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When does meiosis occur in a mammalian egg?
At fertilization
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The mammalian egg is covered by ____.
Cumulus cells and ECM (extra-cellular matrix)
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Where does fertilization occur?
The fallopian tube
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What drives sperm?
chemotaxis
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What kind of sperm can fertilize an egg?
Capacitated AND Acrosome(cap)-reacted sperm
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What are the advantages of having the ECM/Zona Pellucia?
- Eliminates polysperming
- Protects during "journey" thru the fallopian tube
- Selects for healthy sperm
- Eliminates cross-species fertilization
-
What in the fallopian tube also protects against cross-species fertilization?
The pH of the fallopian tube is usually unique to a species
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Define fertilization.
Binding of sperm to an egg resulting in egg-activation.
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What penetrates the egg during fertilization?
The pro-nuclues (haploid nuclues)
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What is the result of sperm capacitation?
- Exposure of transmembrane proteins necessary for egg binding by removal of GLYCOPROTEIN layer
- Acrosomal cap becomes exposed
- Spermatozoa becomes more motile
-
When does sperm capacitaion occur?
After ejaculation and during its ascension to/thru the fallopian tubes
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Why is the glycoprotein needed?
To survive the acidic environment in the female's reproductive system
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What is the acrosome?
Tip of sperm containing enzymes and proteins required for the sperm to digest the zona pellucida
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What are the enzymes in the acrosome for?
digesting the zona pellucida (not for membrane fusion)
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What allows for membrane fusion?
sperm receptors on the egg
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What part of the sperm actually enters the egg?
The pronucleus
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What sperm-egg interactive proteins are on the sperm?
- PH-20
- Beta-1,4-galactosyltransferase
- ADAM2/3
- Izumo
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What is PH-20?
- The "first protein" sperm-egg interaction
- Another protection against cross-species fertilization
- Sperm enzyme(hyaluronidase) that facilitates binding/degredation of zona pellucida and also protects against cross-species fertilization
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What is Beta-1,4-galactosyltransferase and how was it identified initially?
- sperm-zona binding factor (not an enzyme)
- Mouse knockouts
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What is ADAM2/3?
Enzymes required for binding to zona.
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Sperm binding => ____?
Fertilization
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What is the Izumo protein?
IgSF membrane protein expressed in sperm critical/req'd to sperm-egg membrane fusion (not for zona binding)
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What is CD9?
In mammals, transmembrane protein expressed in egg with four domains expressed in egg that binds to integrins (receptors on sperm) and is critical for sperm-egg membrane fusion
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ADAM proteins on sperm cell are expressed as ____.
Heterodimers: adam1b/2
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Describe binding between egg and sperm.
- ADAM1B/2 on sperm bind to Alpha-6Beta-1 on the egg
- CD9 on egg binds to binds to integrins on sperm
- Izumo on sperm binds to receptor on egg.
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How would you identify a binding receptor such as Alpha-6Beta-1?
Find antibodies for Izumo and and immunoprecipitate
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How would image the fertilization process?
Label CD9 (for example) with GFP
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Upon fertilization, describe how polyspermy is prevented.
- 1. Influx of calcium releases enzymes from vesicles
- 2. Enzymes separate outer and inner layer of egg membrane (fertilization/vitelline envelope and plasma membrane) with increased physical distance within 30 sec
- 3. The polarity of the membrane changes setting up an electrical block
- 4. Fertilization envelope becomes biochemically and mechanically stable within minutes filtering out larger particles
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The vitelline membrane _____, which protects the zygote from ____ .
hardens, infection
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What effect does the location of fertilization on the egg have on future development in a mouse?
- It establishes the "great circle" between the vegetal and animal poles.
- The point of sperm entry is the future anterior of the animal.
- Two polar bodies ar located near animal pole
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What proteins make up the vitelline layer?
p160 and redezvin
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What does p160 do?
It anchors the vitelline layer to the plasma membrane
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What does redezvin do?
It forms the scaffold that separates the fertilization envelope from the plasma membrane
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List ways for polyspermy blocking.
