Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Cooling of embryonic cells,isolated blastoderms,and intact embryos of the zebra fish Brachydanio rerio to −196 °c

Single cells from the developing embryo of the zebra fish survive freezing when protected with 1 M DMSO and cooled to ?196 °C in two steps. Cell survival drops from 85 to 26% when clumps of 5–10 cells are similarly frozen, and to 2% when isolated blastoderms are treated in the same way. This drastic decrease in survival is interpreted as an example of the “scale-up problem,” in which diffusional barriers prevent cryoprotectant equilibration and osmotic dehydration in large cell assemblanges.Isolated blastoderms develop considerably in culture, and retain some of this ability following cooling to ?25 °C after protection with DMSO or glycerol.Intact embryos protected with high concentrations of glycerol (2.8 M) tolerate slow cooling to ?196 °C surprisingly well, with most of the embryonic cells morphologically intact and actively extruding lobopodia. Glycerol could, however, only be removed from cells by disrupting the embryo so that diffusional barriers were removed. DMSO (2.8 M) was ineffective in preserving embryos or cells cooled to ?196 °C.     

Effect of stage of development on survival of mouse embryos frozen-thawed rapidly

Embryos were recovered on Day 4 of pregnancy from superovulated random-bred OF₁ Swiss albino mice. They were classified into four categories based on their stage of development: expanding blastocyst, blastocyst, early blastocyst, and compacted morula. They were then cooled at 2 °C/min from ?7 to ?25 °C in a freezing medium containing 1.36 M glycerol and 0.25 M sucrose in phosphate-buffered saline (PBS). At ?25 °C, they were plunged into LN₂ and thawed a few hours later in water at 20 °C. After washing in PBS, recovered embryos were cultured for 20 to 24 hr and the number of embryos that had developed normally was recorded. The results showed a clear effect of the stage of development on survival. Survival of expanding blastocysts and blastocysts was very low (1.4 and 21.8%, respectively) compared to that of early blastocysts and compacted morulae (69.4 and 73.5%). The more differentiated stage of the blastocyst (two kinds of cells) and the presence of a blastocoelic cavity may explain the differences observed under our cooling conditions. As a further test of viability, 93 blastocysts that had developed in culture for 20 hr from 153 frozen-thawed early blastocysts and compacted morulae (60.8%) were transferred to 8 recipient mice. Seven became pregnant, yielding $\text{38}\text{82}$ normal live young (46.3%).     

Permeability of the 17-day fetal rat pancreas to glycerol and dimethylsulfoxide

Experimentally induced diabetes in rats can be reversed by the transplantation of several fresh or frozen-thawed fetal pancreases. An important question to both the mechanistic and practical aspects of cryobiology is the role played by the permeation of protective additives during freezing, thawing, and subsequent dilution. Answers require knowledge of the kinetics of permeation of the specific additive into the cell or tissue. In this paper, we report isotopic measurements of the rate of permeation of 2 M glycerol and 1 and 2 M dimethylsulfoxide (Me₂SO) into 17-day fetal pancreases at 0 and 22 °C. In Me₂SO, equilibrium was achieved in about 10–15 min at 0 °C and in less than 10 min at 22 °C. In glycerol, equilibrium was attained in about 60 min at 22 °C; but at 0 °C permeation was only 65% complete after 180 min. In general, Me₂SO permeated 10–30 times more rapidly than glycerol at 0 °C, and glycerol permeated about 10 times more rapidly at 22 than at 0 °C.The kinetics of permeation were more characteristic of a two-compartment than a single-compartment system. In all probability, the two compartments are the intercellular space and the intracellular space. The permeability data suggest that each compartment occupies about half the total volume.     

Preservation of mouse embryos by two-step freezing

Eight-cell mouse embryos in 1.5 M DMSO were preserved in LN₂ by a two-step procedure. Fifteen minutes exposure at ?20 °C protected the embryos against damage during rapid cooling to -196 °C and during rapid warming and rapid dilution. Since survival was poor on slow warming it is suggested that the method permits the formation of some intracellular ice.     

Ovary-autonomous maternal inheritance at a developmental locus in Drosophila

The sex-linked temperature-sensitive mutation shibire^ts of Drosophila melanogaster shows a maternal effect causing embryonic lethality at 29°C. The maternal influence is due to gene action autonomous to the ovary. Embryos carrying the paternally derived wild-type gene can survive at 29°C but only if heat pulses are begun at least 9 hr after oviposition. The paternal rescue is presumably due to zygotic gene action at this locus beginning part way through embryogenesis. A maternal wild-type genome, however, can produce shi embryos that have sufficient shi⁺ product to support embryogenesis up to the hatching stage even at 29°C.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

Lack of effect of 2.45-GHz microwave radiation on the development of preimplantation embryos of mice

The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm² and 19 mW/cm² on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 °C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.     

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of ?1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at ?1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia.Increments in free glycerol are accompanied by decreases in the insect's total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [¹⁴C] glucose indicate it is also metabolically active at low temperature.Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[³H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.     

