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Advanced concepts in reproduction > Week 5 > Flashcards

Week 5 Flashcards

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1
Q

What are the steps of follicular growth?

A

Oogonium
↓
Primordial follicles
↓
Primary follicles
↓
Preantral follicles
↓
Antral follicles
↓
Ovulatory follicles

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2
Q

What are the oocyte stages of meiosis?

A

Mitosis
↓
Initiation of meiosis
↓
Arrest at prophase I (GV stage)
↓
Resumption of meiosis
↓
Arrest at metaphase II

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3
Q

What is oocyte maturation?

A

The set of nuclear, cytoplasmic and molecular changes that enables the oocyte to be normally fertilised

Considered a critical step which influences the success for fertilisation and subsequent embryo development

Cumulus cell expansion
Nuclear maturation

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4
Q

What are the 2 steps of oocyte maturation?

A
  1. Nuclear maturation
  2. Cytoplasmic maturation
    Synthesis of mRNA and proteins, redistribution of cytoplasmic organelles, and activation of factors regulating maturation
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5
Q

What is meiotic maturation and its first stages of embryo development in mammals?

A

Germinal vesicle stage:
Follicle growth:
Prophase of 1st meiotic division
- Fully grown
- Oocyte

Meiotic maturation:
Metaphase I
Metaphase II
- Ovulated
- Oocyte

Fertilisation:
Pro-nucleus
- One cell
- Zygote

Early development of embryo

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6
Q

How does follicle size affect oocyte maturation?

A

Larger the follicle, the more likely the oocyte is to mature further

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7
Q

How does the oocyte resumption of meiosis take place?

A

LH surge
↓
Resumption of meiosis
↓
First polar body emitted, arrest at metaphase II of meiosis I
(Secondary Oocyte)
↓
Ovulation (most species)
↓
Sperm penetration
↓
Zygote
(pronucleate egg)

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8
Q

What happens after removal of oocyte from follicle?

A

Meiosis resumes spontaneously

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9
Q

What are the layers of the oocyte?

A

Oocyte
↓
Zona pellucida
↓
Corona radiata

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10
Q

What are gap junctions between corona radiata and zona pellucida for?

A

Intact gap junctional communication is critical for maintenance of meiotic arrest

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11
Q

How is meiotic arrest maintained?

A

The concentration of cAMP in the oocyte is regulated by an equilibrium between two enzymes: adenyl cyclase (AC) and the phosphodiesterase (PDE)

Interaction of CNP or NPPC produced by the granulosa cells (GC) and its receptor NPR2, expressed in cumulus cells (and up-regulated by OSF and E2) induces the conversion of GTP into cGMP, which is then transferred into the oocytes via gap junctions

cGMP prevents PDE3 activation within the oocyte and cAMP is then maintained at high levels

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12
Q

During follicular growth where is the oocyte attached?

A

To the cells in the follicle wall by gap junctional communication and the cumulus cells extend their cytoplasmic process through the Zona Pellucida which attach to the oocyte cell membrane

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13
Q

What does cAMP do in the oocyte?

A

Critical for maintaining the oocyte in meiotic arrest at the prophase of meiosis I

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14
Q

What does FSH binding to its receptor lead to?

A

Activation of adenylate cyclase which converts ATP to cAMP in the granulosa cells which is then transferred into the oocytes

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15
Q

What does PDE3A inhibition do?

A

Inhibited by cGMP

Prevents degradation of cAMP

This is regulated by CNP from granulosa cells binding to NPR2 in the cumulus cells leading to production of cGMP.

NPR2 expression is influenced by oestradiol as well as the oocyte secreted factors

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16
Q

What does the LH surge induce?

A

Cumulus cell expansion

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17
Q

What does the LH surge lead to?

A

Meiotic resumption

LHR only found in large antral follicles

Cumulus cells produce hyaluronic acid (HA) in response to LH

This leads to formation of extracellular matrix in the cumulus cells

High molecular weight HA is produced by hyaluonan synthase II (HAS-2) is hydrophilic leading to cumulus cells expansion and gradual separation of COC from follicular wall

HABPs on the surface of sperm bind to HA guiding sperm towards oocyte

Proteins, particularly laminin) accumulate in the mucified mass - signal for sperm-egg recognition

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18
Q

What does the LH surge activate? What does this do?

A

Activates the PKA signalling pathway and induces

The production of the EGF-like factors (i.e. AREG/EREG).

  • EGF-like factors, in turn, will activate a cascade of events mediated by the EGFR (both in mural and cumulus cells)
  • As a result, ERK1/2 will induce other main events:
  1. The up-regulation of transcripts responsible for cumulus expansion, Has2, Tnfaip, Ptx3 and Ptgs2 (which are also positively regulated by the CEEFs derived from the oocyte)
  2. Production of PTGS2, which enhance the response to LH stimuli through the production of PGE2 and by inducing the production of more EGF-like factors and activation of the subsequent cascade
  3. The closure of the gap junctions by AREG/EREG avoids the transfer of cGMP into the oocyte
  4. In parallel, CNP and NPR2 levels decrease and also contribute to the low production of cGMP
  5. Within the oocyte, some transcripts crucial for the transition from the GV to the MII stage are actively translated
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19
Q

What are the steps of the cell cycle?

