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PubMed1988

Biophysical properties of the zona pellucida measured by capillary suction: is zona hardening a mechanical phenomenon?

Following fertilization, the zona pellucida, a glycoprotein shell investing mammalian eggs, becomes more resistant to dissolution by various agents. This decreased solubility, termed zona hardening, does not occur in hamsters. Thus, universal roles of zona hardening in the block to polyspermy and in oviductal interaction with embryos have been discounted. Although these roles probably have mechanical components, zona hardening has been assessed only as decreased solubility. In our studies, mouse and hamster ovulated oocytes were compared with 2-cell embryos by capillary suction. Step changes in pipet pressure were measured manometrically while resulting zona deformations were determined videomicrographically. Both mouse and hamster zonae were more "deformable" in oocytes than embryos. These results suggest that mechanical zona hardening may be a universal phenomenon. In addition to zona hardening following fertilization, a "spontaneous" zona hardening phenomenon has been reported for mouse oocytes (not embryos) cultured in vitro. In our system, this spontaneous decrease in zona solubility by protease was not accompanied by a mechanical change. In contrast, hamster oocyte and embryo zonae became slightly more soluble, and considerably more deformable during 4-h culture. Thus, spontaneous zona hardening is not a universal phenomenon, and, while decreased solubility has been called "hardening", it is not always accompanied by a mechanical change.

Animals↗
PubMed1984

Effects of prolonged antitubulin culture on the ultrastructure of anterior limb bud cells of the chick embryo.

The anterior limb bud mesenchyme cells of stage 24 chick embryos were dissociated by trypsinization followed by gentle pipetting, and placed in a tissue culture medium of F12 containing 10% fetal calf serum and antibiotics. As the cells became nearly confluent, some of them were exposed to colchicine or vinblastine sulfate for durations as long as 48 hr. The control and antitubulin-treated cells were processed for transmission electron microscopy and the ultrastructure of the cells was compared. Annulate lamellae (AL) were observed in small amounts in both control and antitubulin-treated cells. The amount of AL did not markedly differ in the control versus antitubulin-treated cells. Furthermore, few multinucleated cells were observed in antitubulin-treated cultures. These results indicate that prolonged culture of cells in antitubulins need not, in itself, lead to a condition of enhanced AL development as reported in several other studies using various cell types.

Animals↗
PubMed1978

Separation and spectrophotometric determination of theophylline and hydroxyethyltheophylline in a pharmaceutical syrup.

A simple chromatographic procedure was developed for the separation and determination of theophylline and hydroxyethyltheophylline. An aliquot of diluted syrup was pipetted into a chromatographic column containing a strong anion-exchange resin, which retains theophylline and allows the hydroxyethyl derivative to pass through it. Theophylline was subsequently eluted with 1 N HCl and determined by difference spectrophotometry at 284 nm; its derivative was determined at 272 nm.

Solutions↗
PubMed1986

Intranasal administration of peptides: nasal deposition, biological response, and absorption of desmopressin.

The nasal administration of desmopressin [1-(3-mercaptopropionic acid)-8-D-arginine-vasopressin] in humans was investigated. Desmopressin solutions containing 99mTc-labeled human serum albumin were administered intranasally as a spray, using two metered-dose pumps, or as drops, using a rhinyle catheter or a single-dose pipet. Images of the sites of deposition and rates of clearance were monitored quantitatively by gamma scintigraphy. Plasma levels of desmopressin were measured using a highly sensitive and specific radioimmunoassay. The biological response was determined by measuring circulating levels of F VIII, the antihemophilia factor. The sprays were deposited mainly anteriorly, from which small portions were cleared slowly into the nasal pharynx. In contrast, the drops were deposited mostly posteriorly and cleared very rapidly in large portions; some were swallowed immediately. Plasma levels showed that desmopressin was absorbed to a greater extent after administration of the spray with a 2 to 3-fold increase in the relative bioavailability compared with the drops. The biological response was clearly enhanced after spray administration and produced similar increases in F VIII activity. A linear correlation was observed between maximum plasma desmopressin levels and maximum F VIII activity. The use of an intranasal spray device can deposit well-controlled doses within the nasal cavity, which remain there sufficiently long to provide a clear enhancement in absorption and bioavailability.

Absorption↗
PubMed1990

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits.

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

Animals↗
PubMed1993

Biotinylation of proteins on the surface of zona-free mouse oocytes.

In this study, we have labelled proteins on the surface of unfertilized, zona-free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow-bore glass pipette. Surface proteins were labelled using sulfo-NHS-biotin (sulfosuccinimidobiotin), a water-soluble, membrane-impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin-FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using streptavidin-HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of M(r) 82 and 69 kD, but other major proteins of M(r) 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of M(r) 18-100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos.

Animals↗
PubMed2002

Postsynaptic kinase signaling underlies inhibitory synaptic plasticity in the lateral superior olive.

