Lecture 6 - Cellular Aspects of Ovulation and Fertilization Flashcards

Question 1

Q

Meiosis in the female

Answer

A

oogonium (2n)
primary oocytes (2n) (+ polar body)
secondary oocyte (n) (+ polar body)

meiosis between oogonium and primary oocyte

meiosis between primary and secondary oocyte

Question 2

Q

Meiosis in females starts with

Answer

A

oogonium

in men - spermatogonia
in females, have no oogonia because they have already entered meiosis 6 months before they’re born

Question 3

Q

Primary oocyte v Polar body

Answer

A

the primary oocyte gets all the cytoplasm
polar body - the bag of DNA that’s not going anywhere

Question 4

Q

Meiosis in females

Answer

A

doubled the DNA (2n), put half in the oocyte and half into the polar body with no cytoplasm
in the second round of meiosis do the same again - divide the chormosomes up so that a haploid oocyte (with 4x cytoplasm) and get rid of bag of DNA that’s not going anywhere
this whole thing in eutherian mammals starts inutero and then stops
might be arrested there for 60+ years and then resumes on que, which doesn’t start until menarche (in humans this will be arrested for at least 10 years, and in each cycle a different cohort ofprimordial follicles will wake up and the oocytes will resumem meiosis)

Question 5

Q

Meiosis in females

summary

Answer

A

initiates in utero **and then arrested
at/after menarche cohorts of oocytes recruited to develop each month until menopause

Question 6

Q

Meiosis in females

(picture)

Question 7

Q

Meiotic arrest in mammalian oocytes

(picture)

Question 8

Q

Secondary oocyte

Answer

A

haploid
finished meiosis
got rid of 2 polar bodies along the way

Question 9

Q

Telophase I

Answer

A

the point where we split into 1 primary oocyte and a polar body
doesn’t produce 2 cells but a cell and a dead bunch of DNA

Question 10

Q

At first meiotic arrest

Answer

A

not even diakinesis (with recombination of maternal and paternal alleles)
the surge in LH around the middle of the cycle will wake up some - not all - oocytes so they resume meiosis
when the resume meiosis they don’t get to finish

Question 11

Q

When resume meiosis after LH surge

Answer

A

don’t get to finish
wake up, recondense their chromosomes
MI AI TI PII MII - then stop again after 36 hours
don’t complete meiosis II unless they get fertilized by sperm
TII is marked by the ejection of a second polar body
- allows to see if fertilized
- no second polar body unless the egg has been fertilized because can’t complete meiosis without sperm fusion

Question 12

Q

TII is marked by

Answer

A

a second polar body

allows to see if fertilized
no second polar body unless the egg has been fertilized because can’t complete meiosis without sperm fusion

Question 13

Q

The second meiotic arrest

Answer

A

second meiotic spindle stabilized by maturation promoting factor (MPF), a complex of
- cyclin B
- cyclin-dependent kinase 1 (cdk1)
MPF stabilized by cytostatic factor (CSF), a ocmplex of
- Emi2 (which inhibits…)
- anaphase promoting complex/cyclosome (APC/C)

Question 14

Q

Second meiotic spindle ahs gotten us as far as

Answer

A

metaphase II

Question 15

Q

Second meiotic spindle is stabilized by

Answer

A

MPF - stops from going metaphase to anaphase

Question 16

Q

MPF is a complex of

Answer

A

cyclin B
cyclin-dependent kinase 1 (cdk1)

Question 17

Q

MPF stabilized by

Answer

A

cytostatic factor (CSF)

stops the cell progressing

Question 18

Q

Cytostatic factor (CSF) is a complex of

Answer

A

Emi2 - protein which inhibits…
anaphase promoting complex/cyclosome (APC/C)
is stabilizing rather than attacking MPF and spindle stays frozen in time until fertilization happens
something about sperm has to remove all of that so egg can complete meiosis

Question 19

Q

Oocyte maturation in most eutheria

Answer

A

prior to ovulation, oocyte undergoes both cytoplasmic and nuclear maturation
prior to ovulatory LH surge, primary oocyte arrested at prophase I
LH surge stimulates resumption of meiosis; oocyte completes meiosis I, ejects first polar body and then arrests again at metaphase II

(~36h)

Question 20

Q

Prior to ovulation, oocyte is stuck in

Answer

A

prophase I

Question 21

Q

Prior to ovulation the oocyte undergoes

Answer

A

cytoplasmic and nuclear maturation

Question 22

Q

Prior to ovulatory LH surge, primary oocyte arrested at

Answer

A

prophase I

Question 23

Q

LH surge stimulates the resumption of meiosis

Answer

A

oocyte prophase I to metaphase II before it gets released

= nuclear maturation - what’s going with chromosomes
cytoplasmic maturation - reorganization of the organelles and antioxidants

