Oocyte Cytoplasmic Maturation

Question 1

Oocyte cytoplasmic maturation is described as processes that

Answer 1

modify the oocyte cytoplasm that are essential for fertilization and developmental competence.

Question 2

However, oocyte cytoplasmic maturation lacks definable attributes, and there is a paucity of information regarding

Answer 2

this essential component of oocyte maturation.

Question 3

Central to the concept of cytoplasmic maturation are the

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production and presence of specific factors, relocation of cytoplasmic organelles, and post-transcriptional modification of MRNAs that have accumulated during oogenesis.

Question 4

Understanding the control of oocyte maturation requires the recognition of

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mechanisms that control meiotic arrest and those that trigger meiotic resumption in vivo.

Question 5

These have proven difficult to study because

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the entire follicle surrounding the oocyte must remain intact to function normally thus, rendering the oocyte inaccessible for study.

Question 6

Oocyte-granulosa cell communication: It is important to acknowledge that the oocyte plays an active role in

Answer 6

its own development, being responsible for the proliferation, development function of the GC.

Question 7

Ovarian follicle development requires the

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maintenance of a bi-directional communication between follicle components the somatic GC and the oocyte.

Question 8

Junctional communications between the oocyte and GC have been observed in primordial follicles and their presence is maintained

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throughout later stages of follicle growth.

Question 9

Within the follicle, the developing oocyte maintains direct communication with cumulus and mural granulosa cells by way of granulosa cell extensions that

Answer 9

traverse the zona pellucida and form gap junctions, specialized regions in closely apposed membranes of adjacent cells that mediate cell-to-cell communication.

Question 10

These channels provide a means of transferring

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nutrients and molecules essential for oocyte cytoplasmic maturation

Question 11

Ongoing granulosa-oocyte communication is critical for

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oocyte growth the development of maturational competence, and the maintenance of meiotic arrest.

Question 12

Gap junction proteins and their role in follicle maturation: These proteins are also known as

Answer 12

connexins and several have been identified.

Question 13

Connexin-37, Cx37 (also called GJA4) is the major connexin present in

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gap junctions between the oocyte and cumulus cells.

Question 14

Connexin-43, Cx43 (GJA1), is the main gap junction protein in channels between

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granulosa cells.

Question 15

The preovulatory surge of the gonadotropins, particularly LHBI, or following hCG administration in ART cycles, disrupts the

Answer 15

disrupts the cumulus:oocyte communication and brings about the resumption of meiosis/.

Question 16

Kalma and colleagues (2004) demonstrated that LH inhibits

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Cx43 expression and this effect is mediated by both protein kinase A (PKA) and activation of mitogen-activated protein kinase (MAPK).

Question 17

Factors controlling resumption of meiosis: The adenylyl cyclaselcyclic adenosine monophosphate (cAMP)/PKA pathway has been widely accepted as the primary signaling cascade through which

Answer 17

the surge of FSH and LH exert their actions that lead to the resumption of meiosis.

Question 18

During maturation, signals from follicular somatic cells keep the oocyte’s cell cycle arrested at

Answer 18

prophase of meiosis I (see above).

Question 19

Elevated cAMP levels within the pre-ovulatory oocyte support PKA activity, preventing

Answer 19

further downstream signaling in the meiosis activation pathway.

Question 20

Intra-oocyte CAMP and cyclic guanosine monophosphate (cGMP) signaling have been suggested to cooperate in the

Answer 20

maintenance of the oocyte’s meiotic arrest, and the level of both cyclic nucleotides has been observed to decrease prior to GVBD.

Question 21

Epidermal growth factor (EGF)-like growth factors, meiosis-activating sterol and gonadal steroid hormones, are involved in

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the activation of MAPK, possibly via the protein kinase A and protein kinase C pathways.

Question 22

The EGF receptor, RAS, and MAPK pathway may act as intrafollicular mediators of

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gonadotrophin-induced signals from the surface somatic follicle cells surrounding the oocyte to the oocyte that lacks gonadotropin receptors.

Question 23

While it is not known whether the FSH- and LH-induced pathways act in a redundant manner via the same signaling pathway, or their pathways overlap directly, downstream actions of the gonadotropin surge likely

Answer 23

trigger pathways that culminate in both the relief of inhibition of some substrates and the activation of others. Together these changes reactivate meiosis I and induce final maturational events in the oocyte cytoplasm.

Question 24

Identifying the specific somatic cell signal(s) that maintain meiotic arrest is still under investigation. Experimental evidence shows high levels of CAMP are required

Answer 24

to maintain meiotic arrest oocyte.

