli
Germinal
epithelium
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The Testis
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The different compartments:
Seminiferous tubuli
Interstitial cells
The blood-testis barrier
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The Testis - The compartments
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The Testis – The compartments
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Tubuli:
Sertoli cells
Spermatogenesis
Estrogens
InhibinA
Interstitial cells
Blood, lymph
Leydig cells
Testosterone
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Testis- Production of hormones
LDL-Cholesterol
LH
FSH
Estradiol
OH
O
Testosterone
Leydig - cell
Sertoli - cell
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Hormoner og reproduksjon
De viktigste aktørene
Luteniserende hormon
Follikkel stimulerende hormon
humant Chorion Gonadotropin
- LH
- FSH
- hCG
Glykoproteiner bestående av to kjeder (α−β)
α - kjedene er like
β - kjedene er ulike
Sterkt glykosylert (40%) av vekt
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Hormoner og reproduksjon
De viktigste aktørene
LH
α-kjede 92 aa
β-kjede 115 aa
hCG
FSH
92 aa
131 aa
92 aa
118 aa
β - kjeden til LH og hCG er lik fram til
aminosyre 115.
Forskjell i glykosylering mellom LH og hCG
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Testis- Production of hormones
Regulation trough Negative Feedback
Hypothalamus
Hypofyse ⊕
Brain
TRH
GnRH \
\
\
LH
Cholesterol
⊕
Testosteron
FSH
⊕
Østradiol
Inhibin
Leydig – celle - Peritubular-celle - Sertoli-celle - Sædceller
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Hormoner og reproduksjon
Gonadotropin frigjøringshormon
GnRH
Dekapeptid
pyroGLU-HIS-TRP-SER-TYR-GLY-LEU-ARG-PRO-GLT-NH2
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Spermcellens historie
Primordiale kjønnceller migrerer fra plommesekken
til gonaden 3-5 fosteruke.
Primordale kjønncellerligger i dvale i primitive
sædkanaler omgitt av umodene Sertoliceller ligger
fram til pubertet.
Sædkanlene differensierer og spermatogenesen starter
under puberteten.
Spermatogenesen fortsetter i prinsipper til mannen
dør.
(litt strikkmotorpreget)
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The sperm cell
– Structure and basic components
Break-in
tools
Target identification
systems
Amplitude modulators
Genetic packet
Motor
Whip
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The spermatozoa - The tail
Amplitude modulators
Dense fibres
Mitochondria in
a spiral
Middle piece
Microtubules + axonem
Principal piece
Whip
Terminal piece)
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The spermatozoa - The head
Break in tools
Target identification
system
Genetics packet
Acrosome
Equatorial ring
Receptors
Highly condensed DNA
Packed with protamines
Instead of histones
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Meiose
Dannelse av haploide (n) kjønnsceller
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Dipoid celle med 4 kromosompar
1p 1m 2p 2m 3p 3m 4p 4m
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Dipoid celle med 4 kromosompar
2 4 = 16 mulige kombinasjoner
1p
1p
1p
1p
1p
1p
1p
1p
2p
3p
4p
2p
2p
2p
2m
2m
2m
2m
3m
4p
3p
4m
3m
4m
3p
4p
3m
4p
3m
4m
3p
4m
1m
1m
1m
1m
1m
1m
1m
1m
2p
2p
2p
2p
2m
2m
2m
2m
3p
3m
3p
3m
3p
3m
3m
3p
4p
4p
4m
4m
4p
4p
4m
4m
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Diploid celle med 23 kromosompar
223 = 8 388 608
kombinasjoner bare basert på tilfeldig fordeling av
kromosomer
Rekombinering gir en mulighet for ~ 1014 ulike
kjønnceller
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Sperm: Epididymis -Vas deference
Temperature control
32-33 oC NOT 37 oC
Epididymis
Spermatozoa training camp
Progressive motility
Zona Pellucida receptors
Storage (Cauda Epididymis)
Mobilised by rhythmic
contractions of Vas deference
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Spermtransport
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The first 30 hours- Signalling across membranes
– Membrane receptors
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Sperm transport trough the cervix
– Sperm transport from the cervix
• Liquefaction
– Coagulation after ejaculation
– Liquefy after approx 30 min (proteases from the prostate)
• Propulsion trough the cervix
– Progressive linear movement important
– May remain in the cervix for days
– The spermatozoa swim along the longitudinal microstructure of
the mucus
• During the preovulatory period the uterus contains fluid
facilitating sperm movements
• In many species, a sperm depot is formed at the isthmus
(uterus/fallopian tube) and may stay there for days.