Question 24

Q

Oocte completes meiosis I…

Answer

A

ejects first polar body then arrests again at metaphase II

Question 25

Q

Oocyte maturation in dogs

Answer

A

prior to ovulatory LH surge, primary oocyte arrested at prophase I
LH surge stimulates resumption of meiosis then ovulation
in dogs, ovulation then resumption of meiosis
oocyte resumes meiosis 48-54 hours post-ovulation

Question 26

Q

We can never get past MII without sperm fusion because

Answer

A

MPF is stabilized by CSF

none of this matters if egg isn’t released to oviduct
LH surge triggers ovulation - but what triggers the LH surge?

Question 27

Q

Reflex ovulation

Answer

A

primitive trait in Eutheria, persists in: shrews, ground squirrels, rabbits, cats, ferrets, mink, voles, and camels
triggered by mechanical stimulation of the cervix and reproductive tract
neural reflex (cholinergic and noradrenergic) to hypothalamic POA
stimulated kisspeptin neurones which impinge on GnRH neurones (via GPR54)
increase in GnRH pulse amplitude and/or frequency

→ triggers LH surge

Question 28

Q

Reflex ovulation continued

Answer

A

ovulation still driven by an LH surge, but the LH surge doesn’t happen unless there’s been insemination = don’t waste eggs in the hope that a male’s around - get partner then release eggs
even inspecies that don’t have reflex ovulation, the female will change the timing of their ovulation because they will sense they’ve been mated (evidence in humans)
triggered by mechanical stimulation of the cervix and reproductive tract
- eg cats with curved barbs made of keratin so when male withdraws he stimulates loads of prostaglandins and inflammation
neural reflex (cholinergic and noradrenergic) to hypothalamic POA
stimulate kisspeptin neurones which impinge onGnRH neurones (via GPR54)
- stimulate neurones that impinge (have an effect - esp a negative one)
- kisspeptin is a protein that stimulates a receptor - eg GPR54
- causes GnRH neurones to fire
increase in GnRH pulse amplitude/frequency triggers LH surge
neural reflex back to the hypothalamic cortex area stimulates kisspeptin neurones stimulates GnRH, stimulates LH → LH surge → ovulation timed to coincide with sperm being decapacitated to fertilize

Question 29

Q

Spontaneous ovulation

Answer

A

endocrine feedback varies (predominantly estradiol/E₂) stimulates a “spontaneous” preouvlatory LH surge in the late follicular phase (FP)
LH surge and ovulation occur irrespective of whether its been fertilized or not
still requires LH surge, kisspeptin, GnRH, but now the whole thing is under the control of steroid feedback, especially estradiol
in most of follicular phase estradiol exerts negative feedback, but 24-36h where estradiol switches from negative to positive feedback and stimulates the preovulatory LH surge
endocrine feedback from ovaries (predominantly etradiol/E₂) stimulates a spontaneous preovulatory LH surge in the late follicular phase
LH surge and ovulation occur irrespective of mating

Question 30

Q

HPO axis in the follicular phase

(picture)

Question 31

Q

HPO axis in the follicular phase

Answer

A

so estradiol doesn’t always exert positive feedback because it doesn’t
in 13/14 days in humans it will exert long-loop and short-loop negative feedback

Question 32

Q

HPO axis in the ovulatory phase

(picture)

Question 33

Q

HPO axis in the ovulatory phase

Answer

A

when LH and FSH work together to give us a surge in estradiol, exponential increase in estradiol as work our way up to ovulation
as estradiol is high for several days it stimulates kisspeptin and neurones to stimulate GnRH
the GnRH pulse frequency and amplitude goes up and gives us an LH surge which now triggers oocyte maturation, resumption of meiosis, and ultimately follicular rupture and ovulation
what’s bizarre – if we do the same thing in endocrine terms to a male pituitary it won’t respond the same way
if give a male estradiol in an exponential series and keep the levels high for 24/48 hours, he will never show an LH surge

→ says there’s a hardwired sexual dimorphism in the pituitary

Question 34

Q

Kisspeptin mediates the E₂ induction of the LH surge in sheep

(picture)

