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Michail Nomikos,
Yuansong Yu,
Khalil Elgmati,
Maria Theodoridou, Karen Campbell,
Vyronia Vassilakopoulou,
Christos Zikos,
Evangelia Livaniou,
Nazar Amso,
George Nounesis,
Karl Swann,
F Anthony Lai
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ABSTRACT: Objective: To determine the effect of infertility-linked sperm phospholipase Cz (PLCz) mutations on their ability to trigger oocyte Ca 2þ oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCz protein for rescuing failed oocyte activation and embryo development. Design: Test of a novel therapeutic approach to male factor infertility. Setting: University medical school research laboratory. Patient(s): Donated unfertilized human oocytes from follicle reduction. Intervention(s): Microinjection of oocytes with recombinant human PLCz protein or PLCz cRNA and a Ca 2þ -sensitive fluorescent dye. Main Outcome Measure(s): Measurement of the efficacy of mutant and wild-type PLCz-mediated enzyme activity, oocyte Ca 2þ oscillations, activation, and early embryo development. Result(s): In contrast to the wild-type protein, mutant forms of human sperm PLCz display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCz protein reliably triggers the characteristic pattern of cytoplasmic Ca 2þ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage. Conclusion(s): Dysfunctional sperm PLCz cannot trigger oocyte activation and results in male factor infertility, so a potential thera-peutic approach is oocyte microinjection of active, wild-type PLCz protein. We have demonstrated that recombinant human PLCz can phenotypically rescue failed activation in oocytes that express dysfunctional PLCz, and that this intervention culminates in efficient blastocyst formation.
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ABSTRACT: To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca(2+) oscillations during activation.
Test of a laboratory technique.
University medical school research laboratory.
Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles.
Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca(2+)-sensitive fluorescent dye.
Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca(2+) oscillations using a Ca(2+)-sensitive fluorescent dye.
Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca(2+) oscillations. Each transient Ca(2+) concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis.
The occurrence and frequency of cytoplasmic Ca(2+) oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca(2+) oscillations in human zygotes.
Fertility and sterility 01/2012; 97(3):742-7. · 3.97 Impact Factor
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ABSTRACT: The microinjection of cRNA encoding phospholipase Czeta (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCzeta protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLCzeta with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently allows for sets of eggs to be used for PLCzeta quantitation, Ca2+ imaging, and studies of embryo development.
Methods in molecular biology (Clifton, N.J.) 02/2009; 518:17-29.
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ABSTRACT: Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca(2+) oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca(2+)] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca(2+) oscillations in the cytosol that are transduced into mitochondrial Ca(2+) oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP](mito) and [ATP](cyto). We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca(2+)](cyto) and [ATP](cyto) during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.
Developmental Biology 05/2008; 316(2):431-40. · 4.07 Impact Factor
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ABSTRACT: Mammalian eggs and embryos rely upon mitochondrial ATP production to survive and proceed through preimplantation development. Ca(2+) oscillations at fertilization have been shown to cause a reduction of mitochondrial NAD+ and flavoproteins, suggesting they might also cause changes in cytosolic ATP levels. Here, we have monitored intracellular Ca(2+) and ATP levels in fertilizing mouse eggs by imaging the fluorescence of a Ca(2+) dye and luminescence of firefly luciferase. At fertilization an initial increase in ATP levels occurs with the first Ca(2+) transient, with a second increase occurring about 1 h later. The increase in cytosolic ATP was estimated to be from a prefertilization concentration of 1.9 mM to a peak value of 3 mM. ATP levels returned to prefertilization values as the Ca(2+) oscillations terminated. An increase in ATP also occurred with other stimuli that increase Ca(2+) and it was blocked when Ca(2+) oscillations were inhibited by BAPTA injection. Additionally, an ATP increase was not seen when eggs were activated by cycloheximide, which does not cause a Ca(2+) increase. These data suggest that mammalian fertilization is associated with a sudden but transient increase in cytosolic ATP and that Ca(2+) oscillations are both necessary and sufficient to cause this increase in ATP levels.
Developmental Biology 11/2006; 298(1):225-33. · 4.07 Impact Factor
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ABSTRACT: The sperm-specific phospholipase C-zeta (PLCzeta) elicits fertilization-like Ca2+ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLCzeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCzeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLCzeta structure-function, we assessed the ability of PLCzeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca2+ changes in intact mouse eggs. PLCzeta and the closely related PLCdelta1 had similar K(m) values for phosphatidylinositol 4,5-bisphosphate, but PLCzeta was around 100 times more sensitive to Ca2+ than was PLCdelta1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCzeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca2+ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca2+ oscillations. These data suggest that the exquisite Ca2+-dependent features of PLCzeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.
Journal of Biological Chemistry 10/2005; 280(35):31011-8. · 4.77 Impact Factor
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ABSTRACT: The microinjection of cRNA encoding phospholipase Cζ (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCζ protein that is expressed in eggs after injection
of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged
PLCζ with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin
combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The
use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently
allows for sets of eggs to be used for PLCζ quantitation, Ca2+ imaging, and studies of embryo development.
Key wordsLuminescence–luciferase–phospholipase–egg
01/1970: pages 17-29;