UNLABELLED: In the auditory system, inhibitory transmission from the medial nucleus of the trapezoid body (MNTB) to neurons of the lateral superior olivary nucleus (LSO) undergoes activity-dependent long-term depression, and may be associated with developmental elimination of these synapses [Sanes DH, Friauf E (2000). REVIEW: development and influence of inhibition in the laterial superior olivary nucleus. Hear Res 147:46-58]. Although GABA(B) receptor activation and postsynaptic free calcium are implicated in this depression, little is known about intracellular signaling mechanisms in this or other forms of inhibitory plasticity. In this study, we asked whether the calcium dependency of inhibitory depression was associated with the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and/or cAMP-dependent protein kinase A (PKA). Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, permitting for the selective pharmacologic manipulation of individual postsynaptic LSO neurons. Inclusion of a CaMKII antagonist (KN-62) in the internal pipet solution blocked inhibitory synaptic depression. A second CaMKII inhibitor (autocamtide peptide fragment) significantly decreased inhibitory depression. Inclusion of a specific antagonist of protein kinase C (PKC fragment 19-36) in the internal recording solution also blocked inhibitory depression. To test involvement of a cAMP-dependent intracellular cascade, two different manipulations were performed. Inclusion of PKA antagonists (Rp-cAMPS or a cAMP dependent protein kinase inhibitor peptide) prevented inhibitory depression. In contrast, when a nonhydrolyzable cAMP analog (Sp-cAMPS) was permitted to enter the postsynaptic cell, the MNTB-evoked IPSCs became depressed in the absence of low-frequency stimulation. Thus, three key postsynaptic kinases, CaMKII, PKC, and PKA, participate in the activity-dependent depression of inhibitory MNTB-LSO synapses during postnatal development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases.

We have developed an assay procedure to monitor the cleavage of DNA substrates by restriction endonucleases. This procedure uses DNA substrates that are labeled with biotin on one 5' end and with an antigenic group, e.g., fluorescein or digoxigenin, on the other 5' end. After incubation with the restriction enzyme, the reaction is stopped with EDTA and an aliquot is pipetted into the well of an avidin-coated microtiter plate. This immobilizes the unreacted substrate and the biotinylated cleavage product, whereas the other cleavage product labeled with the antigenic group is subsequently washed off. The unreacted substrate is detected by an enzyme-linked immunosorbent assay with an appropriate enzyme-linked antibody. To test our assay we have measured the steady-state rate constants for cleavage of DNA by EcoRI yielding a kcat of 8.6 min-1 and a Km of 150 nM, which are close to values measured with other assays. The advantage of this assay is that it is not only fast and accurate, but also very sensitive. It allows for many samples to be analyzed in parallel and lends itself to automation. Furthermore, this assay can be designed as a competitive assay, when two substrates carrying different antigenic groups are used. The usefulness of such competitive assay is demonstrated by determining the influence of sequence context on the rate of DNA cleavage by EcoRI.

Base Sequence↗
PubMed1996

Upside-down stopped-flow electrofractionation of complex protein mixtures.

The excellent resolution of SDS-PAGE in protein analysis stimulated the creation of various preparative devices. The main approach used in these devices is the construction of a elution chamber in the lower end of the polyacrylamide gel cylinder or plate. Although this continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis, some limitations to this approach exist. There is strong dilution of protein zones by the eluting buffer, which drastically restricts the sensitivity of the determination of minor proteins, and the restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. To overcome these problems, the upside-down stopped-flow electrofractionation system (UDSFE) was designed. The necessary quantity of fraction is drawn with a pipet in a small volume from just above the gel cylinder. This invention improves the possibility of electrofractionation of deluted complex protein mixtures. The efficiency of this technique is demonstrated by purification a protein kinase from rat liver. The method has also been successfully used for purification of error-correcting 3'-5' exonuclease.

Animals↗
PubMed1999

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence.

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.

Amino Acid Sequence↗
PubMed2001

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.

Calcium↗
PubMed2001

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.

Antibodies↗
PubMed2000

Electrophysiologic characteristics of the Ca-permeable channels, ECaC and CaT, in the kidney.

To investigate the molecular mechanism of Ca transport in the kidney, we have isolated Ca-permeable channels, rECaC (rat ECaC) and mCaT (mouse CaT1), from rodent kidney, which are recently reported as Ca-transporting proteins. RT-PCR suggested the presence of CaT1 in medullary tubules. It showed 67% homology with rECaC constructing a family. Whole cellular currents in Chinese hamster ovary (CHO) cells were measured by patch clamp. Expression of both proteins exhibited a similar large cation current, a high permeability to Ca, a time-dependent rapid inactivation, and a "run-down." When the pipet contained EGTA, the inactivation and the run-down did not occur. Addition of db-cAMP activated and following rp-cAMPS recovered the mCaT-induced current significantly, whereas no influence was observed in the rECaC-induced one. We conclude that ECaC and CaT are a molecular family of ion channel with similar characteristics, contributing Ca transport in the kidney.

Animals↗
PubMed1995

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.

Animals↗
PubMed1998

Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants.