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation

Frozen-thawed mouse oocytes were artificially activated with Sr²⁺ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (>90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozenthawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0°C or 37°C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0°C without DMSO or held at 37°C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos. © 1995 wiley-Liss, Inc.     

The effects of cortisol and amino acids on the metabolic activity of rat skin stored in dimethyl sulfoxide buffer at −196 °C

Addition of amino acids to the DMSO buffer reduces the intracellular amino acid depletion of rat skin tissue frozen and stored at ?196 °C.Although prolonged exposure to DMSO progressively inhibits the [2-¹⁴C]glycine and l-[U-¹⁴C]leucine incorporation into the proteins, cortisol and amino acid additions to the buffer medium protect the protein-synthesizing activity. These factors also stimulate the incorporation of [6-³H]-thymidine into DNA. The stimulatory characteristics of cortisol and of amino acids separately are enhanced when both components are added together to the preserving buffer. These effects are noticeable in tissue only exposed to the DMSO buffer without freezing as well as in skin frozen and stored at ?196 °C and subsequently thawed at 40 °C.A stimulatory effect of cortisol and of a free amino acid, supplement to the medium on the α-amino-[1-¹⁴C]isobutyric acid uptake by the cells is only observed in skin exposed for a short period of time to the DMSO buffer, but it is not detectable after longer exposure and after freezing.     

Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or −10°C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m³/N·s) is 7.1×10⁻¹³+216%-11% at 22°C, 8.2×10⁻¹³+235%−21% at 0°C, and 1.7×10⁻¹⁴+19% −16% at −10°C. The reflection coefficient is 1.0+2%−1% at 22°C and 0°C, and 0.9±5% at −10°C. Solute mobility (cm/s) is 5.9×10⁻⁶+6%–11% at 22°C, 3.1×10⁻⁶+12%−11% at 0°C, and 5.0×10⁻⁸ cm/s+59%−40% at −10°C.     

Cromakalim: embryonic effects and reduction of tolbutamide-induced dysmorphogenesis in vitro.

Cromakalim is a K(+) channel opener that causes smooth muscle relaxation by activating ATP-sensitive K(+) (K(ATP)) channels and producing membrane hyperpolarization. Cromakalim counteracts sulfonylurea-induced K(ATP) channel inhibition in adult cells, but little is known regarding its embryonic effects, alone or in combination with sulfonylureas. K(ATP) channels have been demonstrated in the embryo, but their role in normal and abnormal development is unknown. Early-somite mouse embryos were exposed for 24 hr in vitro to cromakalim at concentrations of 0 (Cntl), 1, 10, 100, 200, or 500 microM in 0.125% DMSO. Embryos were also exposed for 24 hr in vitro to a dysmorphogenic tolbutamide concentration (110 microg/ml) combined with a subdysmorphogenic concentration of cromakalim (1 microM). Embryos were evaluated for somite number, heart rate, malformations, and embryonic and yolk sac protein content. Embryos exposed to 1 microM cromakalim were similar to controls. Cromakalim exposure increased malformation rates at concentrations >/=200 microM, decreased heart rates at >/=10 microM, and decreased somite and protein values at 500 microM. Defects involved cranial neural tube, optic vesicle, heart, and somites. A malformation rate of 59% in embryos exposed to 110 microg/ml tolbutamide was reduced to 13% by adding 1 microM cromakalim to the culture medium. Heart rate, somite number, and protein values were also improved by combined exposure to cromakalim and tolbutamide compared with exposure to tolbutamide alone. These results support previous findings with diazoxide (K(+) channel opener) and chlorpropamide (sulfonylurea) and further suggest a potential role for K(ATP) channel effects in sulfonylurea-induced dysmorphogenesis.     

Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Effect of cryoprotective agents on rat cutaneous nerves.

Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Toxic effects of mercury on the hard clam,Meretrix lusoria,in various salinities

1. The 96-hr lc₅₀ values for juvenile hard clams, Meretrix lusoria, were 328, 392 and 194 μg/l Hg in 10, 20 and 30 ppt salinities at 25 ± 1°C, respectively; for adult hard clams 341 and 140 μg/l Hg in 20 and 30 ppt salinities, respectively.2. Acclimatizing the adult clams to low salinity of 10 ppt lessened the toxicity of mercury. However, juvenile animals appeared to be more sensitive to mercury poisoning after 96 hr exposure in 10 ppt salinity.3. All embryos exposed to 40 μg/l Hg and above died within 30 hr. In the control, 44% of hatched embryos had developed into D-stage larvae, while those exposed to 20 μg/l Hg were still in the trochophore stage. Most of the retarded larvae developed into abnormal forms within 30 hr at 28°C in 15 ppt salinity.4. In order to maintain water quality and protect natural resources, the recommended safe level of mercury is 0.046 (0.039–0.053) μg/l Hg, based on the estimated 30-hr EC₅₀ for the clam embryos, with an application factor of 0.01.