Making Embryos Flashcards
what must sperm get through to fertilize egg
protective coat = zona
cumulus cells = from inside follicle, enclose oocyte when egg ovulated, sperm must get through it
describe how many sperms get through the journey
egg ovulated into fallopian tube (oviduct), sperm must travel here, very long journey
more then 50 mil enter but only a few hundred make it to site of fertilization
name and briefly describe the 3 steps of fertilization
1 - penetration through cumulus cell matrix (somatic cells that surround and are ovulated with oocyte, sperm must get through it)
2 - penetration through zona pellucida
3- fusion with egg plasma membrane
describe the cumulus cell matrix - generally
cell adhesion proteins - adherens
cells stuck together vert tightly before ovulation
describe the cumulus cell matrix - expansion - what is it
under goes expansion = excretes substance - secrete matrix = gel like substance
made up of hyaluronan (hylauronic acid matrix, produces thick gel around oocyte)
describe the cumulus cell matrix - expansion - describe pathway
lh (from pituitary) –> release of egf receptor ligands by mural granulosa –> activation of egf receptor on cumulus granulosa
egf = epidermal growth factor
describe the cumulus cell matrix - expansion - describe specifics
ligands released by mural granulosa = egf receptor ligands
then act on receptors on cumulus cells (oocyte, gdf9, bmp15) growth factors secreted by oocyte
work to upregulate genes = has2, tnfaip6, ptgs2
then cumulus cells produce matrix
what helps sperm get through cumulus - explain
sperm hyaluronidase = localized on sperm surface - enzyme
digests, chews through, enzymatic digestion of matrix by enzymes on sperm
describe zona pellucida- proteins
zp1-4 (4 only in primates, 3 in mouse)
describe zona pellucida- secretion
self assemble extracellular
describe zona pellucida- thickness
7 mu m = mouse
human = 15 mu m (quite thick)
what prevents zona assembly
genetic deletion of zp2 or zp3 prevents assembly of zona
what prevents zona assembly - explain experiment/why is it difficult
delete then see what happens to fertilization process but issue is that zone never produced = cannot determine then
describe penetration through zona pellucida - role of zp2
lab at nih
mix mouse egg with human sperm = sperm do not stick
replace mouse zp gene by human homologue and mix transgenic mouse egg with human sperm- delete any zp protein- one at a time
microscopic fluorescent image =
human oocyte with regular sperm = stick
mouse sperm cannot stick onto human egg
if mouse expresses human zp2 = will stick
human sperm stick to human zp2 - important in binding of sperm to zona
describe what could happen if we found out how sperm fuses with egg plasma membrane
birth control methods - if identify proteins that help process of fusion to membrane of sperm
what sperm membrane protein is required for sperm egg fusion
all tm proteins = stick on cell surface
IZUMO
what is izumo
immunoglobulin superfamily member
Detectable after acrosome reaction
has s-s disulphide bond
what happens when izumo knockout
infertile males - needed for male fertility - sperm can still stick to membrane but never go inside egg, capable of doing everything except fusing with membrane, need izumo for final stage for sperm to fuse
fertile females
what egg membrane protein is required for sperm egg fusion
juno
describe membrane localization of juno
around membrane
izumo binds where juno is
describe anti juno experiment
antibody against juno and stain egg
So perm not able to bind to egg
anti juno binds juno so then izumo cannot
izume binds juno (on oocyte membrane)
what is another membrane protein required for sperm egg fusion
cd9
describe cd9
tm protein
member of tetraspanin cell surface protein family
previously identified on various cell types- platelets and lymphocytes
describe cd9 knockout
fertile males
severely sub fertile females = not good enough for birth control methods tho
describe molecular basis of fertilization
Aggregation is key
izumo binds juno - on cell surface
but cd9 helps aggregate juno so clustered on cell surface = bigger target for izumo
Definitely more complicated than this - other proteins that ensure proteins are in proper conformation and bind stably and rapidly
what is izumo and juno - meanings
izumo = japanese marriage shrine
juno = roman goddess of fertility
what is activation of egg
release from developmental arrest
stop at metaphase 2 - arrested, activation = release from arrest = begins development of embryo
AFTER FERTILIZATION
what are the events that must occur for egg activation
block to polyspermy
Completion of second meiotic division
initiation of mitotic cell cycles of embryo
describe initiation of mitotic cell cycles of embryos
formation of female and male pronuclei
dna rep
cell division - reg cell cycle
what is the mechanism of activation of egg
increase in intracellular calcium
what is the mechanism of activation of egg - describe
repeated transient increases of free calcium in cell
released then brought back and keeps going= released into cytoplasm and then stored in stores
repeated surges at regular intervals
triggered by sperm penetration
what is the mechanism of activation of egg - evidence
sperm penetration induces repetitive release of calcium ions (10-15 min interval in mice)
