Download Oogenesis - U of L Class Index

Lecture plan
Dr. Sylvie Bilodeau-Goeseels
Research Scientist
Agriculture and Agri-Food Canada
Lethbridge Research Centre
Tel.: 403 317-2290
Email: [email protected]
Oogenesis
• The female gamete contributes a haploid
set of chromosomes and also a pile of
molecules and organelles that are needed
for development of the embryo until it can
produce them on its own or obtain them
from the environment.
• Oogenesis (some review + some new
material).
• Early embryogenesis.
• In vitro embryo production (bovine).
• How to study gene expression in
mammalian oocytes and embryos.
Oogenesis
• In mammals, oogenesis begins early in
fetal development and ends months or
years later.
• Formation of primordial germ cells (PGC).
• Migration to the future gonad (genital
ridge).
• PGC divide by mitosis during migration.
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Oogenesis
• When in the gonad: called oogonia (diploid=2
copies of chromosomes).
• To contribute a haploid set of chromosomes, they
have to reduce their chromo. content by ½.
• Meiosis
• Preleptotene: Interphase following the last mitotic
division of oogonia.
• Leptotene: Final DNA replication in preparation
for meiosis takes place. (Then called oocytes=
4copies).
Oogenesis
• In mouse embryos: At Day 14, ½ oogonia
(2c) and ½ oocytes (4c).
• In bovine embryos, meiosis starts at around
Day 82 of gestation.
• Around the time of birth, meiosis stops and
some oocytes begin to grow in coordination
with their follicle.
• A fixed reserve of oocytes at birth.*
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Follicle cells
granulosa cells, theca cells, cumulus cells
• Secrete hormones.
• Long cytoplasmic processes from the
follicle contact the oocyte surface via gap
junctions. Nutrients and molecules that
regulate oocyte development are passed
into the oocyte via the gap junctions.
• The cumulus cells accompany the egg at
ovulation.
Resumption of meiosis
• In most mammals, fully grown oocytes in
Graafian follicles resume meiosis just
before ovulation in response to
gonadotropins.
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Oogenesis in Drosophila
melanogaster (fruit fly)
• Drosophila widely used to study
developmental genetics.
• Meroistic oogenesis: oogenesis with nurse
cells (not all insects have nurse cells).
Oogenesis in D. melanogaster
• Meroistic oogenesis: oogenesis with nurse
cells (not all insects have nurse cells).
• Mitosis of an oogonial stem cell produces 2
daughter cells that separate from one
another.
• One of them=stem cell. Other=cystoblast
• Cystoblast divides 4 times, daughter cells
don’t separate. (ring canals).
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Oogenesis in D. melanogaster
• 2 of the 16 cells have 4 ring canals. Both
prepare for meiosis but it is completed in
one of them only= oocyte.
• The other one + the 14 other cells become
nurse cells.
• Oocyte + nurse cells = egg chamber
• Egg chamber surrounded by follicle cells.
Nurse cells of D.melanogaster
• Grow and replicate their DNA but don’t divide.
• May contain 1024 times as much DNA as the
haploid genome.
• Very active in RNA synthesis and RNA is
transferred to the oocyte via ring canals.
• Inject all their cytoplasm in the oocyte.
• Cytoplasmic volume of oocytes can increase
90000 X in 3 days.
Meroistic oogenesis
• Polytrophic ovaries: The nurse cells are
intimately connected to the oocyte.
• Telotrophic ovaries: Have clusters of nurse
cells at one end of the ovary that are
connected to oocytes via cytoplasmic
bridges.
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Oocyte growth in amphibians
• Seasonal synchronous growth
of follicles and oocytes.
• End of growth: volume
increased 27000 X
• Genes coding for rRNA
amplified.
• Lampbrush chromosomes
Role of follicle cells in insects
• Also coupled to the oocyte via gap
junctions so they also transfer molecules to
the oocyte.
• Can synthesize yolk precursors or sequester
yolk precursors for transport to the oocyte.
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Yolk
Early development
• Not chemically defined.
• In amphibians: The yolk precursor
(vitellogenin) is synthesized in the liver and
transported to the ovary via blood.
• Just before ovulation: meiosis resumes,
expulsion of 1st polar body. Meiosis arrests
again.
• After fertilization: meiosis continues,
expulsion of 2nd polar body.
• In reptiles, fishes, birds: Yolk produced in
the liver, transported to ovary via blood.
Early development
• Formation of female and male pronuclei.
• Fusion of pronuclei to form the diploid
zygote nucleus.
• Zygote= one-cell embryo.
• Cleavage initiated.
