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When does meiosis occur in a mammalian egg?
The mammalian egg is covered by ____.
Cumulus cells and ECM (extra-cellular matrix)
Where does fertilization occur?
The fallopian tube
What drives sperm?
What kind of sperm can fertilize an egg?
Capacitated AND Acrosome(cap)-reacted sperm
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
Binding of sperm to an egg resulting in egg-activation.
What penetrates the egg during fertilization?
The pro-nuclues (haploid nuclues)
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
Why is the glycoprotein needed?
To survive the acidic environment in the female's reproductive system
What is the acrosome?
Tip of sperm containing enzymes and proteins required for the sperm to digest the zona pellucida
What are the enzymes in the acrosome for?
digesting the zona pellucida (not for membrane fusion)
What allows for membrane fusion?
sperm receptors on the egg
What part of the sperm actually enters the egg?
What sperm-egg interactive proteins are on the sperm?
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
What is Beta-1,4-galactosyltransferase and how was it identified initially?
- sperm-zona binding factor (not an enzyme)
- Mouse knockouts
What is ADAM2/3?
Enzymes required for binding to zona.
Sperm binding => ____?
What is the Izumo protein?
IgSF membrane protein expressed in sperm critical/req'd to sperm-egg membrane fusion (not for zona binding)
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
ADAM proteins on sperm cell are expressed as ____.
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.
How would you identify a binding receptor such as Alpha-6Beta-1?
Find antibodies for Izumo and and immunoprecipitate
How would image the fertilization process?
Label CD9 (for example) with GFP
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
The vitelline membrane _____, which protects the zygote from ____ .
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
What proteins make up the vitelline layer?
p160 and redezvin
What does p160 do?
It anchors the vitelline layer to the plasma membrane
What does redezvin do?
It forms the scaffold that separates the fertilization envelope from the plasma membrane
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
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)
____ results in ____ zygote and ____ polar bodies because the ____ nucleus is present.
Meiosis, diploid, haploid, sperm
What are three mechanisms for zygotic transcription silencing?
- Chromatin-mediated repression
- Deficiencies in transcription machinery
- Transcriptional repression by rapid cell cycles
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)
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
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)
The maternal factors dominate from one cell to ____ stage, but drop off and give way to the zygotic by the ____ stage.
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
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
Describe Wnt pathway
Negatively regulates POP-1 which negatively regulates E-Cells development resulting in endoderm
Wnt pathway has two other maternal factors, ____ and ____, both of which regulate a zygotic factor: ____.
POP-1 (negative), SKN-1 (positive), end-1
In AB lineages (ecto), two maternal factors are ____, a ligand, and ____, a receptor.
When does mammalian MZT occur, and what is it called?
Between the 1 and 2-celled stage, zygotic gene activation (ZGA)
What are the three phases of mammalian MZT?
- Maternal mRNA degradation
- Replacement with zygotic transcripts (housekeeping - not cell-specific)
- Embryonic-specific mRNA
Give a couple examples of functions regulated by housekeeping transcripts.
- Glucose metabolism
- DNA polymerase
Zygotic mRNA is needed for....
proper cleavage of 2-celled stage embryos
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
Transcription at the ___ pronucleus occurs earlier than at the ___ pronucleus.
MPN lack ____ found in the early ____.
transcriptional repressors, embryo
Why do MPN have earlier transcription?
Sperm are tiny and have no repressors present.
Before fertilization, the DNA is packed with ___ and not ___.
What are protamines?
Arg-rich nuclear proteins which are later replaced by (highly acetylated histones.
What do protamines do?
They inhibit transcription.
The egg can inactivate male genes using ___.
What are the first zygotic genes?
Hsp70.1, MuERV-L, U2afbp-rs, elF-1A
First gene transcript (viral)
translational elongation factor
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
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]
What are some maternal-effect genes?
Mater, Hsf1, Zar1, Npm2, Zfp36I2
Unknown function; required for proper development past 2-celled stage embryo
controls Hsp70.1 expression
Critical for transition from S phase to G2 (1-celled stage)
Chromatin remodeling (acetylation)
RNA-binding protein; bind to AU-rich sequences
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)
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)
How is RNA localized?
- Like a postal service
- zipcode: cis-acting elements - heterogeneous nuclear ribonucleoproteins (hnRNPs)
What is the cortex of the cell?
Region underneath cell membrane where mRNA can be stored (with poly-a tail removed for storage)
mRNA localization occurs in ___ and ___ cells.
Why does mRNA localization occur?
It is more cost-efficient to move RNA to where a protein is needed.
What are a few examples of mRNA localization?
- Bicoid in Drosophila
- Axonal growth cones
- Cultured endothelial cells
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
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
The ___ has the ability to select ___ and ___ splice sites and therefore executes ___ splicing.
5', 3', alternative
The selection of splice sites is governed by:
- cis-acting sequences in the exons and introns
___ indicates the 5' splice site.
___ indicates the 3' splice site.
___ is needed to provide energy for splicing.
___ 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 ___.
Give examples of strong and weak splice sites.
- UUUAG: strong 3' splice site
- AAUAG: week 3' splice site
mRNAs with short half-lives have ___.
AU-rich elements (AREs)
Removal of poly(A) tail thru ARE-dependent pathway requires ___.
pol(A) ribonuclease (PARN)
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
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?
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?
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
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.
A ___ interacts with a cleavage site, protecting it from degradation.
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
Stored maternal transcripts lose their ___. ___ found in maternal transcripts add them back.
poly(A) tail, CPE
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.
__ is a 5' CAP binding protein, and ___ is a 3' poly A binding protein.
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.
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.
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.
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
- 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
- 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
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)
- 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
- 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
- Maintains potency of ESCs
- Expressed in extra-embryonic ectoderm
Knockdown of Sox2 results in ___.
polyploidy and trophectoderm differentiation from ES
- 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
- Myocardial infarction
- liver disease
What are the sperm binding proteins?
What are the egg binding proteins?
- 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