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Sperm capacitation
– Capacitation
• As spermatozoa ascends the female genital tract they are said to
undergo a physiological transformation resulting in the attainment
of a state of “capacitation”
– Ca2+ increase, cAMP increase
– Sperm plasma membrane increase fluidity (cholesterol loss)
• In the capacitated state, the sperm cell can undergo the acrosome
reaction in response to appropriate stimulus.
– De-capacitating factors
• The seminal plasma contains factors that prevent capacitation
– Mask receptors,
– Cholesterol source
– Calmodulin like proteins (Ca2+ binder)
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Oocytten
Metafase-II oocytt
Perivitelline
rom
Zona pellucida
Pol-legeme
Polbody
Oocyttmembran
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Oogenesis
• Nuclear maturation
– Completion of meiosis
• Cytoplasmic maturation
– Acquire the functional capacity of an
mature oocyte
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The history of the oocyte
Primordial germ cells migrate form the Yolk
sac to the primitive gonad in foetal week 3-5.
Differentiates to oogonia and thereafter to
primary oocytes
Starts first meiotic division and is surrounded
by a layer of follicular cells (7. Month)
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Number of oocytes in the ovary
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Preantral follicles
Crosstalk between oocyte
and surrounding follicular
cells
Default pathway (FSH):
Mural granulosa cells
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Oocyte Cumulus cell crosstalk
Follicle Formation
Proliferation
Steroidogenesis
Differentiation
Cumulus Expansion
Ovulation
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Oocyte Cumulus cell crosstalk
Growth
Meiotic arrest
Transcription
Maturation
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Growth of the oocytes
20 µm
~30 follicles/day
start development
60 days
120 µm
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Growth vs competence
20 µm
Ability to undergo
Resumption of meiosis
120 µm
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Oocyte maturation
• Nuclear maturation
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–
–
–
Achievement of meiotic competence
GVBD, resumption of meiosis
Progression to MII
Easy to detect by morphological markers
Germinal Vesicle
Metaphase I stage
Metaphase II stage
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The ovary
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Produksjon av steroider i ovariet i follikelfasen
2 - celle konseptet
LDL-Cholesterol
LH
FSH
Østradiol
O
O
Androstendion
Theca - celle
Granulosa - celle
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Hormoner og reproduksjon
GnRH
Hypothalamus
-
Hypofyse
-
+
-
LH
Activin
FSH
Cholesterol
Inhibin
Androstendion
Theca - celle
Østradiol
Progesteron
Granulosaceller
Oocytter
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Hormoner og reproduksjon
Produksjon av steroider i ovariet i lutealfasen
LDL-Cholesterol
LH (hCG)
Theca lutein - celle
Progesteron
Granulosa lutein - celle
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Oocytten: den omnipotente
• Egget: Nøkkelen
til livet
DNA-fra mor (kan byttes ut)
Metabolsk niste
Mitochondrier (kan byttes ut)
Umodent (GV) egg
m-RNA (informasjonsbærere)
Transkripsjonsfaktorer (regulatorer)
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Meiotic arrest
• Meiosis arrested at prophase I (chiasmata formed)
• The arrest actively maintained by corona cells
who pump in and maintain a high cAMP level in
the oocyte
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Meiotic arrest
• Meiosis arrested at prophase I (chiasmata formed)
• The arrest actively maintained by corona cells who pump
maintain a high cAMP level in the oocyte
• Connexin43 expression in corona cells downregulated by
LH
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Nuclear maturation
GV-stage –foetal life to 1 day prior to ovulation
Metaphase I
Chromosomes aligned on a metaphase plate
Still 4 sets of chromosomes
Metaphase II
Chromosomes aligned on a (MII) metaphase plate
Half the chromosomes expelled in the polar body
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Ready for fertilization
Extracellular matrix
– Interaction with the cumulus mass
– Extracellular matrix
• Mainly hyaluronic acid
– In animals, only hypercativated, acrosome intact spermatozoa can
penetrate the cumulus mass.
– The sperm have receptors for hyaluronic acid which might
modulate sperm movements
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Binding to the Zona pellucida
– The Zona pellucida comprises three major
glycoprotein species,
• ZP1 and ZP2 forms an open porous matrix formed
by interconnecting filaments (ZP2 filaments held
together by ZP1)
• ZP3 plays a role in sperm recognition
– “The sperm receptor”
• ZP3-/- female mice infertile
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The acrosome reaction
– As a consequence of the spermatozoa to the
ZP3 receptor, the cell undergo a secretory event
known as the acrosome reaction.
