scholarly journals Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 505-515 ◽  
Author(s):  
Shinsuke Seki ◽  
Keisuke Edashige ◽  
Sakiko Wada ◽  
Peter Mazur
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Types ◽  
Ice Nucleation ◽  
Nucleation Temperature ◽  
Ice Formation ◽  
Mouse Oocytes ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Mature Mouse

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

Cellular Response of Mouse Oocytes to Freezing Stress: Prediction of Intracellular Ice Formation

1993 ◽  
Vol 115 (2) ◽  
pp. 169-174 ◽  
Author(s):  
M. Toner ◽  
E. G. Cravalho ◽  
M. Karel
Keyword(s):  
Plasma Membrane ◽  
Cellular Response ◽  
Freezing Stress ◽  
Ice Formation ◽  
Coupled Modes ◽  
Mouse Oocytes ◽  
Intracellular Ice Formation ◽  
Stress Prediction ◽  
Intracellular Ice ◽  
Experimental Protocols

Successful protocols for cryopreservation of living cells can be designed if the physicochemical conditions to preclude intracellular ice formation (IIF) can be defined. Unfortunately, all attempts to predict the probability of IIF have met with very limited success. In this study, an analytical model is developed to predict ice formation inside mouse oocytes subjected to a freezing stress. According to the model, IIF is catalyzed heterogeneously by the plasma membrane (i.e., surface catalyzed nucleation, SCN). A local site on the plasma membrane is assumed to become an ice nucleator in the presence of the extracellular ice via its effects on the membrane. This interaction is characterized by the contact angle between the plasma membrane and the ice cluster. In addition, IIF is assumed to be catalyzed at temperatures below -30° C by intracellular particles distributed throughout the cell volume (i.e., volume catalyzed nucleation, VCN). In the present study, these two distinctly coupled modes of IIF, especially SCN, are applied to various experimental protocols from mouse oocytes. Excellent agreement between predictions and observations suggests that the proposed model of IIF is adequate.

Evaluation of Freezing Effects on Human Microvascular-Endothelial Cells (hMEC)

2001 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Endothelial Cells ◽  
Predictive Ability ◽  
Cell Types ◽  
Cell System ◽  
Ice Formation ◽  
Microvascular Endothelial Cells ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Microvascular Endothelial

Abstract There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing. Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing and compare them with post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130°C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics were found to be governed by a membrane permeability Lpg and activation energy ELp of 0.05 (μm/min.atm) and 14.8 (kcal/mole) respectively [R2 = 0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15°C/min with a good agreement [R2 = 0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% ∼ −18°C) and at cooling rates greater than or equal to 25°C/min. At cooling rates above 50°C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN). IIF parameters Ωo, and κo were found to be 6.8 × 10−8 (m2.s)−1 and 8.3 × 10−9 (K5) [R2 = 0.94] respectively. Viability results suggest an inverted U-shape curve between 1 and 50°C/min (with a maximum at 10°C/min). But viability appears to increase again at cooling rates > 50°C/min (i.e. it does not continue to drop) which suggests that the traditional two factor hypothesis may not completely describe viability in this system. Additional cellular destruction was found by lowering the end-temperature to −30°C or below. At this temperature the majority of the cell population was destroyed regardless of the cooling rate.

A Determination of Biophysical Parameters Related to Freezing of an ELT-3 Cell Line

2000 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Cell Line ◽  
Volume Fraction ◽  
Cell Types ◽  
Cellular Level ◽  
Ice Formation ◽  
Biophysical Parameters ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Biophysical Study ◽  
Intracellular Ice

Abstract This study investigates two destructive biophysical mechanisms during freezing (extensive dehydration and intracellular ice formation) at the cellular level in the rodent ELT-3 uterine fibroid cell-line. The osmotically inactive volume fraction (Vb) of ELT-3 cells was approximated to 0.35 of the initial isotonic cell volume (Vo). The water transport characteristics of this cell-line are such that ELT-3 cells are highly permeable with a strong ability to lose water even at low subzero temperatures. The hydraulic reference permeability, Lpg and activation energy, Elp associated with Lp were found to be 0.13 (μm/min.atm) and 19.0 (kcal/mole) [R2 = 0.86] respectively. Intracellular Ice Formation (IIF) occurs at lower temperatures than many cell-types (i.e. TIIF 50% below −15°C) at cooling rates > 25 °C/min. Darkening IIF, which was assumed to occur by Surface Catalyzed Nucleation (SCN), is governed by kinetic Ωo and thermodynamic κo biophysical parameters, which were found to be 6.1×108(m2.s)−1 and 5.3×109(K5) [R2 = 0.94] respectively. At a cooling rate of 100°C/min, twitching IIF (non-darkening IIF) was observed. Viability data from a separate study (Bischof et. al., 2000) indicated that at cooling rates ≤ 1°C/min and ≥ 50°C/min with an end-temperature of −20°C, extensive damage to cells was observed. The current biophysical study shows that extensive dehydration occurs at 1°C/min while substantial IIF (77%) occurs at 50 °C/min. This data suggests that while biophysics can explain some of the destruction occurring at the investigated temperatures, other effects or mechanisms may be playing a role at lower end-temperatures.

Extra- and intracellular ice formation in mouse oocytes

Cryobiology ◽  
2005 ◽  
Vol 51 (1) ◽  
pp. 29-53 ◽  
Author(s):  
Peter Mazur ◽  
Shinsuke Seki ◽  
Irina L. Pinn ◽  
F.W. Kleinhans ◽  
Keisuke Edashige
Keyword(s):  
Ice Formation ◽  
Mouse Oocytes ◽  
Intracellular Ice Formation ◽  
Intracellular Ice
Sign in / Sign up

Export Citation Format

Share Document