parthenogenic stimuli induce calcium release (egg not fertilized, increase intracellular calcium = will trigger beginning of embryonic dev, sex determination in insects)
Inhibiting release of calcium prevents activation of egg
how does the sperm trigger calcium release in egg - 2 hypotheses
1 - sperm acts as ligand to activate intracellular signalling pathway = like growth factor, triggers intracellular signalling
2 - sperm component enters oocyte cytoplasm, triggers ca relase = some factor in sperm so when enters = release factor and causes release of calcium
describe pathway of sperm activating egg - calcium release
1 hypothesis matches with this pathway = g protein coupled receptor pathway
g protein binds gpcr = phospholipase c breaks down phophatidylinositol phosphate (pip2) (causes activation of protein kinase c) to diacylglycerol and ip3 (calcium release)
BUT NO ONE COULD FIND RECEPTOR
describe icsi
method of fertilization
works well
inject sperm into egg directly
does not show any negative effects, like systematic effects that reoccur
what is inconsistent with the membrane receptor model
success of icsi
since normal fertilization dependent on binding of izumo and juno but in icsi that does not happen at all
How does the sperm trigger Ca2+ release in the egg - 3 specific features
1 = injecting plczeta mrna, but not plcdelta, triggers calcium pulses similar to fertilization and dev to blastocyst = same pulses, other isoforms do not produce pulses tho
2 = quantity produced from injected mrna = quantity in single sperm
3 = sperm extracts immunologically depleted of plczeta cannot trigger calcium release in egg
How does the sperm trigger Ca2+ release in the egg - what is the key
sperm specific phospholipase c zeta
explain actual pathway of sperm triggering egg activation
model not correct = the g protein binding to receptor but all downstream activities correct
the phospholipase c was brought in by sperm, special isoform, triggers all downstream reactions
could be a form of birth control maybe bc fert blocked
polyspermy =
DEATH
blocking polyspermic fertilization - 2 mechanisms
happens quickly after fertilization
Membrane block - not sure if in humans, mainly marine animals
zona block = main one
describe what calcium mediates - egg activation
cortical granule exocytosis
before fert = cortical granules are bags of enzymes near membrane
calcium released at fert triggers cortical granule exocytosis
fuse with egg plasma membrane and dumps contents in subzonal space (between oocyte and zona)
chemically modified zp2 so sperm cannot attach and bind and get into egg
what causes zona to block fertilization
cortical granule exocytosis
sperm usually binds to disulfide bond of zp2
but once cortical granules dump contents = enzyme ovastacin cleaves disulfide bonds so now zp2 will not have sperm bind - no longer recognized by sperm
explain zona before and after fertilization
before = sperm bind
during = modified topology, altered zona by ovastacin
after = sperm cannot bind
Calcium triggers fusion of granules to plasma membrane= release enzyme = modifies zona = sperm no longer bind
when can polyspermy happen
polyspermy if sperm already made it inside zona before the reaction - does not happen often
if polyspermy = fertilized by more than one sperm = tetraploidy, diploid and die
what happens during early embryonic development
completion of meiosis 2
chromosome segregation = cohesin and separase
histones replace protamines in sperm chromatin
first mitotic cell cycle
cleavage stages
activation of embryonic transcription
blastocyst
describe completion of meiosis 2 - early embryonic development
2nd polar body extrusion - sister chromatid segregation
formation of pronuclei - haploid
complete 2nd division
must see 2 polar bodies
describe chromosome segregation - early embryonic development
cohesin = rec8, around arms, separase separates arms of homologues during meiosis 1
then separase chews near centromeres = sister chromatids separate
describe activation of separase - generally
cdc20 and apc (anaphase promoting complex, ub ligase) degrades securin (attached to seprase) so now seprase see and degrades cohesin
describe activation of separase - meiosis 1
spindle assembly checkpoint inhibits apc and cdc20 complex
checks to see if everything ok
describe activation of separase - meiosis 2
emi2 = inhibits cdc20 apc complex
at time of fert = calcium degrades emi2
so then separase active nc apc cdc20 complex becomes active (degrades securin)
calcium release activates 2nd meiotic division so sister chromatids seprate
what happens to protamines after fertilization
histones replace protamines in sperm chromatin
how fast do histones replace protamines
~2hrs = occurs very quickly, few hours after fertilization
describe replacement of protamines by histones - whole pathway
sperm dna associated mainly with protamines (during spermatogenesis= replaces histones on sperm chromatin) –> removal of protamines (so when sperm enters egg = dna 90% with protamines but sperm cannot be used for dna rep since highly condense so get rid of protamines) –>
Assembly of histones
–> organization into nucleosomes (just like chromatin of regular cells)
–> assembly of other chromatin associated proteins
