Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.     

Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions

For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 degrees C and three different media: Dulbecco's phosphate buffered saline (DPBS), TCM199 and Emcare, were tested for two different embryo ages: D4 embryos (collected 144 h after hCG treatment) and D5 embryos (collected 168 h after hCG). After slaughter of the donor gilts, embryos were collected and transported at 25 degrees C to the lab where morulas and blastocyst were selected (D4 n = 222; D5 n = 167) and randomly used as controls or distributed over the treatment groups. Developmental stage and embryo diameter were assessed by normal light microscopy, while total number of cells and incidence of apoptosis were assessed using a fluorescent embryo quality staining technique that combines three different dyes: Ethidium Homodimer (EthD-1), TUNEL and Hoechst 33342. Following 24 h storage, D5 embryos had higher rates of hatching (24%) and degeneration (9%) compared to D4 embryos (10 and 4%, respectively; P < 0.05). Embryos stored at 38 degrees C had higher rates of hatching (37%) compared to those ones stored at 25 degrees C (13%) or 18 degrees C (0%; P < 0.01). More embryos hatched when stored in medium Dulbecco's phosphate buffered saline (DPBS) or in TCM199 compared to those stored in Emcare (P < 0.05). A higher percentage of embryos stored at 18 degrees C degenerated compared to those stored at 25 or 38 degrees C (P < 0.01). No significant increase in apoptosis was observed after storage compared to the rates of apoptosis at 0 h (controls) or between the different storage groups. Based on the results we conclude that D4 porcine embryos produced in vivo, selected under normal light microscopy and stored at 25 degrees C in a serum free medium for 24 h will have a suitable developmental stage for ET and a high embryo quality.     

Two step freezing of cow embryos in 1.4 M glycerol

Day 6 1 2 -7 1 2 cow embryos were frozen in 1.4 M glycerol in PBS, at 0.3 degrees C/min to -30 (group I), -35 (group II), and -40 degrees C (group III) before being plunged into liquid nitrogen. They were subsequently thawed by direct transfer to water at 37 degrees C. In Experiment I, embryos frozen and thawed were cultured in vitro, 12 out of 19 embryos (63%) survived and there were no significant (p > 0.05) differences in survival rates among the three freezing groups. In Experiment II, 29 embryos frozen to -30 or -35 degrees C were transferred non-surgically to heifers on day 7. Seventeen of 29 recipients (59%) were pregnant at day 60. Five embryos frozen to -35 degrees C resulted in 5 pregnancies (100%) after thawing and surgical transfer.     

On the stability of key enzymes of energy metabolism in muscle biopsies

Enzymes of energy metabolism were tested for stability depending on different storage conditions (-20, -80 degrees C). To avoid problems due to the different fiber type composition of human muscle, we selected two muscles from rabbit. The m. psoas consists almost exclusively of type 2B fibers, and the m. soleus consists almost exclusively of type 1 fibers. Enzyme activities were measured from small aliquots of these muscles at various time points up to 1 year after sacrificing the animal. Enzymes from anaerobic metabolism were stable for more than 1 year, independent of whether the muscle was stored at -20 or -80 degrees C. Oxidative enzymes, such as succinate dehydrogenase, citrate synthetase, or cytochrome c oxidase (COX) decrease in activity at -20 degrees C and, to a lesser degree, at -80 degrees C. In addition, mitochondria were isolated from freshly taken muscle and stored at -80 degrees C. Oxidative enzymes were surprisingly stable for more than 1 year, with the exception of COX which decreased by 60% of its original activity in mitochondria from m. soleus.     

Thermal difference circular dichroism of Pf1 filamentous virus and effects of mercury(II), silver(I), and copper(II)

The circular dichroism (CD) of Pfl filamentous virus has been examined over the temperature range 0-40 degrees C, in the absence and presence of Hg(II), Ag(I), and Cu(II). Thermal difference CD spectra were obtained by subtraction of spectra recorded above and below a thermally induced structure transition near 12 degrees C. The thermal difference spectra look like they arise from shifts in two exciton bands, one centered at 230 nm and the other at 290 nm. The amplitudes on either side of a crossover at 230 nm are 10 times those of a crossover at 290 nm. It is proposed that the difference spectra result from thermally induced shifts in coupled oscillator interactions between Tyr40 residues of the coat protein and the guanine and cytosine bases of the DNA. Metal ions can reduce or block these shifts. The changes in ellipticities at 220, 237, and 270 nm induced by changing the temperature have inflections near 12 degrees C. Ag(I) and Hg(II), which are known to bind to the DNA bases in Pfl, reduce or eliminate the inflections in the thermal profiles, depending on the metal ion type and concentration. Cu(II) ions do not affect the profiles. The spectral changes and the effects of the metal ions indicate intimate contact between the DNA bases and the protein subunits in the virion.     