- 1. Removal of sperm receptors
- 2. Modfication of the vitelline layer (rigidity and prevention of covalent bonding)
- 3. Egg activation
- 4. Regulation by CGSP1
- 5. Charge change
- 6. Increase membrame/vitelline layer distance
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How is meiosis restarted?
- Phospholipase C pathway: DAG and IP3 messengers released
- IP3 releases calcium in storage which removes the meiotic arrest (i.e. activates meiosis)
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____ results in ____ zygote and ____ polar bodies because the ____ nucleus is present.
Meiosis, diploid, haploid, sperm
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What are three mechanisms for zygotic transcription silencing?
- Chromatin-mediated repression
- Deficiencies in transcription machinery
- Transcriptional repression by rapid cell cycles
-
Chromatin-mediated repression
gene-specific methylation patterns; histones and DNA
-
Deficiencies in transcription machinery
basal (i.e. all necessary) transcriptional proteins are absent
-
Transcriptional repression by rapid cell cycles
Very few genes are transcribed during DNA replication or during mitosis (condensed DNA: no transcription)
-
Describe the initiation of zygotic regulation
- In most species, early development is regulated by maternal factors
- Called midblastula transition or maternal-zygotic transition (MZT)
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Maternal factors drive:
- Axis formation
- Cleavage axis
- Fate determination of early blastomeres
- Cell cycle rate
-
Describe midblastula transition or maternal-zygotic transition
- Cell cycles begin to lengthen
- maternal mRNAs begin to degrade - 3' UTR-specific degredation
-
Describe the initial cell cycle (before lengthening)
- G1 and G0 are not used
- mitosis -> S -> mitosis -> S, etc
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What drives the transition to zygotic control?
- Activation of transcription in the zygote
- Rapid degradation of maternal mRNAs
-
Describe the activation of transcription in the zygote
- Rapid cell divisions allow for the "dilution" of transcriptional repressors when the zygote divides
- Some unknown factor(s) regulate the timing of MZT (histones in some species)
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The maternal factors dominate from one cell to ____ stage, but drop off and give way to the zygotic by the ____ stage.
blastula, gastrula
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Describe MZT in C. elegans
- Zygotic transcription is initially repressed by PIE-1 in cells that will give rise to germ cells (P lineage)
- In the EMS (endo) and AB (ecto) lineages, the RNAP II is phosphorylated (active)
- There are two germ layers: endo and ecto
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In C. elegans, how is the EMS lineage regulated?
- Requires zygotic transcription
- SKN-1 (maternal transcriptional factor): Activates genes in EMS lineage
- Wnt (maternal transcriptional factor): restricts endodermal fate to the E cells derivatives
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Describe Wnt pathway
Negatively regulates POP-1 which negatively regulates E-Cells development resulting in endoderm
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Wnt pathway has two other maternal factors, ____ and ____, both of which regulate a zygotic factor: ____.
POP-1 (negative), SKN-1 (positive), end-1
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In AB lineages (ecto), two maternal factors are ____, a ligand, and ____, a receptor.
Lag-2, Lin-12
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When does mammalian MZT occur, and what is it called?
Between the 1 and 2-celled stage, zygotic gene activation (ZGA)
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What are the three phases of mammalian MZT?
- Maternal mRNA degradation
- Replacement with zygotic transcripts (housekeeping - not cell-specific)
- Embryonic-specific mRNA
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Give a couple examples of functions regulated by housekeeping transcripts.
- Glucose metabolism
- DNA polymerase
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Zygotic mRNA is needed for....
proper cleavage of 2-celled stage embryos
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What does alpha-amanitin do?
It inhibits transcription by repressing RNA polymerase II which results in no cell division
-
____ RNAs are transcribed at the 1-celled stage, but ___________ happens at the 2-celled stage.
Few, a large burst of transcription and translation
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Transcription at the ___ pronucleus occurs earlier than at the ___ pronucleus.
Male, female
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MPN lack ____ found in the early ____.
transcriptional repressors, embryo
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Why do MPN have earlier transcription?
Sperm are tiny and have no repressors present.
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Before fertilization, the DNA is packed with ___ and not ___.
protamines, histones
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What are protamines?
Arg-rich nuclear proteins which are later replaced by (highly acetylated histones.