Question 35

Q

Kisspeptin mediates the E₂ induction of the LH surge in sheep

Answer

A

sheep that have been administered GnRH and estradiol to induce an LH surge
if at the same time we infuse into the hypothalamus an antagonist of kisspeptin à no LH surge
says kisspeptin is needed for estradiol to stimulate an LH surge
maybe there are different kisspeptin neurones in a female brain than in a male brain
that’s why you get a surge in a female and not in a male to the same endocrine stimulus

Question 36

Q

Kisspeptin mediates the E₂ induction of the LH surge in rats

Answer

A

in rodents, the negative feedback steroids are mediated through these KNDy neuronse
the positive surge effect requires estradiol to stimulate kisspeptin neurones
outpouring of kisspeptin upregulates GnRH neurones, get surge in LH
in the rodent brain, the negative feedback effects through the arcuate nucleus and different neurones to the surge effects which are through the AVPV and kisspeptin neurones

Question 37

Q

KNDy (Kisspeptin-Neurokinin B-β-Dynorphin) neurones may mediate positive feedback in humans

(picture)

Question 38

Q

KNDy (Kisspeptin-Neurokinin B-β-Dynorphin) neurones may mediate positive feedback in humans

Answer

A

KNDy neurone also makes kisspeptin but also makes neurokinin B and β-dynorphin
so KNDy neurone is making kisspeptine, but difference is make different products
in humans, don’t understand the LH surge as well but it looks like negative feedback and positive feedback are through these KNDy neurones
KNDy neurones in a different place in the hypothalamus, but it looks like activating different receptors in KNDy neurones either gives you negative feedback (suppression of GnRH) or positive feedback generates surge which leads to an LH surge

Question 39

Q

Consequences of the ovulatory LH surge

Answer

A

midcycle surge in LH triggers ovulation at +36hours (in humans)
when oocyte has undergone nuclear maturation and cytoplasmic maturation it can be released
LH drives (nuclear maturation) resumption of meiosis in the immature, prophase I oocyte
(GVBD – germinal vesicle breakdown)
in oocyte is dark spot called germinal vesicle
looking at meiotic spindle
as the oocyte moves out of prophase I the germinal vesicle breaks down, resumed meiosis
LH stimulates follicle rupture and release of the cumulus-oocyte complex (COC) with mature, MII oocyte
follicle must rip apart – it’s not going to mature in an oocyte cytoplasmically or at the nuclear level when it’s trapped inside the follicle
need follicle to rupture such that cumulus-oocyte complex can be released

Question 40

Q

Consequences of the ovulatory LH surge

midcycle surge in LH triggers ovulation at

Answer

A

+36 hours (in humans)

when oocyte has undergone nuclear maturation and cytoplasmic maturation it can be released

Question 41

Q

Consequences of the ovulatory LH surge

LH drives

Answer

A

(nuclear maturation) resumption of meiosis in the immature, prophase I oocyte

(GVBD – germinal vesicle breakdown)
in oocyte is dark spot called germinal vesicle
looking at meiotic spindle
as the oocyte moves out of prophase I the germinal vesicle breaks down, resumed meiosis

Question 42

Q

Consequences of the ovulatory LH surge

LH stimulates

Answer

A

follicle rupture and release of the cumulus-oocyte complex (COC) with mature, MII oocyte

follicle must rip apart – it’s not going to mature in an oocyte cytoplasmically or at the nuclear level when it’s trapped inside the follicle
need follicle to rupture such that cumulus-oocyte complex can be released

Question 43

Q

Ovulation of cumulus-oocyte complex

Answer

A

3 cell types – inside (black) is oocyte, around the outside are cumulus cells
fluffy cumulus cells on the outside that are fluffy
cumulus cells right next to oocyte as well which is really dense called the coronaradiata (?) which are in intimate contact to the oocyte through the zona pellucida
this whole thing is easier for the oviduct to catch than an egg – sticky cumulus cells

Question 44

Q

Consequences of the ovulatory LH surge

Answer

A

LH surge induces inflammatory cascade
follicle rupture is an inflammatory cascade
may contribute to body temperature going up during ovulation
LH upregulates expression of pro-inflammatory cytokines (eg IL-1 and TNF-α) and PTGS-2 (COX-2):
- prostaglandins cause hyperaemia (increased blood flow to the follicle which is about to rupture) and weaken follicle wall

Question 45

Q

Composition of the follicle wall

(picture)

Question 46

Q

Composition of the follicle wall

Answer

A

the oocyte is above the picture
granulosa cells if looking at the follicle wall are neural granuloma cells
membrane propria – the basement membrane that keeps the theca and granulosa separate until ovulation when they mix up
the membrane propria keeps blood vessels out of the granulosa
granulosa cells are avascular