A

M Phase - meiosis

Interphase:
- G1, S and G2
- Growth, DNA synthesis

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20
Q

What is MPF?

A

Maturation promoting factor

Cyclin-dependent kinase

Exists in an inactive (interphase) and active (M phase) form

Active form causes:
- GVBD
- Chromosome condensation
- Mitotic spindle formation
- Proteolytic enzymes

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21
Q

How is MPF activated?

A

By dephosphorylation of cdc2

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22
Q

What is happening to the oocyte during oocyte growth and before the LH surge?

A

Gap junctions allow transfer of cAMP into the oocyte

High level of PKA makes MPF components phosphorylated and inactive as pre-MPF form

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23
Q

What do we know about MPF?

A

MPF activity during oocyte maturation is detected just before GVBD and increases until metaphase I

It decreased during the anaphase to telophase transition

It increases again until the oocyte reaches metaphase II

When the oocyte becomes arrested at MII, MPF activity remains high

It remains high due to the actions of cytostatic factor (CSF) and the proto-oncogene c-mos

These act to phosphorylate cyclin B and hence increase MPF stability

Upon fertilisation, MPF activity declines rapidly

It remains low in pronuclear zygots until the zygote prepares to undergo the first cleavage division, when once again it is high at metaphase during mitosis

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24
Q

How are oocytes maintains in meiotic arrest?