• focal fusion of the plasma membrane and the
acrosomal membrane
• releases of the most soluble components of the
acrosome vesicle
• vigorous movements of the sperm drives the sperm
trough the zona pellucida into the perivitelline space
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The acrosome reaction
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Sperm-oocyte fusion
– During the course of the acrosome reaction, a
discrete band of plasma membrane around the
equatorial segment of the sperm head suddenly
acquires the capacity to recognise and fuse with
the vitelline membrane of the oocyte
– Prior to the acrosome reaction, the spermatozoa
has no capacity to interact with the oocyte
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Sperm-oocyte fusion
– Several molecules implicated in the fusion
process that are similar to viral fusion proteins
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The first 30 hours- Ca-oscillations
• Sperm entry induces oscillations in the
intracellular free Ca2+ level
• The model:
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The first 30 hours- Oocyte activation
– Cortical granule release
• Appears to be induced by the calcium transients
– Prevents polyspermy by inducing changes in the zona
pellucida
– The defence against polyspermy:
• The slow zona block
• The fast vitelline block
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Pronuclear formation
– The sperm aster
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Sperm aster (centrosome) formation
Unfertilised oocyte
3-6,5 h post insemination sperm astral microtubules
assemble around the base of the sperm head
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Sperm aster (centrosome) formation
Duplication of the
centrosome
(15 h post insemination)
Sperm aster enlarge and
seek out the female pronuclei
Prometaphase
Bipolar array of
microtubules
marks the
first mitotic spindle
Sperm axoneme
still visible
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The sperm
• The sperm provides 3 essential components
– The paternal chromosomes
– The centrosome
– The oocyte activating factor(s) inducing Ca2+
oscillations
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Sperm nucleus decondensation
• The sperm chromatin is packed using protamines
• The sperm chromatin must be reorganised
– Protamines must be replaced with histones
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The first 30 hours- Oocyte activation
– Following sperm-oocyte fusion, the oocyte
becomes activated and initiates the cascade of
events that culminate in the initiation of
embryonic differentiation
• The release of cortical granules
• The resumption of meiosis
• The formation of male and female pronuclei
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Germinal vesicle
Cellekjerne med 4n DNA
Germinal vesicle breakdown
Kjernemembranen forsvinner
Meiosen starter opp igjen
Fra fosterliv
Metafase-I oocytt
Follikkelfase
Metafase-II-oocytt
LH
Ved eggløsning
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Metafase-II oocytt
Befruktning
Siste meiotiske deling
Dannelse av pronuklei
Fusjon av pronuklei
6-8 timer
Mitose
12-16 timer
22-24 timer
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Maternelle vekstfaktorer, kofaktorer
m-RNA etc (oocytt-niste)
Totipotente celler
Ekspresjon av
fetale genom
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Human embryo development
Expression of embryonal genes
Day 2
Day 3
Day 4
Day 5
Day 5/6
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Feil under fertilisering
3PN zygoter
Kan utvikle seg til termin
Enkelte zygoter kan
reorganisere til normal
2n genotyp eller til
2n/3n mosaikk
Feil kromosom segregering
Bare kvinnenes
arvemateriale
Bare mannens
Arvemateriale
Placenta dannes
ikke og utviklingen
stopper ved
implantasjonen
Selve embryoet
dannes ikke
kun anlegg til
placenta
Blæremola
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Hva skjer ?
What can I do?
Who am I?
We won!
We are friends
Anybody here
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Det er fysiologisk med store genetiske avvik tidlig i
embryoutviklingen hos mennesker
Frequency of gametes/zygotes/embryos with wrong number of chromosomes
30% in all cells
80% one or more cells
40%
20% in all cells
100% one or more cells
30% oocytes
10% spermatozoa
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The old view
• Gametogenesis
– A process designed to produce the “best” sperm and
oocytes
– 200 million sperm cells a day
• 1/250 000 000 chance of winning the lottery on a lucky day
– 10-15 oocytes a day
• 4-500 oocytes lost each month
• One dominant follicle ovulates the winner
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The old view – the winner is
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The old view
• Fertilization and early embryo cleavage
• A biological selection procedure
• Only the “best” embryos will develop and implant
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The old view
• Gametogenesis, gamete transport, fertilization and
early embryo development
• Designed to weed out the bad ones
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Gametogenesis
• Are genetically abnormal gametes deselected
during gametogenesis?
• Yes to a certain extent
• Balanced/unbalanced translocations
• Klinefelter - sperm
• …….
• No selection of oocytes during the final maturation
– Follicular dominance not correlated to genetic health of
the oocytes
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Gamete transport
Genetically abnormal gametes deselected ?