-
What do protamines do?
They inhibit transcription.
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The egg can inactivate male genes using ___.
imprinting/methylation
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What are the first zygotic genes?
Hsp70.1, MuERV-L, U2afbp-rs, elF-1A
-
-
MuERV-L
First gene transcript (viral)
-
elF-1A
translational elongation factor
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Describe the first zygotic genes.
- All are transient
- Necessary for normal development from 2-celled stage on (ZGA)
- Necessary for initiation of zygotic transcription, translation, RNA processing, and metabolism
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Describe maternal mRNA degradation
- 3'-UTR sequence specific
- microRNA (miRNA)-driven; from intronic sequences (not specific)
- Maternally-derived proteins (maternal RNAs that are translated upon fertilization)
- AU-rich [not sure if this is correct]
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What are some maternal-effect genes?
Mater, Hsf1, Zar1, Npm2, Zfp36I2
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Mater
Unknown function; required for proper development past 2-celled stage embryo
-
Hsf1
controls Hsp70.1 expression
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Zar1
Critical for transition from S phase to G2 (1-celled stage)
-
Npm2
Chromatin remodeling (acetylation)
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Zfp36I2
RNA-binding protein; bind to AU-rich sequences
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List 4 broad areas of post-transcriptional regulation.
- mRNA processing
- mRNA stability
- mRNA localization
- mRNA translational regulation
-
List a couple methods of post-transcriptional regulation in the nucleus.
- Transcriptional control affecting RNA transcript
- RNA processing control affecting mRNA (on exons)
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List a few methods of post-transcriptional regulation in the cytosol.
- RNA transport and localization (might be stored as needed)
- Translation control
- mRNA degradation control (leads to inactive mRNA)
- protein activity control (can lead to inactive protein)
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How is RNA localized?
- Like a postal service
- zipcode: cis-acting elements - heterogeneous nuclear ribonucleoproteins (hnRNPs)
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What is the cortex of the cell?
Region underneath cell membrane where mRNA can be stored (with poly-a tail removed for storage)
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mRNA localization occurs in ___ and ___ cells.
adult, embryonic
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Why does mRNA localization occur?
It is more cost-efficient to move RNA to where a protein is needed.
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What are a few examples of mRNA localization?
- Bicoid in Drosophila
- Axonal growth cones
- Cultured endothelial cells
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What are the functions of RNA localization?
- High concentration of proteins
- Gradients of morphogen
- Cell lineage specification
- Association with specific subcellular structures
- Localized translation neurons
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Describe RNA localization cis-acting elements
- Most in 3'UTR
- No conserved consensus sequences
- Form secondary clusters and/or clusters of elements
- Serve as RNA-binding protein motifs
-
Approximately ___% of mammalian genes are processed using ___.
75, alternative splicing
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The ___ has the ability to select ___ and ___ splice sites and therefore executes ___ splicing.
5', 3', alternative
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The selection of splice sites is governed by:
- Proteins
- phosphorylation
- hnRNAs
- cis-acting sequences in the exons and introns
-
___ indicates the 5' splice site.
GU
-
___ indicates the 3' splice site.
AG
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___ is needed to provide energy for splicing.
ATP
-
___ is the branch point in splicing and is bound to protein (no RNA)
-
What are the three required sites in splicing?
GU, AG, branch point
-
Describe DexD/H box proteins.
- RNA-dependent ATPases
- Specific to sites
- Change spliceosomal structure
-
Splicing can take place ____.
Co-transcriptionally, i.e. multiple ORFs used to make a single mRNA
-
Sequences can regulate specific splice sites and are found on ___ and not ___.
mRNA, DNA
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Give examples of strong and weak splice sites.
- UUUAG: strong 3' splice site
- AAUAG: week 3' splice site
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mRNAs with short half-lives have ___.
AU-rich elements (AREs)
-
Removal of poly(A) tail thru ARE-dependent pathway requires ___.
pol(A) ribonuclease (PARN)
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How do cap and tail of mRNA provide stability?
Allow protective binding proteins to bind.
-
How can mRNA stability be regulated?
