O-039 Mitochondrial ATP generation in mammalian eggs and Ca2+ signaling

text In metaphase II arrested mammalian oocytes (eggs) and cleavage stage embryos the mitochondria are responsible for nearly all ATP production because glycolysis is inactivated. Luciferase assays show that ATP levels in eggs are strictly dependent upon pyruvate and fatty acid oxidation. The level of ATP in eggs appears to be maximal in conventional medium because the addition of extra mitochondrial substrates to eggs does not increase cytosolic ATP. The only clear elevation of ATP is seen at fertilization and is associated with sperm induced Ca2+ oscillations. Our recent findings suggest that the level of ATP modulates events at fertilization. At fertilization, the egg is activated by sperm derived PLCzeta which triggers a series of Ca2+ oscillations, with each Ca2+ release event causes by inositol trisphosphate (InsP3). Previous studies have shown that mouse eggs are more sensitive to PLCzeta, and generate higher frequency Ca2+ oscillations, than human eggs. Mouse eggs also generate Ca2+ oscillations and activate in response to Sr2+ that directly stimulates InsP3 receptors. In contrast, human eggs that contain the same type of InsP3 receptors do not generate Ca2+ oscillations in response to Sr2+. The difference in sensitivity of Ca2+ release between species can be explained by the fact that mouse eggs are about ten times more sensitive to InsP3 than human eggs. The reason for this difference appears to be due to ATP. The ATP level in unfertilized mouse eggs is about twice that in human eggs. Furthermore, the ability of mouse eggs to Sr2+ medium can be abolished by removing the mitochondrial substrate pyruvate, which reduces the ATP level. Adding back pyruvate to such eggs restores ATP levels promotes Sr2+ induced Ca2+ levels in mouse eggs. These data suggest that the level of ATP, possibly as ATP4-, modulates the sensitivity of the InsP3 receptor and the ability of eggs to generate Ca2+ oscillations. The level of cytosolic ATP may represent a significant ‘egg factor’ in determining the success of fertilization in humans. Enhancing mitochondrial ATP production could be useful in improving activation and embryo development after fertilization, or after artificial egg activation. References: Dumollard et al. (2009) Seminar in Cell and Developmental Biology 20, 346-353 Campbell and Swann (2006) Developmental Biology 298, 225-233 Storey et al. (2021) Molecular Human Reproduction 27, gaaa086

Two mechanisms drive pronuclear migration in mouse zygotes

A new life begins with the unification of the maternal and paternal chromosomes upon fertilization. The parental chromosomes first become enclosed in two separate pronuclei near the surface of the fertilized egg. The mechanisms that then move the pronuclei inwards for their unification are only poorly understood in mammals. Here, we report two mechanisms that act in concert to unite the parental genomes in fertilized mouse eggs. The male pronucleus assembles within the fertilization cone and is rapidly moved inwards by the flattening cone. Rab11a recruits the actin nucleation factors Spire and Formin-2 into the fertilization cone, where they locally nucleate actin and further accelerate the pronucleus inwards. In parallel, a dynamic network of microtubules assembles that slowly moves the male and female pronuclei towards the cell centre in a dynein-dependent manner. Both mechanisms are partially redundant and act in concert to unite the parental pronuclei in the zygote’s centre.

The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs

At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1-4-5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI. Mouse eggs incubated in Ca2+-free, Sr2+-containing medium immediately underwent Ca2+ oscillations but human eggs consistently failed to undergo Ca2+ oscillations in the same Sr2+ medium. We tested the InsP3-receptor (IP3R) sensitivity directly by photo-release of caged InsP3 and found that mouse eggs were about 10 times more sensitive to InsP3 than human eggs. There were no major differences in the Ca2+ store content between mouse and human eggs. However, we found that the ATP concentration was consistently higher in mouse compared to human eggs. When ATP levels were lowered in mouse eggs by incubation in pyruvate-free medium, Sr2+ failed to cause Ca2+ oscillations. When pyruvate was added back to these eggs, the ATP levels increased and Ca2+ oscillations were induced. This suggests that ATP modulates the ability of Sr2+ to stimulate IP3R-induced Ca2+ release in eggs. We suggest that human eggs may be unresponsive to Sr2+ medium because they have a lower level of cytosolic ATP.

The soluble sperm factor that activates the egg: PLCzeta and beyond

PLCzeta(ζ) initiates Ca2+ oscillations and egg activation at fertilization in mammals, but studies in mouse eggs fertilized by PLCζ knockout (KO) sperm imply that there is another slow acting factor causing Ca2+ release. Here, I propose a hypothesis for how this second sperm factor might cause Ca2+ oscillations in mouse eggs.

Development of a genetically encoded sensor for endogenous CaMKII activity

ABSTRACTCaMKII is a crucial oligomeric enzyme in neuronal and cardiac signaling, fertilization and immunity. Here, we report the construction of a novel, substrate-based, genetically-encoded sensor for CaMKII activity, FRESCA (FRET-based Sensor for CaMKII Activity). Currently, there is one biosensor for CaMKII activity, Camui, which contains CaMKII. FRESCA allows us to measure all endogenous CaMKII variants, while Camui can track a single variant. Since there are ~40 CaMKII variants, using FRESCA to measure aggregate activity allows a fresh perspective on CaMKII activity. We show, using live-cell imaging, FRESCA response is concurrent with Ca2+ rises in HEK293T cells and mouse eggs. In eggs, we stimulate oscillatory patterns of Ca2+ and observe the differential responses of FRESCA and Camui. Our results implicate an important role for the variable linker region in CaMKII, which tunes its activation. FRESCA will be a transformative tool for studies in neurons, cardiomyocytes and other CaMKII-containing cells.