Fertilisation and Reprogramming to Totipotency Practice Questions

This flashcard set covers the essential biological processes from fertilisation through the transition to totipotency, including molecular signaling, chromatin remodeling, and epigenetic reprogramming.

What does the term 'engender' mean in the context of cellular potency?

To give rise to.

How is 'cellular potency' defined in developmental biology?

The range of commitment options open to a cell.

What is a 'totipotent cell'?

A cell that can engender an entire individual.

What characterizes a 'plenipotent' cell?

It can engender any cell in adults plus extra-embryonic lineages, but not an entire individual.

What is the capacity of a 'pluripotent' cell?

It can engender any cell in an adult but not an entire individual.

Define 'multipotent' cells.

Cells that can engender a limited number of cell types, such as adult stem cells or cord blood stem cells.

What is a 'unipotent' cell?

An immature precursor giving rise to only one cell type.

Which state is considered the maximum cellular potency?

Totipotency.

What specific type of information does a totipotent cell possess that allows it to produce an individual?

It has the sperm information that choreographs differentiation at the right time and right space.

When does the state of totipotency naturally arise?

After sperm and egg fuse in fertilisation.

What are two ways totipotency can occur?

Naturally as an emergent property of the gamete to embryo transition or artificially in nuclear transfer cloning.

How many chromosomes are contained in a human spermatozoon?

A haploid complement of chromosomes.

What is the first step a sperm must take to reach the oocyte's plasma membrane?

Penetrate the zona pellucida.

What is the 'zona pellucida'?

A glycoprotein matrix surrounding the oocyte that is not a living cell.

How many glycoproteins comprise the zona pellucida in mice?

$3$ Glycoproteins.

What are the four glycoproteins that make up the human zona pellucida?

ZP1, ZP2, ZP3, and ZP4.

What event marks the beginning of the gamete-to-embryo transition?

When a sperm and an oocyte (egg) unite.

What event marks the conclusion of the gamete-to-embryo transition?

When the paternal and maternal chromosomes mingle for the first time.

Why is a one-cell embryo or fertilised egg considered totipotent?

Because it has the maximum degree of cellular potency.

After which stage does an individual cell in a mouse embryo lose totipotency?

After the two-cell stage.

If the two cells of a mouse two-cell embryo are experimentally separated, what can they develop into?

Each one can autonomously develop into a complete offspring.

What happens if you separate a blastomere from a four-cell mouse embryo?

It will not develop into an entire individual on its own.

Between which embryonic stages does the window of totipotency close in the mouse?

Between the two-cell and four-cell stages.

What physical factors are hypothesized to be related to the loss of totipotency?

Cytoplasmic volume or cell size.

What is the collective name for the entire process of the gamete-to-embryo transition?

Oocyte activation.

How long does the gamete-to-embryo transition typically unfold in a mouse?

Approximately $18$ to $20$ hours after fertilisation.

At what stage are mature oocytes arrested prior to fertilisation?

Metaphase II.

What is the 'perivitelline space'?

The gap between the oocyte's plasma membrane and the zona pellucida.

What is the approximate diameter of mouse and human mature oocytes?

$90 ext{ microns}$.

What protein causes the arrest at metaphase II?

Endogenous meiotic inhibitors 2 (EMI2), also known as Fbxo43.

Is the protein EMI2 found in mitotic cells?

No, it is not present in mitotic cells.

Which complex does EMI2 stabilize to maintain meiotic arrest?

Maturation Promoting Factor (MPF).

What is the molecular composition of the heterodimer MPF?

Cyclin B and CDK1 (also known as CDC2).

What are the consequences of high MPF activity regarding chromosomes?

It results in phosphorylation of separase (preventing cleavage) and condensins (keeping chromosomes condensed).

Which kinase targets H1, H3, myosin, and condensins during metaphase II arrest?

MPF kinase.

How does EMI2 stabilize MPF?

By inhibiting CDC20.

What is the role of CDC20 in the cell cycle?

It is a co-activator of the Anaphase Promoting Complex (APC).

What biochemical activity does the APC perform?

It is an E3 ubiquitin ligase that ubiquitinates Cyclin B for destruction by the proteasome.

Which pathway leads to the phosphorylation and stabilization of EMI2?

The MOS kinase pathway (or MOS pathway).

What chemical element is absolutely required for the activity of EMI2?

Zinc ($ext{Zn}^{2+}$).

What effect does a zinc chelating agent have on a mature oocyte?

It inactivates EMI2, causing the cell cycle to resume.

What are the intermediates in the MOS kinase cascade?

MOS kinase ightarrow MAP kinase kinase (MKK) ightarrow MAP kinase (MAPK) ightarrow RSK.

Which kinase at the end of the MOS cascade phosphorylates EMI2?

RSK.

What does 'ZBR' stand for in the context of EMI2?

Zinc-binding region.

What is the physiological trigger for metaphase II exit during fertilisation?

A change in intracellular calcium concentration ($riangle[ ext{Ca}^{2+}]_i$).

What is the resting concentration of free calcium inside a mammalian egg?

Around $100-200 ext{ nM}$.

To what concentration does free calcium rise during fertilization in mammals?

About $1 ext{ } ext{µM}$.

What pattern does the calcium rise take in mammalian eggs?

Oscillations.

What specific molecule is delivered by the sperm to trigger calcium release?

Phospholipase C-Zeta ($ext{PLC} ext{ζ}$).

What is the substrate for the enzyme $ext{PLC} ext{ζ}$?

