Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs

Abstract 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.

Download Full-text

Selective and Continuous Elimination of Mitochondria Microinjected Into Mouse Eggs From Spermatids, but Not From Liver Cells, Occurs Throughout Embryogenesis

Genetics ◽

10.1093/genetics/156.3.1277 ◽

2000 ◽

Vol 156 (3) ◽

pp. 1277-1284 ◽

Cited By ~ 8

Author(s):

Hiroshi Shitara ◽

Hideki Kaneda ◽

Akitsugu Sato ◽

Kimiko Inoue ◽

Atsuo Ogura ◽

...

Keyword(s):

Early Stage ◽

Liver Mitochondria ◽

Blastocyst Stage ◽

Mus Musculus Domesticus ◽

Newborn Mice ◽

Selective Elimination ◽

Specific Factors ◽

Pcr Method ◽

Mammalian Eggs ◽

Mouse Eggs

Abstract Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mtspr strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mtspr mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Download Full-text

Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs

Biochemical Journal ◽

10.1042/bj3460743 ◽

2000 ◽

Vol 346 (3) ◽

pp. 743-749 ◽

Cited By ~ 32

Author(s):

Keith T. JONES ◽

Miho MATSUDA ◽

John PARRINGTON ◽

Matilda KATAN ◽

Karl SWANN

Keyword(s):

Phospholipase C ◽

Sea Urchin ◽

Tissue Extracts ◽

Sea Urchin Egg ◽

Boar Sperm ◽

Specific Activities ◽

Mammalian Eggs ◽

Mouse Eggs

A soluble phospholipase C (PLC) from boar sperm generates InsP3 and hence causes Ca2+ release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca2+ oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca2+ oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P2, the ability of sperm extracts to release Ca2+ was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca2+-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCβ1, -γ1, -γ2, -∆1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca2+ in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca2+ oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P2 in eggs.

Download Full-text