what controls replacement of protamines by histones after fertilization
all of this controlled by egg
histones come from egg and also has enzymes needed to organize chromatin into nucleosomes for replication (transcriptionally active and can be segregated etc)
how long is first mitotic cell cycle
~20 hrs
quite long
slow compared to frogs or worms
describe first mitotic cell cycle generally
once 2nd meiotic division over and protamines replace histones = 2 sets of dna, oocyte and sperm form nuclei and replication begins then 1st mitotic division, 2 cell embryo to 4 cell embryo
describe first mitotic cell cycle specifically
formation of pronuclei then dna rep and 1st mitosis
during first cell cycle = chromosomes form pro nuclei = one for egg and one for sperm, not normal nuclei tho, during 1st cell cycle = genetic info from mom and dad kept separate become mixed during 1st mitosis, at 2 cell embryo
standard cell cycle after
describe development during cleavage stage
period of development before implantation into uterus but after fert
cleavage divisions (dna rep and mitosis without cell growth) produce an embryo with 50-100 cells
no increase in mass of embryo
describe development during cleavage stage - length and stuff
lasts approx 5 days
6-7 divisions
15-20 hrs / divison
describe development during cleavage stage = END RESULT
compaction morula - cells become flattened and increase surface area of contact
Blastocyst
describe activation of embryonic transcription - gen
essential for dev of embryo
active transcription during growth but none during maturation, transcription stops until species specific stage
describe activation of embryonic transcription - when specifically
major activation occurs at 2 cell stage in mouse
and 4 cell stage in humans
and 8 cell stage in cows
describe activation of embryonic transcription - oocyte vs embryonic
oocyte mrna and protein = degrades, happens right at fertilization, as embryo becomes genetically active
replaced by embryonic mrna and protein , gene expression, extended out for several cycles
what is thought to make an embryo better
cleavage process = fast dev rapid at cleavage stage thought to be best embryo - use one that was fastest on film
what is blastocyst
embryo consists of 50-100 cells and separate lineages begin to differentiate
4-5 days after dev = ready to implant
describe lineages of blastocyst - generally
Blastocyst = ball of cells filled with fluid
blastocyst cavity = blastocele
3 cell lineages = descendants of each become diff cells
trophectoderm
inner cell mass = epiblast and primitive endoderm
who figured out the different cell types at blastocyst stage
richard Gardner
janet rossant
anne mclaren
figured out which cells in embryo are coming from which cells in blastocyst
describe what primitive ectoderm gives rise to
= epiblast
all tissues of embryo
amnion
allantois
yolk sac mesoderm
(only cells that give rise to rest of tissues)
describe what primitive endoderm gives rise to
yolk sac endoderm = membrane surrounding embryo during its dev
describe what primitive trophectoderm gives rise to
= trophoblast
fetal portion of placenta
describe cell lineage relationships - shape in mouse vs humans
mouse= looks dif than human
morphological rearrangement could lead to different arrangement = push down kinda
after implantation after proliferating and moving around and other morphological rearrangements
which genes help go from cells of morula –> trophectoderm
cdx2
v important for te, make or keep
which genes help go from cells of morula –> icm
oct4
sox2
nanog
generate icm, key players in differentiation process
which genes help go from cells of icm –> primitive endoderm
gat4,6
TURN OFF nanog
which genes help go from cells of icm –> epiblast
oct4
sox2
nanog
keep same ones
how do inner and outer cells acquire different fates - hypotheses
inside outside model = cells adopt inner or outer positions, te = outside cells, icm = inside cells, FATE DETERMINED BY POSITION
polarity model = cells preselected by means of differential gene expression to be ether icm or te
but not matter what = cells on inside icm and cells on outside te
describe when hippo on
hippo on = leads to cascade of phosphorylation reactions triggered by kinases = leads to phosphorylation of yap/taz so then yap/tza degraded (ub)
so cannot get into nucleus = no expression of target genes
describe when hippo off
no phopshorylation so yap/taz stabilized
yap/taz enters nucleus and works together with tead = complex binds to dna and regulates transcription - activates downstream genes
describe specifying icm vs te - generally
in te cells = hippo off, yap stable and activates transcription of target gnes
icm (epiblast +primitive endoderm) = yap degrades and cannot get into nucleus
describe specifying icm vs te - specifics - apical domain
amot = in outside cells that are not in contact with any other cells
amot recruited to apical domain
amot cannot recruit the machinery that phosphorylated and inactivates yap
exposed membrane attracts amot and inactivated amot = cannot activate hippo = frees up yap and goes into nucleus = yap on
describe specifying icm vs te - specifics - basolateral domain
no apical domain so amot can recruit machinery that phosphorylates and inactivates yap
no apical domain so amot goes elsewhere
so yap does not go into nucleus