Question 47

Q

Composition of the membrana propria in bovine follicles

Answer

A

laminins
collagens
classified cells as primordial, preantral, antral (healthy/growing) and antral (atretic/dying)
looked at which proteins are expressed
all have the same laminins
always have type IV collagen α1 and α2, immature follicles have additional collagens which are lost when the follicle starts to weaken and loosen up to prepare for ovulation
means if we want a follicle to rupture we have to break these proteins down or the follicle won’t be able to come apart
if you want the follicle to rupture we have to break these down

Question 48

Q

Consequences of the ovulatory LH surge

Answer

A

LH surge upregulates expression of matrix metalloproteinases (MMPs)
- break down the extracellular matrix in the presence of selenium
- MMP4 is collagenase (double check)
suppresses expression of tissue inhibitors of metalloproteinases (TIMPs)
- TIMPS inhibit MMPs
- so LH upregulates enzymes and downregulates their inhibitors so we can start to digest the follicle wall
block with indomethacin (aspirin)
- aspirin is anti-inflammatory, and this is an inflammatory cascade

Question 49

Q

Sperm capacitation

Answer

A

sperm decapacitated in the epididymis (deactivated) so need to wake up
woken up only when in female tract that has been estrogen-primed (must be ready for fertilizatition)
within E₂-primed, female reproductive tract, sperm undergo capacitation
hyperactivation of the flagellum (slow à whiplash movements of flagellum)
change in membrane properties (loss of “decapacitation factors”) – capable of undergoing the acrosome reaction
gets ready for acrosome reaction – spermatozoan has a nucleus above which there is a bag of enzymes – the acrosome
gets ready to lose the acrosomal enzymes
loses decapacitation factors – the extra cholesterol is washed off in the female tract

Question 50

Q

Sperm decapacitated in the

Answer

A

epididymis (deactivated) so need to wake up

woken up only when in the female tract that has been estrogen-primed (must be ready for fertilization)

Question 51

Q

Within E2-primed, female reproductive tract, sperm undergo capacitation

Answer

A

hyperactivation of the flagellum (slow à whiplash movements of flagellum)
change in membrane properties (loss of “decapacitation factors”) – capable of undergoing the acrosome reaction
gets ready for acrosome reaction – spermatozoan has a nucleus above which there is a bag of enzymes – the acrosome
gets ready to lose the acrosomal enzymes
loses decapacitation factors – the extra cholesterol is washed off in the female tract

Question 52

Q

Sperm capacitation (picture)

Answer

A

left is uncapacitated sperm
as it undergoes capacitation first thing we see is hyperactivity - starts to swim really fast in circles
also gets ready to release acrosome enzymes – to burst the acrosome

first thing the sperm has to do is get through the cloud of cumulus cells

Question 53

Q

Cumulus cell penetration

cumulus cells held together by unsulphaged glycosaminoglycan

Answer

A

hyaluronan

digested by hyaluronidase released from the initial (sacrificial) sperms’ acrosomes
- initial sperm gets there, releases acrosome with hyaluronidase to break up cumulus cells but have noc hanve of fertilizing the egg now
cumulus cells dissociate and expose the zona pelucida for the next wave of sperm to reach

Question 54

Q

Zona binding and penetration

Answer

A

second wave of sperm binds to multiple zona pellucida (ZP) receptors:
- ZP3 and ZP2 in mouse
- ZP3 and ZP4 in human
once the sperm binds to the zona, there’s an increase in calcium inside the sperm
- increases in calcium are about exocytosis
- that’s what causes the acrosome to react in the second wave of sperm
- release enzymes like acrosin which digest ZP
ZP binding (via Ca2+) triggers sperm membrane fusion/exocytosis – acrosome reaction: release of enzymes (eg acrosin) to digest ZP

Question 55

Q

Once the sperm binds to the zona there’s an increase in

Answer

A

calcium inside the sperm

increases in calcium are about exocytosis
that’s what causes the acrosome to react in the second wave of sperm
release enzymes like acrosin which digest ZP

Question 56

Q

ZP bindig (via Ca²⁺) triggers

Answer

A

sperm fusion/exocytosis

acrosome reaction: release of enzymes (eg acrosin) to digest ZP

Question 57

Q

Sperm-oocyte interactions

Answer

A

sperm lies tangential to the oolemma in the perivitelline space
- lies tangential to the oocyte membrane
- perivitelline space is the space between the oocyte and the zona
sperm head engulfed by microvilli on the oolemma
fusion between the oolemma and the equatorial + post-acrosomal membranes of the sperm
- fuse not along entire length of the sperm, just specific regions
lots of sperm do this but just want one to fertilize the egg
- as soon as the sperm arrives there’s a massive increase of calcium inside the egg

