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Reproductive and Developmental Biology > Lecture 6 - Cellular Aspects of Ovulation and Fertilization > Flashcards

Lecture 6 - Cellular Aspects of Ovulation and Fertilization Flashcards

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1
Q

Meiosis in the female

A
  1. oogonium (2n)
  2. primary oocytes (2n) (+ polar body)
  3. secondary oocyte (n) (+ polar body)

meiosis between oogonium and primary oocyte

meiosis between primary and secondary oocyte

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2
Q

Meiosis in females starts with

A

oogonium

  • in men - spermatogonia
  • in females, have no oogonia because they have already entered meiosis 6 months before they’re born
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3
Q

Primary oocyte v Polar body

A
  • the primary oocyte gets all the cytoplasm
  • polar body - the bag of DNA that’s not going anywhere
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4
Q

Meiosis in females

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)
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5
Q

Meiosis in females

summary

A
  • initiates in utero **and then arrested
  • at/after menarche cohorts of oocytes recruited to develop each month until menopause
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6
Q

Meiosis in females

(picture)

A
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7
Q

Meiotic arrest in mammalian oocytes

(picture)

A
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8
Q

Secondary oocyte

A
  • haploid
  • finished meiosis
  • got rid of 2 polar bodies along the way
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9
Q

Telophase I

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
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10
Q

At first meiotic arrest

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
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11
Q

When resume meiosis after LH surge

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
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12
Q

TII is marked by

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
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13
Q

The second meiotic arrest

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)
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14
Q

Second meiotic spindle ahs gotten us as far as

A

metaphase II

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15
Q

Second meiotic spindle is stabilized by

A

MPF - stops from going metaphase to anaphase

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16
Q

MPF is a complex of

A
  • cyclin B
  • cyclin-dependent kinase 1 (cdk1)
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17
Q

MPF stabilized by

A

cytostatic factor (CSF)

  • stops the cell progressing
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18
Q

Cytostatic factor (CSF) is a complex of

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
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19
Q

Oocyte maturation in most eutheria

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)

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20
Q

Prior to ovulation, oocyte is stuck in

A

prophase I

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21
Q

Prior to ovulation the oocyte undergoes

A

cytoplasmic and nuclear maturation

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22
Q

Prior to ovulatory LH surge, primary oocyte arrested at

A

prophase I

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23
Q

LH surge stimulates the resumption of meiosis

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
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24
Q

Oocte completes meiosis I…

A
  • ejects first polar body then arrests again at metaphase II
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25
Q

Oocyte maturation in dogs

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
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26
Q

We can never get past MII without sperm fusion because

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?
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27
Q

Reflex ovulation

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

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28
Q

Reflex ovulation continued

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
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29
Q

Spontaneous ovulation

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
30
Q

HPO axis in the follicular phase

(picture)

A
31
Q

HPO axis in the follicular phase

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
32
Q

HPO axis in the ovulatory phase

(picture)

A
33
Q

HPO axis in the ovulatory phase

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

34
Q

Kisspeptin mediates the E2 induction of the LH surge in sheep

(picture)

A
35
Q

Kisspeptin mediates the E2 induction of the LH surge in sheep

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
36
Q

Kisspeptin mediates the E2 induction of the LH surge in rats

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
37
Q

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

(picture)

A
38
Q

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

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
39
Q

Consequences of the ovulatory LH surge

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
40
Q

Consequences of the ovulatory LH surge

midcycle surge in LH triggers ovulation at

A

+36 hours (in humans)

  • when oocyte has undergone nuclear maturation and cytoplasmic maturation it can be released
41
Q

Consequences of the ovulatory LH surge

LH drives

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
42
Q

Consequences of the ovulatory LH surge

LH stimulates

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
43
Q

Ovulation of cumulus-oocyte complex

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
44
Q

Consequences of the ovulatory LH surge

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
45
Q

Composition of the follicle wall

(picture)

A
46
Q

Composition of the follicle wall

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
47
Q

Composition of the membrana propria in bovine follicles

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
48
Q

Consequences of the ovulatory LH surge

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
49
Q

Sperm capacitation

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
50
Q

Sperm decapacitated in the

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)
51
Q

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

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
52
Q

Sperm capacitation (picture)

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

53
Q

Cumulus cell penetration

cumulus cells held together by unsulphaged glycosaminoglycan

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
54
Q

Zona binding and penetration

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
55
Q

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

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
56
Q

ZP bindig (via Ca2+) triggers

A

sperm fusion/exocytosis

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

57
Q

Sperm-oocyte interactions

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
58
Q

Sperm lies tangential to the

A

oolemma in the perivitelline space

  • lies tangential to the oocyte membrane
  • perivitelline space is the space between the oocyte and the zona
59
Q

Sperm are engulfed by

A
  • microvilli on the oolemma
60
Q

Fusion between the oolemma and the

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
61
Q

Oocyte activation

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
62
Q

Oocyte activation

within 5 minutes of sperm fusion, dramatic increase in

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
63
Q

Sperm injects

A

phospholipase C (PLC) zeta

64
Q

Oocyte activation and PLC zeta

(picture)

A
65
Q

Oocyte activation and PLC zeta

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
66
Q

The second meiotic arrest

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
67
Q

Second meiotic spindle stabilized by maturating promotion factor –MPF – a complex of

A
  • cyclin
  • cyclin-dependent kinase 1 (cdk1)
68
Q

MPF stabilized by cytostatic factor (CSF), a complex of

A
  • Emi2 which inhibits
  • anaphase promoting complex / cyclosome (APC/C)
69
Q

Meiotic resumption post fertilization

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)
70
Q

The block to polyspermy

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
71
Q

A block to polyspermy

an exception to every rule

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
72
Q
A

Reproductive and Developmental Biology (8 decks)

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