Phosphatidylinositol 4,5-bisphosphate ($ext{PIP}_2$).

What are the two products of $ext{PIP}_2$ cleavage by $ext{PLC} ext{ζ}$?

Inositol 1,4,5-trisphosphate ($ext{IP}_3$) and diacylglycerol (DAG).

How does $ext{IP}_3$ induce calcium release?

It diffuses and interacts with calcium channels in the endoplasmic reticulum (ER), causing them to open.

What calcium-binding protein is activated by the rise in intracellular calcium?

Calmodulin (Cam).

Which specific calmodulin-dependent kinase is primary in eggs?

Calmodulin kinase 2 gamma ($ext{CaMKII} ext{γ}$).

What is the target of activated $ext{CaMKII} ext{γ}$ in the oocyte?

It phosphorylates EMI2, leading to its degradation.

What happens to the APC once EMI2 is removed?

It is no longer inhibited and becomes active, degrading Cyclin B.

Does fertilisation use zinc removal as its physiological trigger for meiotic exit?

No, it is caused by the change in calcium levels.

What was the fertility status of female transgenic mice ubiquitously expressing $ext{PLC} ext{ζ}$?

They were highly subfertile with very small litter sizes.

What pathological development occurred in mice with ectopic $ext{PLC} ext{ζ}$ expression?

Ovarian cysts and enormous ovarian tumors.

What does the term 'oocyte activation' imply about the sperm's role?

The sperm induces the egg to resume embryonic development via specific signaling.

What constitutes 'paternal chromatin remodeling'?

The removal of sperm nucleoproteins (decondensation) and the deposition of maternal histones (recondensation).

How can researchers visualize the global distribution of antigens in embryos?

Confocal immunofluorescence (IF) microscopy on fixed samples.

What happens to the volume of the sperm chromatin during decondensation?

It increases/expands.

What is the major nuclear protein in sperm that must be removed?

Protamine.

What percentage of sperm nuclear protein is protamine in mice?

$98$ to 99 ext{%}.

What percentage of sperm nuclear protein is protamine in humans?

Approximately 85 ext{%}.

In what cellular stages is protamine restricted?

Spermatids and sperm.

What are the chemical characteristics of protamine?

It is an arginine and cysteine rich basic protein.

What physical shape does DNA take when wrapped around protamine?

A toroidal (donut-like) structure.

What force, facilitated by cysteine residues, allows for the dense packing of the sperm nucleus?

Disulfide bonds.

Which two genes encode protamines in mice and humans?

PRM1 and PRM2.

How is PRM2 synthesized compared to PRM1?

PRM1 is synthesized in mature form ($50$ amino acids); PRM2 is synthesized as a precursor and processed ($63$ amino acids).

What metal ion does PRM2 bind?

A single zinc ion ($ext{Zn}^{2+}$).

Where is histone-based chromatin typically located in sperm?

At promoters, especially of developmentally important genes.

What is a 'bivalent mark' in chromatin?

The presence of both H3K4me3 (active) and H3K27me3 (repressive) marks.

What is the functional purpose of a bivalent mark?

To keep genes silenced but poised for differentiation.

Which protein complex is associated with H3K27me3-mediated gene silencing?

Polycomb Group (PcG) repressor complex.

Does sperm chromatin remodeling require DNA replication?

No, it is S-phase independent.

What is the name of the acidic chaperone that removes protamines in Xenopus laevis?

Nucleoplasmin.

Name the three mammalian orthologs of nucleoplasmin.

Npm1 (nucleophosmin), Npm2, and Npm3.

In which disease are Npm1 translocations involved?

Cancer, such as non-Hodgkin lymphoma.

What is the phenotype of Npm2 null knockout mice?

Normal decondensation but abnormal pronuclei.

When does histone deposition initiate in mouse oocytes after sperm entry?

About $20$ minutes after sperm entry.

Which histone variant is the major one during the gamete-to-embryo transition?

H2A.X.

Which chaperone is responsible for depositing the H3.3 histone variant?

Hira.

How is H3.3 deposition restricted after the 4-cell stage?

It becomes restricted to euchromatin (initially present in both heterochromatin and euchromatin).

What membrane-bound structures encapsulate parental genomes after remodeling?

Pronuclei (pn).

When do pronuclei typically become visible in a mouse zygote?

After about $4.5$ hours.

Which pronucleus is usually larger in mice?

The male pronucleus.

From which organelle is the pronuclear membrane derived?

Endoplasmic reticulum (ER).

What is the term for the process where chromosomes from separate pronuclei come together?

Syngamy.

Which histone modification is high throughout the 1-cell embryo in both parental sets?

$H4K12ac$ (and other histone acetylation).

Which paternal marks are delayed in the one-cell embryo?

H3K4me3, H3K9me2, H3K27me3.

What does ChIP-seq stand for?

Chromatin Immunoprecipitation sequencing.

What does ATAC-seq reveal?

Genome-wide chromatin accessibility.

What does 'tagmentation' involve in the CUT&TAG method?

Simultaneously cleaving DNA and adding adapters for sequencing.

Which enzyme is used in CUT&TAG?

Hyperactive Tn5 transposase fused to Protein A.

How does DNA methylation ($5mC$) occur?

Via DNA methyltransferases (Dnmts) during gametogenesis.

How is the maternal genome primarily demethylated after fertilisation?

Passively demethylated.

How is the paternal genome demethylated after fertilisation?

Major active demethylation via oxidation.