Cleavage
• Cleavage = cell division
– Karyokinesis: division of the nucleus
– Cytokinesis: division of the cytoplasm
Patterns of cleavage: determined by the amount
and distribution of yolk and orientation of the
mitotic apparatus.
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Classification of eggs
• Alecithal eggs: Eggs with little or no yolk.
• Ex.: most mammalian eggs.
• Isolecithal eggs: Modest quantities of evenly
distributed yolk.
• Ex.: echinoderms, annelids, mollusks
– The mitotic apparatus near the center.
– First few cleavages result in blastomeres of equal
size.
– Cleavage through the entire egg = complete or
holoblastic cleavage.
Classification of eggs
• Centrolecithal eggs: Yolk in the center.
• Ex.: most arthropods
• Telolecithal eggs:
• Moderately telolecithal: Yolk displaces mitotic
apparatus to the animal hemisphere. Unequal
holoblastic cleavage. Ex.: amphibians,
• Extremely telolecithal:Yolk fills both hemispheres.
Mitotic apparatus in a small disc of cytoplasm.
Meroblastic (incomplete) cleavage. Fish, reptiles,
birds.
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Radial cleavage
• 1st cleavage through a/v
axis.
• 2nd cl. right angle to 1st.
• 3rd cl. equatorial.
• 4th cl. Meridional
• 5th cl. equatorial
Radial cleavage
• After several cleavages, embryos with
holoblastic cleavages = solid cluster of
blastomeres = morula.
• Fluid filled cavity forms= blastocoel.
• Embryo = blastula
Bilateral holoblastic cleavage
• When the embryo is not
symmetrical.
• Ex.: amphibians
• Mitotic apparatus
displaced in animal
hemisphere.
• Cleavage furrows retarded
by yolk.
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Bilateral meroblastic cleavage
• Yolk restricts mitotic
apparatus and c. furrows
to a small yolk-free
zone=blastodisc
• Cl. not complete
• Rest of zygote=yolk sac.
• Subgerminal and
blastocoel cavities.
Rotational cleavage
• In mammals.
• One of the 1st 2
blastomeres rotates
90o before 2nd cl.
• 2nd cl. meridional in
one cell and
equatorial in other.
Superficial cleavage
• In centrolecithal eggs.
• Ex. Drosophila
• Division of nuclei without
cytokinesis.
• Migration to the periphery.
• Cl in a thin layer of
superficial cytoplasm.
• Cytokinesis after 14 cl.
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Caenorhabditis elegans
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•
•
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Caenorhabditis elegans
• Location and lineage of
every cell known.
• Embryos laid at 30 cells,
hatch with 558 cells.
• Adult has 959 somatic
cells + 2000 germ cells.
• No further division in
adult.
Small soil nematode
Pseudocleavage
Rotational cleavage.
Invariance of lineage.
Bovine early embryogenesis
• 1st cleavage app. 30 h post insemination.
• 2nd cleavage 10-12 h after 1st one.
• 16-cell stage: polarization of blastomeres
initiated.
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Compaction
• 32-cell stage.
• 1st major morphogenetic event.
• Increase in interblastomeric contact and
communication.
• Boundaries between cells not visible.
• Embryo = uniform mass = morula.
• Cell-contact induced polarization increased.
Cavitation
•
•
•
•
Formation of fluid-filled cavity.
2nd major morphogenetic event.
Embryo prepares itself for implantation.
Trophectoderm derived from the polar
outer cells and the inner cell mass is
derived from the apolar inner cells of the
morula.
• Trophectoderm cells acquire gene products
necessary to generate the blastocoelic fluid.
Cavitation
• Required for implantation.
• Trophectoderm initiates implantation via
direct contact with the uterus. Contributes
to the extra-embryonic membranes.
• Inner cell mass: progenitors of the embryo
proper.
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Expansion
• Accumulation of fluid, cavity expands,
embryo = expanded blastocyst.
• The z.p. eventually breaks, the embryo
comes out = hatched blastocyst.
Maternal zygotic transition (MZT)
• Condensed chromo.=no RNA synthesis.
• Eggs contain stockpiles of ribosomes,
messenger RNA, transfer RNA, proteins.
• These molecules are sufficient for embryos
to cleave and survive until the embryonic
genome is activated.
• MZT: development under maternal control
becomes under the control of the embryo.
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Maternal zygotic transition
• The majority of maternal mRNA molecules
accumulated during oogenesis are degraded
and gradually replaced by new zygotic
mRNA molecules.
• Changes in the protein synthesis pattern.
Maternal zygotic transition
• Development beyond stage of MZT
requires mRNA synthesis.
• Translation is required right after
fertilization.