- Removal of 5' cap
- Shortening of poly(A) tail
- cis-acting sequences (mostly in 3' UTR)
- Trans-acting factors
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The exosome is a cellular structure for ___ and is found in ____.
RNA processing and degradation, the nucleus and the cytoplasm
-
Describe a few types of RNA decay.
- Deadenylation-mediated: non-specific
- Nonsense-medited: more specific; requires PTC (protein complex)
- ARE-mediated: specific, i.e. sequence targetted
-
How do you up-regulate the binding of TTP protein?
phosphorylation
-
How do you down-regulate the binding of of TTP protein?
Make the TTP protein from scratch
-
Describe deadenylation (non-specific) mRNA decay pathway.
- Dadenylation (removal of elongation factor)
- Decapping and removal of poly(A) tail
- Degradation by exonuclease and exosome
-
What protects the mRNA during translation?
The ribosome
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Describe ARE (specific) mRNA decay pathway.
- ARE sequence recruits exosome, destabilizes translation process, and allows PARN to deadenylate
- Decapping enzyme removes cap after PARN is done
- Degradation by exonuclease and exosome
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Describe how specific RNA degradation differs from non-specific.
- ARE sequences used for targetting
- One enzyme for removing cap
- Two proteins bring exosome to site
- Different exonuclease used
- Exosome binds while poly(A) tail still present
- Specific takes precendence (happens first if both types of RNA are present)
-
Where does the processing of RNA occur?
In the nucleus (degredation can occur anywhere)
-
An ___ degrades from the end, while a ___ degrades from the middle.
exonuclease, endonuclease
-
A ___ interacts with a cleavage site, protecting it from degradation.
stabilizer
-
Which method of degradation is used?
It depends on where the mRNA is localized.
-
mRNAs that are localized to specific areas are ____.
not translated in transit, but once they reach their final destination
-
Translationally dormant maternal mRNAs contain ___ signal and ___, which signals ___ after fertilization which results in ___.
poly(A) signal, CPE, cytoplasmic polyadenylation, upregulation of translation
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Stored maternal transcripts lose their ___. ___ found in maternal transcripts add them back.
poly(A) tail, CPE
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Explain the steps for translation initiation of maternal transcript.
- Phosphorylation of CPEB
- Release of maskin (which had bound eIF4E and CPEB)
- cytoplasmic polyadenylation specificity factor (CPSF) binds
- poly(A) polymerase (PAP) binds and adds poly(A) tail
- poly(A) binding protein I (PABPI) binds to protect poly(A) tail
- eIf4G proteins bind to cap, eIF4E
-
Translation of maternal mRNA has a ___ model with aid of proteins.
Closed loop
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__ is a 5' CAP binding protein, and ___ is a 3' poly A binding protein.
eIF4G, PABP
-
What are the benefits of a closed-loop model of translation?
- Protects against mRNA degredation
- Increases translational efficiency due to small ribosome recognition
- Only mature mRNAs (w/5'CAP and 3' polyA tail) are translated; truncated mRNAs are not.
-
Why is it good not to translate truncated mRNA?
It is a waste of resources to translate something that won't function as desired.
-
A ___ poly(A) tail results in a greater efficiency of translation.
longer
-
Translation can be blocked by:
- preventing poly(A) tail adenylation or deadenylation
- masking mRNA (steric hindrance - blocking protein is very large)
- Repression of 60S (large) ribosomal subunit
-
Repression of the 60S ribosomal subunit is rarely used in development because ____.
it usually causes death of the organism
-
Blocking of translation usually (but not always) occurs by proteins binding to the UTR in the ___ region.
3'
-
5' end blocking can include ___.
- blocking the start codon
- Blcoking cap, which blocks elongation factors
-
In mRNA masking, what proteins can change mRNA conformation?
Ybox proteins, CPEB
-
A low concentration of Ybox proteins ___ translation.
promotes
-
CPEB binds to ___ which binds ___ which ___ translation initiation.
maskin, eIF4E, prevents
-
Embryonic deadenylation in frogs requires:
- cis-acting elements including AREs
- Proteins including poly(A) specific RNase and embryonic deadenylation element binding protein (EDENBP)
-
Stem cells must be able to:
- Undergo cell renewal
- Give rise to many cell types (except sperm stem cells)
- Repopulate a tissue in vivo
-
What are the three "grades" of mammalian stem cells?