Question 25

The downstream pathway(s) by which cAMP prevents meiotic maturation is incompletely understood, but ultimately CAMP levels within the oocyte affect

Answer 25

the activity of the protein kinase maturation promoting factor (MPF).

Question 26

The origin of the intra-oocyte cAMP also remains undetermined; it could be produced by the oocyte itself or it is

Answer 26

produced by the follicle cells that surround it and diffuses into the oocyte via the gap junctions.

Question 27

If the oocyte does depend on externally produced cAMP then gap junction dissociation after the LH surge would lead to

Answer 27

a breakdown in this communication pathway, a decrease in the oocyte cAMP Concentration and reinitiation of meiosis.

Question 28

Nuclear maturation appears to require reversible protein phosphorylation, a process recognized as

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a major intracellular regulatory mechanism in a wide range of eukaryotic cellular events.

Question 29

Protein kinases bring about phosphorylation (addition of phosphate), and protein phosphatases remove

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phosphate leading to protein dephosphorylation.

Question 30

The principal kinases and phosphatases that have been shown to play a role in the oocyte maturation process are the following:

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A: Maturation Promoting factor (MPF) B: Mitogen-Activating Protein Kinase (MAPK) C: Serine/Threonine Protein Phosphatases (PP) D: Regulation of the Prophase I to Metaphase II transition

Question 31

Maturation Promoting Factor (MPF) is a complex of two major proteins:

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a. p34cdc? (also known as cdk1): a serine/threonine kinase that is negatively regulated by Wee1 kinase and requires dephosphorylation by cdc25 for activity. b. Cyclin B: requires phosphorylation for activity. It has been speculated that continual phosphorylation of cyclin B is necessary for stability and activity of MPF and that dephosphorylation results in MPF degradation.

Question 32

MPF activity fluctuates during the progression of oocyte meiosis, being

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undetectable in GV-intact, detectable at GVBD during anaphase l and telophase I.

Question 34

MPF levels increase again

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peaking at metaphase Il (MII).

Question 35

The exact regulators of MPF activity remain obscure; however we do know that it is stabilized during the metaphase ll arrest via the activity of an

Answer 35

unidentified cytostatic factor (CSF).

Question 38

Mitogen-Activated Protein Kinase (MAPK) also known as extracellular regulated kinase (ERK) is another serine/threonine kinase implicated in the regulation of

Answer 38

oocyte meiosis.

Question 39

In mouse oocytes, MAPK activity increases following

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GVBD and throughout maturation between MI and MIl.

Question 43

MAPK has been suggested to have a critical role in microtubule assembly which is important for

Answer 43

meiotic spindle formation and chromatin organization.

Question 45

MAPK is also involved the arrest of the oocyte in

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metaphase of meiosis II.

Question 46

Together with the c-mos proto-oncogene protein kinase (Mos) MAPK is essential for

Answer 46

establishing CSF activity that enforces a metaphase Il arrest by inhibiting the pathway leading to MPF degradation.

Question 49

Serine/Threonine Protein Phosphatases (PP) remove phosphate groups from

Answer 49

phosphoproteins thus antagonizing protein kinases oocyte maturation.

Question 51

GV-intact oocytes contain both protein phosphatase-1 (PP1) and protein

Question 52

nuclear and cytoplasmic in location

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respectively

Question 53

PP1 is involved in regulating nuclear envelope dissolution and its specific inhibition has been demonstrated to

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stimulate GVBD.

Question 55

PP2A associates with meiotic spindles and its inhibition blocks

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meiotic spindle formation in the oocyte.

Question 56

Processes occurring in the oocyte during the transition from GV-intact to the MIl stage of maturation can be divided into at least

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four specific events, all of which are regulated by either MPF or MAPK phosphorylation and/or PP2A dephosphorylation.

Question 57

The transition from GV-intact to the MIl stage of maturation can be divided into at least 4 events:

Answer 57

i. GVBD is initiated by phosphorylation of nuclear lamins proteins located under the nuclear envelope important for nuclear membrane integrity. ii. Concomitant with GVBD dissolution of the nucleolus occurs. This process has not been investigated during meiosis but studies during mitosis suggest that nucleolar protein phosphorylation is involved. iii. Chromosome condensation is then stimulated by phosphorylation of chromosomal proteins including histones. iv. The fourth event involves microtubule polymerization originating from the centrosomes or microtubule organizing centers and leading to formation of the meiotic spindle.

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b

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i.

