Detection of DNA Double-Stranded Breaks in Mouse Oocytes.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis.

The DNA Damage Response in Fully Grown Mammalian Oocytes.

DNA damage in cells can occur physiologically or may be induced by exogenous factors. Genotoxic damage may cause cancer, ageing, serious developmental diseases and anomalies. If the damage occurs in the germline, it can potentially lead to infertility or chromosomal and genetic aberrations in the developing embryo.

Human oocytes harboring damaged DNA can complete meiosis I.

Objective: To determine whether human oocytes possess a checkpoint to prevent completion of meiosis I when DNA is damaged.

Design: DNA damage is considered a major threat to the establishment of healthy eggs and embryos. Recent studies found that mouse oocytes with damaged DNA can resume meiosis and undergo germinal vesicle breakdown (GVBD), but then arrest in metaphase of meiosis I in a process involving spindle assembly checkpoint (SAC) signaling.

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme.

Cyclin A2 modulates kinetochore-microtubule attachment in meiosis II.

Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes.

Maternal age-dependent APC/C-mediated decrease in securin causes premature sister chromatid separation in meiosis II.

Sister chromatid attachment during meiosis II (MII) is maintained by securin-mediated inhibition of separase. In maternal ageing, oocytes show increased inter-sister kinetochore distance and premature sister chromatid separation (PSCS), suggesting aberrant separase activity. Here, we find that MII oocytes from aged mice have less securin than oocytes from young mice and that this reduction is mediated by increased destruction by the anaphase promoting complex/cyclosome (APC/C) during meiosis I (MI) exit.

Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis.

DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.

In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI).

Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.

Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I.

The DNA damage response in mammalian oocytes.

DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumor formation.

Micro-injection of Morpholino oligonucleotides for depleting Securin in mouse oocytes.

Gene silencing techniques have brought new insights into mammalian oocyte and embryo development. More specifically, the use of Morpholino oligonucleotides which sterically inhibit translation from target mRNAs thereby compromising gene function, allowed the identification of important oocyte regulators and especially factors involved in meiotic cell cycle control. Here we describe the method of application of Morpholino oligonucleotides in mouse oocyte research.

Oocytes progress beyond prophase in the presence of DNA damage.

In the female germline, DNA damage has the potential to induce infertility and even to lead to genetic abnormalities that may be propagated to the resulting embryo [1, 2]. The protracted arrest in meiotic prophase makes oocytes particularly susceptible to the accumulation of environmental insults, including DNA damage. Despite this significant potential to harm reproductive capacity, surprisingly little is known about the DNA damage response in oocytes.

Securin regulates entry into M-phase by modulating the stability of cyclin B.

Timely progression into mitosis is necessary for normal cell division. This transition is sensitive to the levels of cyclin B, the regulatory subunit of the master mitotic kinase, Cdk1. Cyclin B accumulates during G2 and prophase when its rate of destruction by the anaphase promoting complex (APC) is low.

Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein.

Oocyte maturation in mouse is associated with a dramatic reorganisation of the endoplasmic reticulum (ER) from a network of cytoplasmic accumulations in the germinal vesicle-stage oocyte (GV) to a network of distinctive cortical clusters in the metaphase II egg (MII). Multiple lines of evidence suggest that this redistribution of the ER is important to prepare the oocyte for the generation of repetitive Ca2+ transients which trigger egg activation at fertilisation. The aim of the current study was therefore to investigate the timecourse and mechanism of ER reorganisation during oocyte maturation.

Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1).

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1-Cul1-F-box/betaTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD).

The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes.

Cdk1-cyclin B1 kinase activity drives oocytes through meiotic maturation. It is regulated by the phosphorylation status of cdk1 and by its spatial organisation. Here we used a cyclin B1-green fluorescent protein (GFP) fusion protein to examine the dynamics of cdk1-cyclin B1 distribution during meiosis I (MI) in living mouse oocytes.

Fertilization and InsP3-induced Ca2+ release stimulate a persistent increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes.

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation.

Ca2+ signalling and cortical re-organisation during the transition from meiosis to mitosis in mammalian oocytes.

In mammals, the mature ovulated egg is arrested in metaphase II of the first meiotic division. The signal that triggers the transition from meiosis to mitosis is provided by the fertilising sperm and takes the form of a series of Ca(2+) oscillations. The pattern of Ca(2+) oscillations is imposed by maternal control mechanisms that ensure Ca(2+) transients occur during M-phase of meiosis II and during the first mitotic division.

Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production.

At fertilisation, repetitive increases in the intracellular Ca2+ concentration, [Ca2+]i, drive the completion of meiosis and initiate the development of the quiescent egg into an embryo. Although the requirement for an ATP supply is evident, the relative roles of potential ATP sources remains unclear in the mammalian egg, and the specific role of mitochondria in [Ca2+]i regulation as well as in the sperm-triggered [Ca2+] oscillations is unknown. We have used fluorescence and luminescence imaging to investigate mitochondrial activity in single mouse eggs.

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei.

In mammals, the sperm triggers a series of cytosolic Ca(2+) oscillations that continue for approximately 4 hours, stopping close to the time of pronucleus formation. Ca(2+) transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca(2+) signalling is not known.

Cell cycle-dependent regulation of structure of endoplasmic reticulum and inositol 1,4,5-trisphosphate-induced Ca2+ release in mouse oocytes and embryos.

The organization of endoplasmic reticulum (ER) was examined in mouse eggs undergoing fertilization and in embryos during the first cell cycle. The ER in meiosis II (MII)-arrested mouse eggs is characterized by accumulations (clusters) that are restricted to the cortex of the vegetal hemisphere of the egg. Monitoring ER structure with DiI18 after egg activation has demonstrated that ER clusters disappear at the completion of meiosis II.