Question 58

Q

Sperm lies tangential to the

Answer

A

oolemma in the perivitelline space

lies tangential to the oocyte membrane
perivitelline space is the space between the oocyte and the zona

Question 59

Q

Sperm are engulfed by

Answer

A

microvilli on the oolemma

Question 60

Q

Fusion between the oolemma and the

Answer

A

equatorial + post-acrosomal membranes of the sperm

fuse not along entire length of the sperm, just specific regions

lots of sperm do this but just want one to fertilize the egg

as soon as the sperm arrives there’s a massive increase of calcium inside the egg

Question 61

Q

Oocyte activation

Answer

A

within 5 minutes of sperm fusion, dramatic increase in free [Ca2+]I in the oocyte
- calcium levels go up where the sperm enters and goes across the egg
- tells us that the sperm upon arrival is injecting something into the egg that elevates calcium
asynchronous – wave of elevated Ca2+ spreads from point of sperm fusion
sperm injects phospholipase C (PLC) zeta

Question 62

Q

Oocyte activation

within 5 minutes of sperm fusion, dramatic increase in

Answer

A

free [Ca²⁺]_i in the oocyte

calcium levels go up where the sperm enters and goes across the egg
tells us that the sperm upon arrival is injecting something into the egg that elevates calcium
asynchronous – wave of elevated Ca2+ spreads from point of sperm fusion
sperm injects phospholipase C (PLC) zeta

Question 63

Q

Sperm injects

Answer

A

phospholipase C (PLC) zeta

Question 64

Q

Oocyte activation and PLC zeta

(picture)

Answer 57

A

sperm lying tangential to the egg – doesn’t stimulate the egg but injects PLC zeta that will hydrolyze pip2 (?) to give is IP3 which gives us a calcium signal and diacylglycerol (?) which activates protein kinase C (?)
everything that happens next happens because the sperm injected phospholipase C and raised calcium in the egg

Answer 58

A

second meiotic spindle stabilized by maturating promotion factor –MPF – a complex of
- cyclin
- cyclin-dependent kinase 1 (cdk1)
MPF stabilized by cytostatic factor (CSF), a complex of
- Emi2 which inhibits
- anaphase promoting complex / cyclosome (APC/C)
(MPF stabilizing meiotic spindle, CSF stabilizing MPF) (calcium activates calmodulin kinase II)
(sperm injects phospholipase C which gives you IP3 which elevates intracellular calcium, that activates carmodulin kinase II which destroys Emi2 and the anaphase promoting complex is no longer inhibited. the APC can now attack cyclin B1, cyclin B1 gets ubiquitinated and destroyed, which means that the second meiotic spindle is now destabilized and go from metaphaseII to anaphaseII)
all of this happened because the sperm injected phospholipase C

Answer 59

A

cyclin
cyclin-dependent kinase 1 (cdk1)

Answer 60

A

Emi2 which inhibits
anaphase promoting complex / cyclosome (APC/C)

Answer 61

A

elevated [Ca2+]I activates calmodulin kinase II (CaMK2)
CaMK2 phosphorylates Emi2 – targeted for degradation
disinhibits APC/C – can degrade cyclin B1
destroys MPF – destabilizes second meiotic spindle (so can progress to anaphase II)

Answer 62

A

bad for lots of sperm to get in
2 places we can block polyspermy
- obvious is zona – the sperm when it arrives injects phospholipase C, which elevates calcium which reactivates the egg
- calcium should also trigger exocytosis which causes the egg to release granules that are already around the periphery and they harden the zona pellucida so no more sperm can come through
to avoid polyploidy, only one haploid sperm can fertilize the oocyte
subsequent sperm are excluded by the cortical reaction and zona hardening
elevated [Ca2+]I trigger exocytosis of cortical granules (containing enzymes) into the perivitelline space

Answer 63

A

in pigs, 30-65% of oocytes show evidence of polyspermy but are still diploid
extra sperm tend to be trapped in perivitelline space because the block to polyspermy in the pig is not the zona pellucida but the level of the vitelline membrane (oolemma) – the membrane around the egg that doesn’t accept sperm binding
suggested mechanisms – relatively slow zona hardening and/or secondary block to polyspermy on the oolemma