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ANAT 381 > Making Embryos > Flashcards

Making Embryos Flashcards

1
Q

what must sperm get through to fertilize egg

A

protective coat = zona
cumulus cells = from inside follicle, enclose oocyte when egg ovulated, sperm must get through it

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2
Q

describe how many sperms get through the journey

A

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

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3
Q

name and briefly describe the 3 steps of fertilization

A

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

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4
Q

describe the cumulus cell matrix - generally

A

cell adhesion proteins - adherens
cells stuck together vert tightly before ovulation

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5
Q

describe the cumulus cell matrix - expansion - what is it

A

under goes expansion = excretes substance - secrete matrix = gel like substance
made up of hyaluronan (hylauronic acid matrix, produces thick gel around oocyte)

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6
Q

describe the cumulus cell matrix - expansion - describe pathway

A

lh (from pituitary) –> release of egf receptor ligands by mural granulosa –> activation of egf receptor on cumulus granulosa
egf = epidermal growth factor

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7
Q

describe the cumulus cell matrix - expansion - describe specifics

A

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

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8
Q

what helps sperm get through cumulus - explain

A

sperm hyaluronidase = localized on sperm surface - enzyme
digests, chews through, enzymatic digestion of matrix by enzymes on sperm

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9
Q

describe zona pellucida- proteins

A

zp1-4 (4 only in primates, 3 in mouse)

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10
Q

describe zona pellucida- secretion

A

self assemble extracellular

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11
Q

describe zona pellucida- thickness

A

7 mu m = mouse
human = 15 mu m (quite thick)

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12
Q

what prevents zona assembly

A

genetic deletion of zp2 or zp3 prevents assembly of zona

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13
Q

what prevents zona assembly - explain experiment/why is it difficult

A

delete then see what happens to fertilization process but issue is that zone never produced = cannot determine then

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14
Q

describe penetration through zona pellucida - role of zp2

A

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

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15
Q

describe what could happen if we found out how sperm fuses with egg plasma membrane

A

birth control methods - if identify proteins that help process of fusion to membrane of sperm

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16
Q

what sperm membrane protein is required for sperm egg fusion

A

all tm proteins = stick on cell surface
IZUMO

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17
Q

what is izumo

A

immunoglobulin superfamily member
Detectable after acrosome reaction
has s-s disulphide bond

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18
Q

what happens when izumo knockout

A

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

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19
Q

what egg membrane protein is required for sperm egg fusion

A

juno

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20
Q

describe membrane localization of juno

A

around membrane
izumo binds where juno is

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21
Q

describe anti juno experiment

A

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)

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22
Q

what is another membrane protein required for sperm egg fusion

A

cd9

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23
Q

describe cd9

A

tm protein
member of tetraspanin cell surface protein family
previously identified on various cell types- platelets and lymphocytes

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24
Q

describe cd9 knockout

A

fertile males
severely sub fertile females = not good enough for birth control methods tho

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25
Q

describe molecular basis of fertilization

A

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

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26
Q

what is izumo and juno - meanings

A

izumo = japanese marriage shrine
juno = roman goddess of fertility

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27
Q

what is activation of egg

A

release from developmental arrest
stop at metaphase 2 - arrested, activation = release from arrest = begins development of embryo
AFTER FERTILIZATION

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28
Q

what are the events that must occur for egg activation

A

block to polyspermy
Completion of second meiotic division
initiation of mitotic cell cycles of embryo

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29
Q

describe initiation of mitotic cell cycles of embryos

A

formation of female and male pronuclei
dna rep
cell division - reg cell cycle

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30
Q

what is the mechanism of activation of egg

A

increase in intracellular calcium

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31
Q

what is the mechanism of activation of egg - describe

A

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

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32
Q

what is the mechanism of activation of egg - evidence

A

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

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33
Q

how does the sperm trigger calcium release in egg - 2 hypotheses

A

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

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34
Q

describe pathway of sperm activating egg - calcium release

A

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

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35
Q

describe icsi

A

method of fertilization
works well
inject sperm into egg directly
does not show any negative effects, like systematic effects that reoccur

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36
Q

what is inconsistent with the membrane receptor model

A

success of icsi
since normal fertilization dependent on binding of izumo and juno but in icsi that does not happen at all

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37
Q

How does the sperm trigger Ca2+ release in the egg - 3 specific features

A

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

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38
Q

How does the sperm trigger Ca2+ release in the egg - what is the key

A

sperm specific phospholipase c zeta

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39
Q

explain actual pathway of sperm triggering egg activation

A

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

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40
Q

polyspermy =

A

DEATH

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41
Q

blocking polyspermic fertilization - 2 mechanisms

A

happens quickly after fertilization
Membrane block - not sure if in humans, mainly marine animals
zona block = main one

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42
Q

describe what calcium mediates - egg activation

A

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

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43
Q

what causes zona to block fertilization

A

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

44
Q

explain zona before and after fertilization

A

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

45
Q

when can polyspermy happen

A

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

46
Q

what happens during early embryonic development

A

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

47
Q

describe completion of meiosis 2 - early embryonic development

A

2nd polar body extrusion - sister chromatid segregation
formation of pronuclei - haploid
complete 2nd division
must see 2 polar bodies

48
Q

describe chromosome segregation - early embryonic development

A

cohesin = rec8, around arms, separase separates arms of homologues during meiosis 1
then separase chews near centromeres = sister chromatids separate

49
Q

describe activation of separase - generally

A

cdc20 and apc (anaphase promoting complex, ub ligase) degrades securin (attached to seprase) so now seprase see and degrades cohesin