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iv

Question 72

It is becoming increasingly evident that in the oocyte and other cell systems, phosphorylation or dephosphorylation cannot be considered as

Answer 72

isolated intracellular phospho-regulated events.

Question 74

Rather, the important issue is likely the degree of phosphorylation that state of equilibrium between

Answer 74

kinase and phosphatase activity.

Question 77

A clear understanding of cytoplasmic maturation has yet to be elucidated however it is essential torender oocytes capable of undergoing

Answer 77

sperm-induced activation, release of intracellular stores of calcium, and resumption of the second meiotic division.

Question 79

Some of these cytoplasmic events have been identified and include the following:

Question 81

a. Reorganization of endoplasmic reticulum (ER), the main stockpile of calcium in the oocyte

Question 82

Increase in the number of 1

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4

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C. Changes in the biochemical properties of IP3 receptors (sensitivity to IP3)

Question 85

d. Increase in the concentration of calcium ions stored in the ER

Question 86

e. Redistribution of calcium-binding ER proteins.

Question 87

f. Intracellular trafficking of cortical granules may also be considered a component of cytoplasmic maturation. The migration of cortical granules toward the periphery of the oocyte is a process that occurs during oocyte development and is important for release of enzymatic contents upon sperm penetration.

Question 91

g. Oocyte-derived glutathione accumulation in the ooplasm is another example of a maturational change. Once the sperm gains entry to the ooplasm its nucleus must decondense to support normal processing of the male pronucleus. This process involves reduction of disulfide bonds between sperm chromatin-associated protamines and is regulated by glutathione. Indeed, formation of the male pronucleus is rarely observed in GV-intact oocytes due to reduced levels of glutathione in immature oocytes.

Question 93

normal processing of the male pronucleus. This process involves reduction of disulfide bonds between sperm chromatin-associated protamines and is regulated by glutathione. Indeed

Question 95

formation of the male pronucleus is rarely observed in GV-intact oocytes due to reduced levels of glutathione in immature oocytes.

Question 97

i.

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h. There are most likely several additional specific proteins that fluctuate during nuclear maturation

Question 99

and are critical for supporting cytoplasmic maturation. In addition to the production and

Question 100

accumulation of specific proteins during oogenesis

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oocytes also acquire surplus mRNÁs

Question 101

that are not necessarily translated but are important during initial stages of embryogenesis

Answer 101

prior

Question 102

to the switch from maternal to embryonic control of development (ZGA; see below).

Question 103

Mitochondria are the most abundant organelles in the cytoplasm and have an important role in

Question 104

human o0cyte maturation. They undergo redistribution in a manner coordinated with the

Question 105

progression of maturation (Liu et al.

Answer 105

2010). Therefore

Question 106

maturation during oogenesis and nuclear maturation include transcription

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mechanisms that

Question 107

regulate mRNA stability (post-transcriptional regulation)

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translation

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processing of specific factors that support early embryogenesis. These are areas that require

Question 109

further investigation to better understand the shortcomings of in vitro maturation of oocytes.

Question 110

j. In vitro maturation (IVM) of human oocytes has been applied with varying success since 1965

Question 111

and resulted in the first live birth in 1989 (Cha et al.

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1991; Nogueira et al.

Question 112

2013). IVM refers to the maturation of immature oocytes in culture after their recovery from small

Question 113

antral follicles at the stage prior to follicle selection and dominance. IVM requires little or no FSH

Question 114

administration prior to oocyte recOvery

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and this approach may benefit women with an increased

Question 115

risk for ovarian hyperstimulation syndrome after IVF. However

Answer 115

pregnancy rates following IVM are

Question 116

suboptimal compared to IVF. The success of IVM is associated with the source of oocytes

Question 117

selected and the cuiture conditions in vitro (Telfer

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2019). The acquisition of developmental

Question 118

competence by the oocyte occurs during folicle growth and is an absolute requirement for the

Question 119

oocyte to resume meiosis

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to support fertilization events

Question 120

vitro culture conditions must mimic dynamic and complex conditions within the ovary to ensure

Question 121

the development of healthy

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competent oocytes with maintained DNA integrity and stability

Question 122

this remains a significant technical challenge.

Question 123

k. Morphological parameters with which to judge oocyte competency have been examined.

Question 124

Examples include data illustrating central rather than peripheral positioning of the germinal

Question 125

vesicle in competent mouse oocytes (Brunet & Maro

Answer 125

2007) and computer-assisted polarization

Question 126

1.32

Question 127

microscopy (polscope) examination of meiotic spindle integrity and polar body location to predict

Question 128

fertilization and developmental potential in human o0cytes (Wang et al.

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2001).