Production of the mutant E strain of Rickettsia prowazekii by mutagen exposure

The possibility of obtaining the mutants of R. prowazekii, strain E, by exposing these organisms to the action of N-methyl-N-nitro-N-nitrosoguanidine was studied; this substance, used in doses of 5-10 micrograms, showed a mutagenic effect on rickettsiae suspended in physiological saline, after their exposure for 10-20 minutes at 37 degrees C. The mutants thus obtained proved to be resistant to erythromycin and rifampicin and were characterized by heterogeneity in the degree and stability of their antibiotic resistance. The effectiveness of selection was increased if mutagen-treated rickettsiae were selected after the first passage in chick embryos. The induced mutants differed from the original rickettsial strain by their lower infectiosity for chick embryos.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimal equilibration conditions for practical vitrification of two-cell mouse embryos

The objective of the study reported here was to elucidate the optimal equilibration conditions for carrying out vitrification of two-cell mouse embryos, using a solution containing 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol (DAP213) as a cryoprotectant. Embryos were subjected to an equilibration process under 20 conditions of a combination of different temperatures (10 to 37 degrees C) and times (5 to 90 sec), and viability of the embryos was assessed by the rate of development into blastocysts and into live fetuses. As a result, these rates of development into blastocysts did not differ from those for unfrozen embryos. The rate of development of frozen-thawed embryos into live fetuses under conditions of 30 sec. at 20 degrees C, which was selected as having by highest operability, was 55.2%, comparable to the value (65.0%) for unfrozen embryos. Thus, the optimal equilibration condition for vitrification of two-cell mouse embryos, using DAP213 solution, was 30 sec at 20 degrees C, under which embryo viability was maximized, and this equilibration process was considered useful as a practical two-cell embryo freezing process in the vitrification method.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.     

Histone synthesis by cleavage arrested sea urchin eggs.

A change in Hl histone synthesis occurs in blastulae, from Hlm to a faster moving Hlg in acrylamide gel electrophoresis. The experiments below indicate that this shift occurs in the absence of normal cleavage. Hybrid embryos containing paternal Hl histone markers along with homospermic embryos were studied. Both were labeled with L-[3H]lysine. Some cultures were kept at 11 degrees C to inhibit cleavage. It was found that Hlm and Hlg are synthesized sequentially in time by embryos grown at 20 degreet C as well as by those grown at 11 degrees C. The hybrid data establish that Hlm is translated at least in part from mRNA newly transcribed from paternal DNA. This observation also holds for cleavage inhibited hybrid embryos. Hlg is made by both hybrid and homospermic embryos during the later phases of development at both 11 and 20 degrees C. These results confirm and extend those of Seale et al. (1973), Ruderman et al. (1974) and Easton et al. (1974).     

Alterations in ultrastructural morphology of two-cell bovine embryos produced in vitro and in vivo following a physiologically relevant heat shock

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at approximately 28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5 degrees C (cow body temperature), 41.0 degrees C (characteristic temperature for heat-stressed cows), or 43.0 degrees C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0 degrees C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5 degrees C or 41.0 degrees C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Sensitivity to chilling of medaka (Oryzias latipes) embryos at various developmental stages

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.     

Identification of low-temperature-regulated genes in the fire blight pathogen Erwinia amylovora

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 degrees C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for beta-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 degrees C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 degrees C, and two mutants with impaired growth at 18 degrees C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.     

Survival of 16-celled and morula stage rabbit embryos frozen to -196 degrees C,.

Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to -196 degrees C. The cooling rate (from a room temperature to 0 degrees C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developed in vitro, when they were frozen to -196 degrees C, applying the ice-seeding at -4 degrees C in the presence of 12.5% DMSO, after being cooled to 0 degrees C at the rate of 7-9 degrees C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate of in vitro development (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to -196 degrees C applying seeding at -4 degrees C after being cooled to 0 degrees C at the rate of 1 degrees C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and 1% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12th day of pregnancy.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.