Stimulation of acetylcholine synthesis by blockade of presynaptic muscarinic inhibitory autoreceptors: Observations in rat and human brain preparations and comparison with the effect of choline

The central presynaptic muscarinic inhibitory autoreceptor has been monitored by measuring the effects of muscarinic agents on acetylcholine (ACh) synthesis by rat and human neocortical tissue prisms. Quinuclidinyl benzilate (QNB), the antimuscarinic which of 20 tested caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range of concentrations of 0.1 μM–10 μM. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB was to increase synthesis to only 112% of control (value without drug) which was much less than in rat (to 140% of control). ACh synthesis is reduced to 50% of control in neocortex from Alzheimer patients so none of the antimuscarinics tested seem to be potentially capable of appreciably reversing this deficit. A high concentration of choline (10 mM) stimulated synthesis in rat prisms to about the same extent as QNB. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). This suggests that an elevated intracellular concentration of choline is likely to be much more effective than an antimuscarinic agent in stimulating synthesis in Alzheimer brain.     

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.     

Functional studies after storage at - 190 degrees C of isolated and cultivated pituitary cells from pig and rat

For the first time, it is shown here that enzymatically dispersed pituitary cells of animals survive freezing and storage at -190 degrees C in liquid nitrogen. Frozen/thawed pituitary cells from both rat and pig are able to form monolayer aggregates in culture, and to produce hormones similar to that observed with unfrozen cells. The production of both basal and LHRH (luteinising hormone releasing-hormone)-induced bioassayable LH (luteinising hormone) were measured before and after cry-opreservation. Though after cryopreservation the number of cells was reduced by about 50%, a highly significant amount of both basal and LHRH stimulation-induced release of LH was measured in cultures from frozen/thawed pituitary cells from both species.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

Freeze-fixation at high subzero temperatures.

Previous attempts to determine the distribution of ice in frozen tissues at high sub-zero temperatures generally called for the further cooling of the tissues in question to facilitate freeze-drying, freeze-substitution, and freeze-fracture replication. Direct cryomicroscopic determinations, free from uncertainties stemming from changes in sample temperature could, it seemed, only be made in certain special cases. We have presented an isothermal “freeze-fixation” procedure designed to permit, instead, the postthaw retention of the freezing pattern and the conventional processing, afterward, of the thawed specimen. The method demands the exposure of the frozen tissues to fixative solutions incapable of dissolving ice. Frozen specimens are immersed in aqueous fixative solutions prepared in each instance (1) to freeze at a temperature equal to that at which fixation is to be conducted, (2) to contain quantities of finely divided ice sufficient to guarantee the maintenance of a constant water activity. Frozen frog and rat hearts and skeletal muscle tissues were exposed to formaldehyde, formaldehyde/ glutaraldehyde, and glutaraldehyde solutions at ?2, ?5, and ?10 °C, the temperatures being maintained in each case to ± 0.1 °C, or better. Tissues withdrawn at intervals were thawed, postfixed, dehydrated, embedded, and sectioned. The sections demonstrated the retention, after thawing, of structural features characteristic of the frozen state. The small hearts we exposed to formaldehyde were fixed throughout in 3 hr at ?2 ° and in 20 hr at ?5 °C. The action of osmium tetroxide was investigated. The method appears to be well-suited to numerous experimental applications.     

Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.     

The effect of oestradiol on the acid-soluble nucleotides of rat uterus

1. Techniques have been developed to measure the concentrations of the ribonucleotides of the immature rat uterus in vivo. Tissue was frozen rapidly in liquid nitrogen, ground to a fine powder, dispersed in frozen perchloric acid and thawed slowly. Nucleotides were separated from other acid-soluble constituents on short columns of polyethyleneimine-cellulose and the mixture was resolved into individual nucleotides by two-dimensional thin-layer chromatography on polyethyl-eneimine-cellulose plates. 2. The nucleotides of immature rat uterus consisted of approximately 75% of ATP-ADP, 10-12% each of GTP-GDP and UTP-UDP and less than 2% of CTP. 3. Injection of oestradiol (5mug) promoted a linear decrease in the amounts of purine nucleotides to approximately 60% of control values in 4-5h, followed by a return to greater than control values in 8-10h. Concentrations of the pyrimidine nucleotides remained constant for 4-6h and then increased to 200% of control at 12h after hormone treatment.     

Metabolically Active Synaptosomes Can Be Prepared from Frozen Rat and Human Brain

Abstract: Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue-derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1-5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter-related studies.     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins

DNA microarrays are powerful tools for high throughput analysis of gene expression; however, they do not measure protein expression. Current methods for producing protein arrays require sophisticated equipment or extensive protein modification. We developed a low overhead, customizable assay platform called frozen protein arrays that can detect native proteins in protein lysates. Frozen protein arrays were formed from a block of frozen histologic embedding compound containing an array of wells. The wells were filled with samples, which freeze and bond to the block. Cryosections were cut and transferred to nitrocellulose-coated slides. The reproducibility, linearity, and sensitivity was confirmed using frozen protein arrays filled with prostate specific antigen. Frozen protein arrays could detect native tissue proteins. The alpha1 subunit of NaK-ATPase was detected in rat kidneys with a coefficient of variation of 4.3-6.6%. Frozen protein array analysis indicated that the protein abundance decreased by 48.7% following renal ischemia, similar to the 40% decrease by Western blotting. We conclude that frozen protein arrays are a low cost, moderate size platform for arraying samples including protein lysates. Production of many identical frozen protein arrays is easy, inexpensive, and requires only small sample volumes. The method is gentle on proteins as they remain frozen during production.     