A total of 498 porcine embryos at various stages of development collected from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blastocysts (HB) were used to determine the effect of exposure to concentrated solutions of ethylene glycol as cryoprotective additives (CPAs) on embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethylene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solution of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. To vitrify the embryos, they were exposed to 2.0 M EG for 5 min and then were pipetted directly into short columns of 8.0 M EG-PVP contained within (1.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, the embryos were immediately mixed with galactose within the straws by shaking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method 1: Embryos in the EG-PVP-galactose mixture were expelled from the straws and rinsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cultured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. After 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method II, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB vitrified embryos diluted by Method III and transferred into a recipient, four developed into live piglets; two other recipients failed to litter although one had been pregnant for 65 days. These results demonstrate that porcine embryos can be successfully cryopreserved by rapid cooling in EG-PVP and by careful dilution of the CPA after warming.

Animals↗
PubMed1998

Vitrification of mature mouse oocytes in a 6 M Me2SO solution supplemented with antifreeze glycoproteins: the effect of temperature.

Oocytes have been successfully cryopreserved using rapid and slow freezing procedures. However, variability in the success of replicates has limited its practical application. In the present study, mature mouse oocytes were vitrified in 6 M dimethyl sulfoxide supplemented with 1 mg/ml antifreeze glycoproteins (AFGP) (solution known as VSD + AFGP) from the blood of Antarctic notothenioid fish. Such AFGPs have been used to protect mammalian cells during hypothermia and cryopreservation. However, the degree of protection afforded is a contentious issue. Stepwise addition of cryoprotectant was performed either at room temperature (19-21 degreesC) or on ice (2-4 degreesC), at the final stage of which oocytes were pipetted into 0.25 ml plastic insemination straws and held in liquid nitrogen vapor at -140 degreesC for 3 min before being plunged into liquid nitrogen. Thawing involved holding the straw in the air for 10 s and then in water at 20 degreesC for 10 s before dilution of the VSD solution with 1 M sucrose. Viability was assessed by in vitro fertilization; results have been quoted as median (range). Statistical analyses were performed using Kruskall-Wallis and Mann-Whitney U tests (P < 0.05). Of the oocytes cryopreserved following exposure to VSD + AFGP at room temperature (n = 518, 15 experimental runs), 78% (0-94%) retained normal morphology and, of these, 53% (0-100%) cleaved to two cells. Of these two-cell embryos, 56% (0-100%) went on to develop to blastocyst. The overall percentage development to blastocyst, i.e., number of blastocysts/total number of oocytes treated x 100, was 20% (0-76%). Exposure of oocytes to the VSD + AFGP on ice prior to cryopreservation yielded significantly improved rates of fertilization (94%, 82-100%) and overall development to blastocyst (66%, 24-89%) when compared with oocytes cryopreserved following exposure to the VSD + AFGP at room temperature. Rates of normality (86%, 35-95%) and development to blastocyst (89%, 64-100%) were also improved. Cryopreservation in 6 M dimethyl sulfoxide supplemented with 1 mg/ml AFGP resulted in poor rates of survival which were highly variable when exposure to cryoprotective agent (CPA) was performed at room temperature. Lowering the temperature of exposure to CPA prior to cryopreservation resulted in improved viability.

Animals↗
PubMed2000

Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose.

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

Animals↗
PubMed2001

The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts.

A total of 678 bovine blastocysts, which had been produced by in vitro maturation, fertilization, and culture, were placed into plastic straws and were vitrified in various solutions of ethylene glycol (EG) + polyvinylpyrrolidone (PVP). Part of the straw was loaded with TCM199 medium + 0.3 M trehalose as a diluent; the diluent portions of the straw were prefrozen to either -30 or -196 degrees C. Then, the embryos suspended in the vitrification solution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degrees C and then agitated to mix the diluent and cryoprotectant solution for 5 min followed by culture in TCM199 + 10% FCS + 5 + microg/ml insulin + 50 microg/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitrification, the PVP concentration, and the temperature of exposure to EG + PVP prior to vitrification. Survival and hatching rates of the blastocysts exposed to 40% EG in four steps at 4 degrees C were higher than those of embryos exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% and 71.5 +/- 4.7%, respectively; P < 0.05). The same indices were superior following vitrification-thawing of the blastocysts in 40% EG + 20% PVP than it was in 40% EG + 10% PVP (76.1 +/- 5.5% vs 63.7 +/- 1.8%; P < 0.05; and 61.6 +/- 6.0% vs 70.5 +/- 4.7%; P < 0.01, respectively). Exposure to the vitrification solution (40% EG + 20% PVP) at higher temperatures (37.5 degrees C vs 4 degrees C) reduced both survival and hatching rates (45.8 +/- 6.9% vs 83.9 +/- 4.4% and 41.5 +/- 1.8% vs 64.0 +/- 4.7%, respectively; P < 0.001). These results indicate that blastocysts vitrified after prefreezing the diluent portions of the straws do favor developmental competence of in vitro produced embryos.

Animals↗