Maternal zygotic transition
• Timing varies from species to species.
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Hamster-mouse: 2-cell stage.
Human: 4-8-cell stage.
Pig: 8-10-cell stage.
Cow and sheep: 8-16-cell stage
Sea urchin: blastula
Xenopus: 4000 cells
Maternal mRNAs
• How does the oocyte distinguishes between
mRNAs that are for use during oogenesis
and mRNA that are to be stored and used
after fertilization only?
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Maternal mRNAs
• Level of polyadenylation: Stored mRNAs
don’t have polyA tail.
• PolyA tail added when the transcripts are
needed.
• Sequences controlling extent and timing of
polyadenylation in the 3’untranslated
region.
Localization of mRNA
• Some mRNAs and proteins are localized to
particular regions of the egg.
• Differentially distributed to blastomeres
during cleavage.
• Determine the fate of blastomeres to which
they are distributed.
Localization of mRNAs
• Ex. in amphibian oocytes: Vg1, localized at the
vegetal pole. Component of the mesoderminducing signals produced by the vegetal
blastomeres.
• In Drosophila oocytes: Numerous localized
transcripts identified. Some involved in
determining the dorsal-ventral axis or anteriorposterior axis.
• In C. elegans, localized mRNAs and proteins
affect the fate of the first cells.
Embryonic polarization
• Can occur
–
–
–
–
During oogenesis
As a consequence of fertilization
During cleavage
Later in development
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Polarization of amphibian embryos
• Initiated at fertilization.
• Sperm-egg fusion can occur
only in the animal pole.
• Pigment granules accumulate
around sperm entry point.
• After fert., egg cortex rotates.
• Rotation reveals grey crescent.
• Site of sperm entry= ventral
side.
• Grey crescent=dorsal side.
Polarization of C. elegans embryos
• Anteroposterior polarity determined by
point of sperm entry = posterior.
• Egg contains P granules initially randomly
distributed.
• Concentrate in the posterior end during
cytoplasmic rearrangement.
• Concentrate in region of new P cell.
Polarization of Drosophila embryos
• Begins during oogenesis.
• Egg has dorso-ventral axis at ovulation.
• Cells on dorsal surface—epidermis or
amnioserosa.
• Cells of ventral side—ventral epidermis,
mesoderm and nervous system.
Gastrulation
• Cells of the blastoderm are translocated to
new positions in the embryos to produce
the 3 primary germ layers:
– Ectoderm: Epiderm, nervous tissues.
– Mesoderm: Muscles, bones and connective
tissues.
– Endoderm: Organs of the gut and accessory
glands.
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In vitro embryo production
• Why produce embryos in vitro?
– Human—Infertility treatment
1st IVF live birth in 1978
In vitro embryo production
• 3 steps:
– In vitro maturation of oocytes.
– In vitro fertilization of the oocytes.
– In vitro development of the embryos.
- Animals- To obtain progeny from an infertile animal
- Reproduction of endangered species
- Research
- Biotechnologies
In Vitro Maturation of Oocytes
• Oocytes recovered by aspiration or slashing
of follicles (ovaries from slaughterhouse).
• Selected and washed 3 times.
• Cultured in TCM-199 + serum + FSH
+pyruvate.
• 22-24 h, 5% CO2 39o C.
Nuclear Maturation of Oocytes
• Oocytes are arrested at the diplotene stage
of first meiotic division until LH surge.
• When oocytes are cultured, meiosis
resumes spontaneously.
• After 22-24 h of culture, oocytes are at the
metaphase II stage.
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In Vitro Fertilization
• Oocytes fertilized with frozen-thawed
semen.
• Sperm prepared by swim up procedure.
• Wash and count.
• In 50 µl drops, 10-12 oocytes per drop, 1
million sperm cell/ml.
• 18-20 h, 5% CO2, 39oC.
In Vitro Development
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Zygotes stripped of cumulus cells.
Transferred to drops of SOF medium.
Culture at lower O2 concentration.
After 24 h, embryos at the 2-4 cell stage,
some 6-cell.
• After 48 h, embryos at the 8-cell stage,
transferred to fresh SOF.
• Blastocysts on Days 8 and 9.
Synthetic Oviductal Fluid
Medium
• Contains salts, energy sources, PVA, Lglutamine and amino acids.
• BSA added at the 8-cell stage.
• No serum, no coculture.
• Other media also used.
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How to study gene expression in
embryos.
Examples of questions
Limitation to embryo production
in vitro.
• If 100 oocytes are incubated with sperm,
75-80 will cleave.
• Approximately 30% will form blastocysts.
• How do embryos develop?
• Which mRNAs and proteins accumulated in the
oocytes play a role in early development?
