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. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. We have investigated Ca2+ -induced cyclin destruction in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. We find no evidence for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. We conclude that increases in cytosolic Ca2+ induce transient changes in autofluorescence and that the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+ -induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction.

Animals↗

PubMed2004

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. The transition from meiosis to mitosis involves a major re-organisation. The unfertilised egg is polarised with the meiotic spindle located in the cortex of the animal pole and clusters of endoplasmic reticulum in the vegetal hemisphere. By the time of the first mitotic division some 20h later the spindle has formed in the centre of the embryo and is surrounded by endoplasmic reticulum. These changes in organisation have implications for the inheritance of ER in meiotic and mitotic cell divisions and may reflect different roles and requirements for Ca(2+) in meiosis and mitosis.

Animals↗

PubMed2004

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. Simultaneous imaging of mitochondrial redox state (NADH and flavoprotein autofluorescence) and [Ca2+]i revealed that sperm-triggered [Ca2+] oscillations are transmitted to the mitochondria where they directly stimulate mitochondrial activity. Inhibition of mitochondrial oxidative phosphorylation caused release of Ca2+ from the endoplasmic reticulum because of local ATP depletion. Mitochondrial ATP production is an absolute requirement for maintaining a low resting [Ca2+]i and for sustaining sperm-triggered [Ca2+] oscillations. Luminescence measurements of intracellular [ATP] from single eggs confirmed that mitochondrial oxidative phosphorylation is the major source of ATP synthesis in the dormant unfertilised egg. These observations show that a high local ATP consumption is balanced by mitochondrial ATP production, and that balance is critically poised. Mitochondrial ATP supply and demand are thus closely coupled in mouse eggs. As mitochondrial ATP generation is essential to sustain the [Ca2+] signals that are crucial to initiate development, mitochondrial integrity is clearly fundamental in sustaining fertility in mammalian eggs.

Adenosine Triphosphate↗

PubMed2004

Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCzeta.

During the first cell cycle Ca2+ oscillations are regulated in a cell cycle-dependent manner, such that the oscillations are unique to M phase. How the Ca2+ oscillations are regulated with such cell cycle stage-dependency is unknown, despite their importance for egg activation and embryo development. We recently identified a novel, sperm-specific phospholipase C (PLCzeta; PLCzeta) that triggers Ca2+ oscillations similar to those caused by sperm. We show that PLCzeta-induced Ca2+ oscillations also occur exclusively during M phase. The cell cycle-dependency can be explained by PLCzeta's localisation to the pronuclei, which depends specifically upon a nuclear localisation signal sequence. Preventing pronuclear localisation of PLCzeta by mutation of the nuclear localisation signal, or by inhibiting pronuclear formation/import, can prolong Ca2+ oscillations or allow them to occur during interphase. These data suggest a novel mechanism for regulating a PLC through nuclear sequestration and may explain the cell cycle-dependent regulation of Ca2+ oscillations following fertilisation.

Amino Acid Substitution↗

PubMed2004

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs.

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCalpha and PKCgamma in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1-3 microM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.

Animals↗

PubMed2004

Atrial septal perforation from TIPS stent migration.

Transjugular intrahepatic portosystemic shunts (TIPS) are safe and effective for the treatment of portal hypertension. Cardiac complications are unusual. This study reports a case of TIPS stent migration to the right atrium causing perforation of the atrial septum in a patient with end-stage liver disease. The shunt was removed transvenously but attempts at transvenous occlusion of the septal perforation were unsuccessful. The patient went on to undergo combined open-heart surgery with septum repair and liver transplantation. The case highlights a rare complication of TIPS and methods for treatment including transvenous removal, transvenous repair, and combined cardiotomy-liver transplantation surgery.

Adult↗

PubMed2004

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. Microinjection of cyclin B1-GFP accelerated germinal vesicle breakdown (GVBD) and, as previously described, overrides cAMP-mediated meiotic arrest. GVBD was pre-empted by a translocation of cyclin B1-GFP from the cytoplasm to the germinal vesicle (GV). After nuclear accumulation, cyclin B1-GFP localised to the chromatin. The localisation of cyclin B1-GFP is governed by nuclear import and export. In GV intact oocytes, cyclin export was demonstrated by showing that cyclin B1-GFP injected into the GV is exported to the cytoplasm while a similar size dextran is retained. Import was revealed by the finding that cyclin B1-GFP accumulated in the GV when export was inhibited using leptomycin B. These studies show that GVBD in mouse oocytes is sensitive to cyclin B1 abundance and that the changes in distribution of cyclin B1 contribute to progression through MI.

Animals↗

PubMed2004