Totipotent, pluripotent, multipotent
-
Describe totipotent stem cells.
Zygote only since no others give rise to extraembryonic tissue
-
Pluripotent
- Inner mass cells thru 8-cell stage
- Give rise to any fetal or adult cell type, but cannot develop into fetal or adult animal since they cannot contribute to extraembryonic tissue
-
Multipotent
- Give rise to various cells of a specific tissue type
- e.g. skin stem cells or hematopoietic stem cell (HSC)
-
With each cell division, stem cells ____.
lose the capacity of giving rise to multiple lineages (i.e. become less potent)
-
True or false: All adult tissues possess stem cells.
Actually, we don't know the answer. It is thought that adult heart cells do not have stem cells.
-
Describe embryonic stem cells (ESCs)
- 8-celled stage from IMC
- Pluripotent
-
The pluripotency of ESCs is maintained by:
- Epigenetic mechanisms (chromatin packaging)
- Transcriptional/translational mechanisms
-
List a few epigenetic mechanisms
- Imprinting (inactivation of DNA)
- Chromatin changes (e.g. methylation of histones ACTIVATES specific genes)
- X-inactivation
- Patterning by Hox genes
-
__ studies shows that some transcription factors such as ___ are required for potency.
Loss of function; Oct3a/4, Nanog, Sox
-
How do transcription factors related to potency work?
- Keep expression by positive feedback loops
- Suppress genes involved in lineage determination/differentiation
- Activate genes invovled in chromatin regulation (e.g. histone modification, DNA methylation)
-
Describe Oct3/4 (POU5F)
- Concentration dependent
- Present at 4-8 cell stage embryo
- At low level, represses differentiation
- At high level, differentiation of primitive endo and mesoderm
-
Knockout of Oct3/4 results in ___
absent ICM and trophectoderm induction
-
Describe Nanog
- Expressed in ESCs which keeps them undifferentiated at high levels of expression
- Prevents formation of primitive endoderm (intended to let embryo travel to uterus)
-
Knockout of Nanog results in ___.
ESCs developing into blastocyst stage and then dying
-
Describe Sox2
- Maintains potency of ESCs
- Expressed in extra-embryonic ectoderm
-
Knockdown of Sox2 results in ___.
polyploidy and trophectoderm differentiation from ES
-
Differentiation involves:
- Silencing of genes by formation of heterochromatin
- Activation of differentiation genes
-
What are terminally differentiated cells?
Adult cells that don't divide (e.g. skin cells)
-
Describe the steps of reproductive/therapeutic cloning.
- Unfertilized egg has DNA removed
- Egg injected with adult cells to be cloned
- Cell division leads to early embryo
- Reproduction: embryo placed in foster mother
- Therapeutic: embryonic cells transferred to culture dish
-
Describe iPS process
- Adult cells taken
- Introduction of three key genes/proteins
-
What is graft vs. host disease?
Transplantation is rejected by patient
-
Describe autologous stem cell therapies
- Origin: host
- Recipient: same person
- No rejection
-
Describe allogenic stem cell therapies
- Origin: donor
- Recipient: different person
- Possible rejection
-
Give some examples of human medical applications of stem cell research
- Diabetes
- Myocardial infarction
- parkinson's
- liver disease
-
What are the sperm binding proteins?
- PH-20
- Beta-1,4-galactosyltransferase
- ADAM1b/2/3
- Izumo
-
What are the egg binding proteins?
-
Preventing polyspermy
- Sperm must travel "long" way thru fallopian tube - many sperm weeded out
- Only capacitated(activated) and acrosome-reacted sperm can fertilize egg
- ECM/zona pellucia is an extra layer and uses binding proteins for recognition
- sea urchin: change in electric potential (fast block)
- cortical granule reaction: mechanical (slow block)
-
Describe cortical granule reaction
- Initiated by calcium ion concentration gradient
- Dissolve protein posts increasing space between envelope and membrane.
- Clip binding receptors of any attached sperm
- Hardens fertilization envelope
- Coating formed around egg
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