• What triggers MZT?
• What proteins are necessary for compaction,
cavitation?
• What energy sources do the embryos use?
• Do they have all the enzymes necessary to
metabolize all energy sources?
How to study gene expression in
mammalian embryos.
Examples of questions
• What new mRNAs are synthesized by the
embryos that were not synthesized by the oocytes?
• What genes are expressed in embryos and not in
any other cell types?
• Are different mRNAs present in embryos
produced in vitro compared to embryos produced
in vivo?
• What genes are expressed in the trophectoderm
only?
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Northern blots
RT-PCR
Differential display
Subtractive hybridization
Arrays
RNAi (to study gene function)
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Northern blots
• RNA is extracted from embryos, separated
according to size on a gel and transferred to a
membrane. The membrane is hybridized with a
labelled probe from a gene of interest.
• Advantage: Simple to perform.
• Inconvenient: RNA from large numbers of
embryos required. Only abundant transcripts can
be analyzed.
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•
REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION
•
RNA is extracted from embryos, reverse transcribed to cDNA. Then two synthetic
oligodeoxynucleotides, which can anneal to sequences flanking the sequence of interest are used to
amplify a target DNA segment through repeated cycles of heat denaturation of DNA, annealing of
the primers to complementary sequences and extension of the annealed primers with a thermostable
DNA polymerase. This results in the exponential accumulation of large amounts of a specific DNA
fragment of defined length and sequence.
•
•
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Reverse primer
3'____5'
A 5' _____________________________ 3'
•
•
•
B 3'______________________________5'
5'___3'
Forward primer
•
Forward primer is complementary to strand B
•
Reverse primer is complementary to strand A
•
•
Advantages:
- Does not require large quantities of material, cDNA can be amplified from single oocytes or
embryos in certain cases.
•
•
- Can be quantitative if a standard is included. Can be in real time.
Inconvenient: Can only study expression of genes of known sequences
Reverse transcription-polymerase
chain reaction
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Differential Display-RT-PCR
100
80
actin
catalase
ß-catenin
cytochrome b
Na/K ATPase
U2
U3
5S rRNA
12S rRNA
28S/18S rRNA
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40
20
0
Egg
• PCR-based method to compare RNA pools from
two or more samples.
• Lower primer: oligo-dT primers
• Upper primer = short random primers
• Amplification in the presence of a radio-labelled
nucleotide for detection.
• Differentially expressed cDNA can be cut out of
gels and sequenced= identification of new genes.
2 -5
cell
Morula
Developmental
Stage
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Subtractive hybridization
• PCR-based method.
• To identify mRNA
unique to a cell type.
• Two hybridizations.
• Only molecules present in
the tester only can be
amplified.
DNA microarrays
• Small solid supports onto which the sequences
from thousands of genes are immobilized in an
orderly fashion.
• mRNA of tissue of interest is hybridized to the
array.
• The amount of cDNA bound to each site on the
array is indicative of the level of expression of
these genes.
• Analyzed by software.
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EST sequencing
• Sequencing of short cDNA fragments
RNA interference
• Transfect cells with
small interfering RNAs.
• Formation of RISC.
• The antisense siRNA
guide the RISC to
complementary RNA
molecules.
• RISC cleaves the
mRNA=gene silencing.
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Sources
• Slack J. 2001. Essential Developmental Biology. Blackwell Sciences
Ltd. Oxford UK.
• Browder LW, Erickson CA, Jeffery WR. 1991. Developmental
Biology. 3rd Edition, Saunders College Publishing. Orlando FL, USA.
• Wassarman PM, Albertini DF. 1994. The mammalian ovum. In: The
Physiology of Reproduction. Vol. 1. editied by E. Knobil and JD
Neill. Raven Press. New York.
• Le Moigne A. 1979. Biologie du Développement. Masson, Paris.
• Johnson J. et al. 2004. Germline stem cells and follicular renewal in
the postnatal mammalian ovary.
Sources
• Bilodeau-Goeseels, S., Schultz GA. 1997. Biol Reprod 56, 13231329.
• Schultz GA et al. 1992. Reprod Fertil Dev 4, 361-371.
• Vigneault C. et al. 2004. Biol Reprod 70, 1701-1709.
• De Sousa PA et al. 1998. Mol Reprod Dev 51, 112-121.
• Natales DR. Et al. 2000. Mol Reprod Dev 55, 152-163.
• Giese K et al. 1999. Differential Display. In PCR applications.
Protocols for functional genomics. MA Innis, DH Gelfand, JJ
Sninsky, Eds. Academic Press. San Diego, CA pp 297-306.
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