Cryopreservation of rat and human liver slices by rapid freezing

The cryopreservation of human liver slices is a promising way to enhance the ability to test the metabolism of drug candidates. This study demonstrates the use of a novel technique for the cryopreservation of both rat and human liver slices. In this technique the slices are treated with Me2SO and sandwiched between aluminum plates separated by a thin gasket. The device is then submerged in liquid nitrogen to freeze the slices, which can then be stored until use. To thaw the slices, the apparatus is submerged in a water bath at 37 degrees C. Slices frozen and thawed in this manner were compared to those frozen in conventional cryovials. The viability of the slices was determined by incubating them in 12-well plates and measuring urea synthesis, ethoxycoumarin metabolism, and cytosolic enzyme leakage (LDH and ALT). The viability of rat slices frozen between plates approached that of fresh slices and was consistently higher than slices frozen in cryovials. Slices from two human samples gave similar results. The technique was found to work over a wide range of Me2SO concentrations (4.5 to 22% was tested) with an optimal concentration between 10 and 15%.     

Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. Optimal conditions for fertilization including suitable medium and sperm:egg ratio were determined. Sperm was diluted in modified Kurokura's 'Extender 2'containing DMSO as cyroprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 2°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min⁻¹; −4°C–80°C: 11°C min⁻¹; from −80°C they were plunged directly into liquid nitrogen (− 196°C). Frozen samples were thawed in a water bath at 40°C. Fertilization rates achieved were much higher in water than in other solutions. Optimal ratios of frozen sperm:egg:water (1:20:20 in volume) and optimal number of frozen spermatozoa:egg (10⁵ spz: 1 egg) were determined. In such conditions, a strong positive correlation (c =+0.846) was found between the post-thaw motility and the fertility of frozen sperm securing high fertilization (99.6%, percent of control). No significant difference was found between fertilization and hatching rates achieved using frozen-thawed common carp sperm.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients.

Partial ovaries from mice, hamsters, rabbits, Japanese monkeys and rats have survived deep-freezing and returned to a normal morphological state after being thawed and transplanted into the rat uterine cavity. This report describes the ice-free cryopreservation of mouse and other ovaries at -196 degrees C by vitrification. The vitrification solution was based on the solutions reported by Rall & Fahy [16]. After ovaries had been exposed to the vitrification solution, they were frozen, with their suspending medium, by liquid nitrogen. After freezing, the ovaries were thawed in 37 degrees C water. The viability of the previously frozen ovarian tissue was tested by transplanting it into the uterine cavity of pseudopregnant rats. Seven days after transplantation, the ovaries were removed with the rat uterus, and stained with haematoxylin and eosin for histological examination. Survival of the frozen-thawed the ovaries in the rat uterine cavity demonstrates that these ovaries can tolerate exposure to osmotic dehydration and vitrification in a concentrated solution of cryoprotectant and are then immunologically acceptable to the uterine cavity.     

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.     

Freezing-altered palatability of Bradford pear to Japanese beetle: evidence for decompartmentalization and enzymatic degradation of feeding deterrents

The basis for resistance of Bradford callery pear, Pyrus calleryana Decaisne Bradford, [Rosaceae] to the Japanese beetle, Popillia japonica Newman, was investigated. Chloroform-dipping rendered leaves palatable, initially suggesting that deterrent waxes had been removed. However, extracts containing surface waxes were not deterrent. Subsequent experiments showed that increased palatability of solvent-dipped leaves is associated with enzymatic tissue browning, characteristic of polyphenol oxidases, rather than simply release of phagostimulants from surface disruption of damaged leaves. Frozen and thawed leaves showed similar browning, becoming increasingly palatable for several hours after thawing. Palatability changes were temperature- and aerobic-sensitive, further evidence that oxidizing enzymes are involved. Juice from leaves that had been frozen and thawed stimulated feeding on glass fiber disks, whereas fresh leaf juice did not. Survival and fecundity were much higher for beetles fed frozen and thawed or chloroform-dipped Bradford pear leaves than for beetles fed normal leaves. We hypothesize that decompartmentalization of deterrent compounds, possibly phenolics, followed by enzymatic oxidation and altered leaf chemistry may explain the increased palatability of chloroform-dipped or frozen and thawed Bradford pear tissue to P. japonica. This approach may be helpful in identifying specific compounds responsible for resistance of woody plants to generalist insects.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Myelin basic protein in frozen and unfrozen bovine brain: a study of autolytic changes in situ.

Abstract— Frozen and unfrozen bovine brains were used to determine the extent of in situ degradation of myelin basic protein. The following three parameters were investigated. (1) The time interval between death and sampling of the tissue, (2) the effective temperature of the tissue during this interval, and (3) the effect of freezing and thawing on the subsequent autolysis of myelin basic protein. Polyacrylamide gel electrophoresis was carried out on unfrozen white matter solubilized with phenol-formic acid–water. The resulting electrophoretic pattern showed no qualitative changes in the myelin basic protein after tissue incubation at 4° or 23°C for up to 24 h. When myelin basic protein was extracted, purified and quantitated, there was no apparent decrease within 24 h of incubation at 23°C. However, if the tissue was frozen and thawed prior to incubation, there was a rapid disappearance of myelin basic protein such that only 10% remained after 24 h of incubation. Basic protein extracted from frozen or unfrozen tissue that had undergone autolysis for up to 24 h was found to be encephalitogenic in guinea pigs. Electron microscopy of frozen and thawed material showed separation and fraying of myelin lamellae. It is postulated that the above morphological changes probably render the basic protein readily accessible to proteolytic enzymes.