50
Q

describe activation of separase - meiosis 1

A

spindle assembly checkpoint inhibits apc and cdc20 complex
checks to see if everything ok

51
Q

describe activation of separase - meiosis 2

A

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

52
Q

what happens to protamines after fertilization

A

histones replace protamines in sperm chromatin

53
Q

how fast do histones replace protamines

A

~2hrs = occurs very quickly, few hours after fertilization

54
Q

describe replacement of protamines by histones - whole pathway

A

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

55
Q

what controls replacement of protamines by histones after fertilization

A

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)

56
Q

how long is first mitotic cell cycle

A

~20 hrs
quite long
slow compared to frogs or worms

57
Q

describe first mitotic cell cycle generally

A

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

58
Q

describe first mitotic cell cycle specifically

A

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

59
Q

describe development during cleavage stage

A

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

60
Q

describe development during cleavage stage - length and stuff

A

lasts approx 5 days
6-7 divisions
15-20 hrs / divison

61
Q

describe development during cleavage stage = END RESULT

A

compaction morula - cells become flattened and increase surface area of contact
Blastocyst

62
Q

describe activation of embryonic transcription - gen

A

essential for dev of embryo
active transcription during growth but none during maturation, transcription stops until species specific stage

63
Q

describe activation of embryonic transcription - when specifically

A

major activation occurs at 2 cell stage in mouse
and 4 cell stage in humans
and 8 cell stage in cows

64
Q

describe activation of embryonic transcription - oocyte vs embryonic

A

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

65
Q

what is thought to make an embryo better

A

cleavage process = fast dev rapid at cleavage stage thought to be best embryo - use one that was fastest on film

66
Q

what is blastocyst

A

embryo consists of 50-100 cells and separate lineages begin to differentiate
4-5 days after dev = ready to implant

67
Q

describe lineages of blastocyst - generally

A

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

68
Q

who figured out the different cell types at blastocyst stage

A

richard Gardner
janet rossant
anne mclaren
figured out which cells in embryo are coming from which cells in blastocyst

69
Q

describe what primitive ectoderm gives rise to

A

= epiblast
all tissues of embryo
amnion
allantois
yolk sac mesoderm
(only cells that give rise to rest of tissues)

70
Q

describe what primitive endoderm gives rise to

A

yolk sac endoderm = membrane surrounding embryo during its dev

71
Q

describe what primitive trophectoderm gives rise to

A

= trophoblast
fetal portion of placenta

72
Q

describe cell lineage relationships - shape in mouse vs humans

A

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

73
Q

which genes help go from cells of morula –> trophectoderm

A

cdx2
v important for te, make or keep

74
Q

which genes help go from cells of morula –> icm

A

oct4
sox2
nanog
generate icm, key players in differentiation process

75
Q

which genes help go from cells of icm –> primitive endoderm

A

gat4,6
TURN OFF nanog

76
Q

which genes help go from cells of icm –> epiblast

A

oct4
sox2
nanog
keep same ones

77
Q

how do inner and outer cells acquire different fates - hypotheses

A

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

78
Q

describe when hippo on

A

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

79
Q

describe when hippo off

A

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

80
Q

describe specifying icm vs te - generally

A

in te cells = hippo off, yap stable and activates transcription of target gnes
icm (epiblast +primitive endoderm) = yap degrades and cannot get into nucleus

81
Q

describe specifying icm vs te - specifics - apical domain

A

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

82
Q

describe specifying icm vs te - specifics - basolateral domain

A

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

83
Q

describe specifying primitive ectoderm vs primitive endoderm- through what

A

fibroblast growth factor signalling

84
Q

describe specifying primitive ectoderm vs primitive endoderm - gen

A

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

85
Q

describe specifying primitive ectoderm vs primitive endoderm - specifics

A

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

86
Q

describe what yap/hippo differentiates between

A

morula to icm ORRR TE

87
Q

describe what fgf differentiates between

A

icm to epiblast ORRRR primitive endoderm

88
Q

what is x chromosome inactivation

A

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

89
Q

describe x chromosome inactivation historical foundations

A

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,

90
Q

describe timing and heritability of x chrom inactivation - experiment

A

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

91
Q

describe timing and heritability of x chrom inactivation - generally

A

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

92
Q

describe timing and heritability of x chrom inactivation - women

A

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

93
Q

describe mechanism of x chrom inactivation - generally

A

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

94
Q

describe mechanism of x chrom inactivation - pathway

A

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

95
Q

describe mechanism of x chrom inactivation - proteins

A

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

96
Q

describe Timing and lineage specificity of X-chromosome inactivation- mouse

A

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

97
Q

describe Timing and lineage specificity of X-chromosome inactivation- human

A

Random x inactivation in placenta and embryo

98
Q

describe Timing and lineage specificity of X-chromosome inactivation- marsupial

A

paternal x inactivated
aLWAYS IN all tissues

99
Q

describe Timing and lineage specificity of X-chromosome inactivation- humans and explain more

A

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

100
Q

describe what happens to some genes- x chrom inactivation

A

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

101
Q

describe what happens to some genes- x chrom inactivation XO

A

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

102
Q

describe what happens to some genes- x chrom inactivation XXY

A

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)

103
Q

describe anti izumo experiment

A

binds to izumo and blocks from binding anything else - no fertilization

104
Q

describe genes that specific different lineages

A

all tfs
could specify or just maintain lineage

105
Q

describe specifying icm vs te - experiment kinda

A

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

ANAT 381 (14 decks)

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