Analysis of global gene expression following mouse blastocyst cryopreservation.

BACKGROUND: The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression. METHODS: Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays. RESULTS: Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts. CONCLUSIONS: Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation.

BACKGROUND: The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification. METHODS: A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures. RESULTS: Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively. CONCLUSIONS: Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010.

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes.

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology.

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.

The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCzeta.

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+ concentration. In mammals the Ca2+ changes at fertilization consist of intracellular Ca2+ oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3 and Ca2+ at fertilization. One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+ oscillations induced by the sperm. We have recently shown that this soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLCzeta(zeta). We describe the evidence that led to the identification of PLCzeta and discuss the issues relating to its potential role in fertilization.

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes.

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.

The role of calcium in ABA-induced gene expression and stomatal movements.

There is much interest in the transduction pathways by which abscisic acid (ABA) regulates stomatal movements (ABA-turgor signalling) and by which this phytohormone regulates the pattern of gene expression in plant cells (ABA-nuclear signalling). A number of second messengers have been identified in both the ABA-turgor and ABA-nuclear signalling pathways. A major challenge is to understand the architecture of ABA-signalling pathways and to determine how the ABA signal is coupled to the appropriate response. We have investigated whether separate Ca2+-dependent and -independent ABA-signalling pathways are present in guard cells. Our data suggest that increases in [Ca2+]i are a common component of the guard cell ABA-turgor and ABA-nuclear signalling pathways. The effects of Ca2+ antagonists on ABA-induced stomatal closure and the ABA-responsive CDeT6-19 gene promoter suggest that Ca2+ is involved in both ABA-turgor signalling and ABA-nuclear signalling in guard cells. However, the sensitivity of these pathways to alterations in the external calcium concentration differ, suggesting that the ABA-nuclear and ABA-turgor signalling pathways are not completely convergent. Our data suggest that whilst Ca2+-independent signalling elements are present in the guard cell, they do not form a completely separate Ca2+-independent ABA-signalling pathway.

Calcium and mitosis.

Calcium is thought to be involved in regulating mitotic transitions. The basis for this view is set out. Recent data from experiments on sea urchin embryos is discussed. The relative simplicity of the embryonic cell cycle and the relative ease with which cell physiology can be done in sea urchin embryos has allowed the clear demonstration that the phosphoinositide-calcium-calmodulin signalling pathway is required for and regulates mitosis entry and anaphase onset. The relevance of the sea urchin work to mitosis in other cell types is briefly discussed.