A

cAMP modulators
- Forskolin
- Invasive adenylate cyclase
- Dibutyryl cAMP

MPF inhibitors
- Butyrolactone I
- Bohemine
- 6-DMAP
- Roscovitine

cGMP modulator
- CNP

PDE inhibitors
Non-specific PDE inhibitors
- Hypoxanthine
- IBMX
- Theophylline

PDE3 inhibitors
- Cilostamide
- Cilostazol
- Milrinone
- Org9935

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25
Why are oocytes maintained in meiotic arrest?
1. To improve the oocyte cytoplasmic maturation 2. Practical feasibility for fertilisation time
26
What does high cAMP in the oocyte do?
Maintains meiotic arrest at prophase of 1st meiotic division - GV
27
What does LH surge during pre-ovulatory stage lead to?
Increased level of cAMP in cumulus cells by activation of PKA
28
What does the LH surge induce the expression of?
Hyaluronan in cumulus cells resulting in cumulus cells, resulting in cumulus cells expansion and disruption of gap junctional communication with the oocyte
29
What does the reduced level of cAMP result in?
Germinal vesicle breakdown - GVBD
30
What is MPF formed of?
2 subunits Cdc2 and P34 which are phosphorylated and inactive in immature oocyte
31
When does MPF become activated?
After LH surge which leads to dephosphorylation of all subunits by MAPKs
32
What is the structure of a spermatozoon?
Sperm Head: Acrosomal vesicle Cell membrane Nucleus Sperm Neck: Centriole Mid-piece: Mitochondria Axoneme Tail End-piece
33
What are the basic facts of fertilisation?
The process of unison of mature male gamete (sperm) with mature female gamete (ovum) to produce new cell of life which is called (zygote) through chain of events in the oviduct (fallopian tubes) Disruption of any event will cause fertilisation failure Deposition of semen into female tract must be closely synchronised with ovulation to ensure the incidence of fertilisation The site of fertilisation on most farm animals is (ampullary-isthmic junction) while in human it is the ampulla region Time of ovulation different in different animals.
34
When do cows ovulate?
10-12 hours after end of oestrus phase
35
When do sheep ovulate?
Late of oestrus phase
36
When do goats ovulate?
Few hours after end of oestrus phase
37
When do swine ovulate?
Mid-oestrus phase
38
When do humans ovulate?
Day 14 after initiation of menstrual cycle
39
What are the basic facts of fertilisation for cows?
Oocyte fertile life: 8-12 hrs Site of semen deposit: Vagina Volume of ejaculate: 4ml Sperm concentration (billion/ml): 1 No of sperm reaching site of fertilisation: 4200-27500
40
What are the basic facts of fertilisation of sheep?
Oocyte fertile life: 16-24hrs Site of semen deposit: Vagina Volume of ejaculate: 1ml Sperm concentration (billion/ml): 2 No of sperm reaching site of fertilisation: 600-5000
41
What are the basic fertilisation facts of pigs?
Oocyte fertile life: 8-10hrs Site of semen deposit: Cervix and Uterus Volume of ejaculate: 125ml Sperm concentration (billion/ml): 0.2 No of sperm reaching site of fertilisation: Few
42
What are the basic facts of fertilisation of humans?
Oocyte fertile life: 6-24hrs Site of semen deposit: Vagina Volume of ejaculate: 3.5 Sperm concentration (billion/ml): 0.120 No of sperm reaching site of fertilisation: Few
43
Whats the structure of a metaphase II oocyte?
Cumulus Zona pellucida Plasma membrane Ooplasm Polar body Metaphase plate
44
What happens in fertilisation?
Sperm penetrates the cumulus oophorus Sperm contacts zona pellucida and acrosome reaction is triggered by ZP3 Sperm penetrates into ZP and adheres to and fuses with plasma membrane Meiosis is complete and 2nd Polar Body extrudes
45
What is the fertilisation sequence?
Attachment to zona pellucida Binding to zona pellucida (species specific receptors) Acrosome reaction Penetration Fusion with the plasma membrane Oocyte activation - Resumption of meiosis - Block to polyspermy
46
How does sperm travel through Zona pellucida?
Mechanical hypothesis Enzymatic hypothesis Possibly a combo of the two mechanisms. Likely more of one in certain species and vice versa
47
What happens in the binding of sperm to the oocyte?
A carb mediated event Species specific - Sperm receptors on egg, ZP1, ZP2, ZP3 - EBPs on sperm BUT This restriction can be overcome by removal of ZP (hamster test)
48
49
What is ZP3?
A structural component of the ZP A sperm receptor Inducer of acrosome reaction
50
What happens to the zona pellucida as the oocyte grows?
Thickens
51
What is the acrosome reaction?
A calcium dependent fusion reaction between the outer acrosomal membrane and the overlying sperm plasma membrane releasing acrosomal contents to participate in zona penetration
52
What is the acrosome?
A relatively large, golgi derived, lysosome-like organelle that overlies th nucleus in the apical region of the sperm head
53
What does the acrosome reaction involve?
Multiple fusions between outer acrosomal membrane and plasma membrane, extensive formation of hybrid membrane vesicles and exposure of inner acrosomal membrane and acrosomal contents Only sperm that have completed the acrosome reaction can penetrate the ZP and fuse with the egg plasma membrane
54
What happens in the sperm fusion with the oocyte?
Sperm in the perivitelline space Sperm fusing with vitelline membrane Sperm being engulfed by oocyte processes Sperm incorporated in the vitellus and nucleus beginning to decondense
55
What is the integrin alpha6ß1 been described as?
A potential fertilin and cyritestin receptor, two members of the ADAM family of proteins present on sperm. Shown to associate with tetraspanins in many cell types The role of alpha1 integrin as an ADAM protein receptor has however been challenged Because the binding of ADAM proteins to the egg is blocked by CD9 mAB, it is likely that the ADAM receptor, whatever its nature, is a CD9-associated molecule. This would be consisten with the ability of CD9 and other tetraspanins to assemble discrete micro domains The association of a putative Izumo receptor with tetraspanins is entirely speculative
56
How does the sperm activate the egg?
Causing calcium oscillations signalling event Providing centriole to contribute to initial cell division To form male and female pronuclei which arrange on the first mitotic spindle for the first division of the zygote NB. ALTHOUGH SPERM MITOCHONDRIA ENTER THE EGG CYTOPLASM THEY ARE SUBSEQUENTLY DESTROYED AND DO NOT CONTRIBUTE TO MITOCHONDRIAL DNA. THEREFORE MITOCHONDRIAL DNA IS ENTIRELY FROM THE MATERNAL SIDE.
57
What is the mechanism of PLC-zeta induced Ca2+ release?
PLC-zeta from spermatozoa is considered to be the responsible activator It activates phospholipase C which catalyses the hydrolysis of PIP2 to diacylglycerol (DAG) and inositol triphosphate (IP3) IP3 acts on its receptor in the ER to cause an intracellular release of Ca
58
How does oocyte respond to stimulation by sperm?
Cortical reaction induces a rise in Ca2+ in cytoplasm that stimulates cortical granules to release their contents outside the egg - Enymes - Polysaccharides This results in formation of the fertilisation envelop leading to a slow block polyspermy
59
What is a block to polyspermy?
Due to release of cortical granule content cross-links zona proteins to make them impenetrable Prevents multiple penetration Cannot overcome simultaneous penetration Therefore depends on limited sperm number in vicinity of oocyte
60
What is the journey of the sperm?
1. Immediate transport 2. Cervix 3. Uterus 4. Oviduct 5. Fertilisation
61
Where are the sites of sperm selection in the female?
Cumulus Isthmus Utero-tubal junction Uterus Cervix Vagina Zona pellucida
62
What biological mechanisms during fertilisation and preimplantation embryo development can promote sex ratio skewing?
A. The ability of the reproductive tract to preclude or retard the progression of one sex-specific population B. The oocyte may preferentially select one population during sperm attachment C. The ability of a particular population of an embryo to undergo selective implantation

Advanced concepts in reproduction (9 decks)

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