Sperm
– Prior to ejaculation – unknown
– After ejaculation – the female genital tract
• Strong selection of correct phenotype
• Weak selection of normal haploid sperm
• Oocytes - unknown
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Fertilization
• Are genetically abnormal gametes deselected during
fertilization?
• Seemingly only on phenotype
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Conventional IVF vs ICSI
ICSI bypass a lot of biological checkpoints
Seemingly without creating problems
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ICSI vs IVF
• Major differences between IVF- ICSI
• The oocyte is a robust little machine
– Can use different biological programs to start the
embryo development
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Hints from the past
• Average fecundity in humans 19-20%.
• Young fertile couples trying to conceive:
– In 60% of the cycles hCG could be detected in serum
in the luteal phase
• hCG secreting embryos
– In 60% of these cases, only transient synthesis of hCG
• Embryos dying
• Large embryo wastage at the time around
implantation
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The CEMAS II Study
København, Gøteborg
Embryo culture until day 3
Blastomere separation
FISH analysis
127 embryos
474 blastomerer
FISH analysis of
X, Y 13, 16, 18, 21, 22
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The CEMAS II Study- Results
Number of with all blastomeres normal
Normal embryos (%)
47 (37 %)
Abnormal embryos (%)
67 (53 %)
Inconclusive embryos (%)
13 (10 %)
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The first cleavages: A dangerous game
No checkpoint control during the first mitosis
Number of abnormal cells increase during the first cleavages
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Aneuploidy rate
30%
10-15%
Fertilization
Zygote
4–cells, 2 days
35%
50%
8-cells, 3 days
>50%
0,6 %
10-15%
Blastocyst 5/6 days
100% ?
30-50%
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Aneuploidy rate
• What happens to all the aneuploid cells?
• Dependent on the cell type?
– Germ line stem cell markers as early as 8-cell stage
• Dies out?
– What if it was an important stem cell?
– Other stem cells can be recruited?
• Rerouted to less important functions?
– Placenta, membranes..
• Persists….mosaicism
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Phenotype vs genotype
• What if some of the phenotypic variation is
secondary to early embryo aneuploidy
•
•
•
•
Chromosomes involved
Frequency i.e. proportion of cells affected
Cell lineages affected
Mosaicism
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Placing the quilt
• Humans have a low fecundity (20%)
• Humans have low genetic quality of their
gametes
• Early human embryo development
imperfect
• Who shall we blame for this?
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I’m dreaming of
a guy walking
on his two feet
and with brains
A. afarensis
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The big brain
• Because we needed it?
• Or because it was attractive?
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Larger brain
Narrow pelvis
Premature birth
Child care
Needs a father
How to keep him
Bond him with sex
A lot of sex
A few pregnancies
Bad sperm,
bad eggs
Quality control downstream
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My hypothesis
• Humans are not designed to be efficient
breeders – on the contrary
– Low genetic quality of gametes and embryos
– Synchronisation of menses
• Bonding was more important to establish
sufficient large cooperative groups
– Sex and small talk
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Implications for ART
• Weak correlation between genotype and
phenotype in human gametes.
• Biological quality control at/after
implantation
• Human gametes and embryos can tolerate a
lot of abuse without this leading to
malformations in the offspring
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Embryokloning - embryosplitting
Embryo splitting
Skjer naturlig
(eneggede tvillinger/trillinger)
Kan også utnyttes bevisst
Naturens egen metode
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Eneggede flerlinger
• Separate morkaker og fosterhule
(diamnionale/dichorionale - di/di )
• Forskjellig morkake, samme
fosterhule (mo/di)
• Deler fosterhule og morkake
• mo/mo
• Deler organer (siamesiske
tvillinger)
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Flerlinger
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Inneholder totipotente celler
Befruktning
Pronuklei stadiet
2-celler, et døgn
8-celler, 3 dager
4–celler, 2 dager
Trofoektoderm
Indre cellemasse
Morula, 4 dager
Blastocyst, 5/6 dager
Inneholder multipotente celler
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Generering av Embryonale stamceller (ES-celler)
Trofoektoderm
Indre cellemasse
Isolert indre cellemasse
Fibroblaster som ”feeder” celler
Dyrkning og stadige nye
omsettinger av cellekulturen
i løper av 3-4 uker
Kultur av Embryonale stamceller (ES-celler)
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Kloning – somatisk cellekjerne overføring
Isolert cellekjerne
overføres til egg uten eget
DNA
Cellekultur av somatiske celler
Egg uten eget
arvemateriale
Fusjonering av
cellekjerne og egg
Donor egg
DNA
8-celles
pre-embryo
Embryonale stamceller
Indre cellemasse
Blastocyst
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