describe specifying primitive ectoderm vs primitive endoderm- through what
fibroblast growth factor signalling
describe specifying primitive ectoderm vs primitive endoderm - gen
epiblast = characterized by expression of nanog
pe = characterized by expression of gata6
Initially all cells express both nanog and gata but then sort out = in either = express one, once made decision = sort themselves out
describe specifying primitive ectoderm vs primitive endoderm - specifics
controlled by interaction of fgf and its receptor
epiblast cells = cells that produce fgf4 = secrete ligand, become epiblast cells
cells that receive ligand, have fgf receptor (receptive to fgf4 made by epiblast cells) = ones with ligand receptor complex activation become primitive endoderm
describe what yap/hippo differentiates between
morula to icm ORRR TE
describe what fgf differentiates between
icm to epiblast ORRRR primitive endoderm
what is x chromosome inactivation
in males = the single x chrom remains active
in females = one x chrom becomes inactivated in all cells, could be x from mom or dad, do not know
goal = equalize gene dosage of x chrom encoded genes between males and females
describe x chromosome inactivation historical foundations
Murray Barr & E G Bertram = first noticed barr body = female dogs had it, dark staining body
Susumu Ohno = one of x chroms in females is the barr body
mary lyon = timing and heritability of x chrom inactivation,
describe timing and heritability of x chrom inactivation - experiment
transgenic mice cells, one x = labelled with red and other with green
in some cells = red chrom active and in others = green chrom active
see big clones - clumps of cells = inactivation happens early, before proliferation
describe timing and heritability of x chrom inactivation - generally
inactivation of one x occurs early = during embryonic dev
early decision and all retain same decision, perpetrated and maintained - all offspring of cells maintain it
describe timing and heritability of x chrom inactivation - women
females = mosaics for x chrom linked genes
controlled by one x chrom and other x chroms = can have both types, ex = cats
Half of cells have dad x inactive and other half have mom x inactive
These x chroms encode slightly diff genes
describe mechanism of x chrom inactivation - generally
xic = x inactivation center, region of chrom known for inactivation, power to silence genes, take this region and insert into autosome and silence genes around it
xist = included in gene, long non coding rna
Micrograph = in situ hybridization = see that one x chrom silent, xist makes rna transcripts, associated with one crhom = the silent x chrom
describe mechanism of x chrom inactivation - pathway
xist rna expressed from one x chrom
xist rna coats that x chrom (in cis)
xist coated x is transcriptionally inactive, expressed xist gene = makes rna that coats and helps silence chrom
Initially on both x’s then one - localized to inactive x
describe mechanism of x chrom inactivation - proteins
xist = recruits silencing proteins to help xist bind to chrom
proteins nucleated by xist
xist rna supramolecular complex = coat and turn off gene expression
active x does not express xist
through coordinated action of xist rna and big supramolecular complex
describe Timing and lineage specificity of X-chromosome inactivation- mouse
paternal x inactivated preferentially in placenta, in xx individuals = x from mom active and x from dad inactive
random x inactivation in neonate = embryo, random, 50/50
describe Timing and lineage specificity of X-chromosome inactivation- human
Random x inactivation in placenta and embryo
describe Timing and lineage specificity of X-chromosome inactivation- marsupial
paternal x inactivated
aLWAYS IN all tissues
describe Timing and lineage specificity of X-chromosome inactivation- humans and explain more
both x active in preimplantation cells, cleavage and up to blastocyst stage
x inactivation occurs around time of implantation = shortly after
choice made early - after one week x that is inactivated stably maintained
describe what happens to some genes- x chrom inactivation
some genes escape inactivation = not all fully silenced = up to 20% escape inactivation
human - gpm6b, ca5b, kdm5c (demethylase) partially conserved
mouse = some of same genes escape inactivation in humans and mice - kdm6a
describe what happens to some genes- x chrom inactivation XO
most die
some survive = small amount
somatic differences = compared to xy and xx
Absence of x has phenotypic effect
physiologically diff = than in ppl with x
genes on inactive x important
describe what happens to some genes- x chrom inactivation XXY
middle x = leaky
very tall
gene expression from 2nd x chrom= responsible for various characteristics of klinefelter syndrome (apart from those related to sperm production)
describe anti izumo experiment
binds to izumo and blocks from binding anything else - no fertilization
describe genes that specific different lineages
all tfs
could specify or just maintain lineage
describe specifying icm vs te - experiment kinda
stain with antibodies against yap and cdx2 (expressed in te cells)
yap present in same cells expressing cdx2
no yap in nuclei of icm = hippo on and cascade of phosphorylations = yap degrades
