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PubMed1992

Effects of cryopreservation on survival and development of interphase- and mitotic-stage 1-cell mouse embryos.

The effects of cryopreservation with 1,2-propanediol on two groups of 1-cell mouse embryos were studied in terms of survival after thawing, growth in vitro until the blastocyst stage and development in vivo assessed by the number of implantations and living fetuses. The two groups were divided according to different stages in the cell cycle: cells in (i) interphase with two distinct pronuclei or (ii) mitosis just prior to the first cleavage division. Zygotes in the interphase stage proved to be more resistant to freezing and thawing procedures, showing a significantly higher survival rate after thawing than zygotes in mitosis (78.5 versus 61.3%, P < 0.05). Blastocyst formation was similar in the two experimental groups: 72.7% for interphase and 60.8% for mitosis (P = 0.06), but for both groups fewer blastocysts formed when compared with the control group (86.7%) (P < or = 0.01). The implantation rates were not statistically different: 54.2% for the interphase cells and 47.4% for the control group and 44.0% for the mitotic cells and 49.4% for the control group. The formation of living fetuses was similar between the experimental and control groups: 36.5% for the interphase group (40.0% for its control group) and 22.6% for the mitotic group (38.8% for its control group). We conclude that freezing embryos during nuclear division is detrimental for their survival after thawing.

Animals↗
PubMed1992

The age of pronucleate mouse ova influences their development in vitro and survival after freezing.

The influence of pronuclear age on embryo development in vitro and on survival and post-thaw development after cryopreservation was investigated. Pronucleate mouse ova were removed from the oviductal environment at three different times: 18 h after human chorionic gonadotrophin (HCG) (just after fertilization), 23 h after HCG (during DNA replication) and 28 h after HCG (just before cleavage). Control ova were either cultured in vitro or transferred in vivo without any treatment or exposed to dimethyl sulphoxide (DMSO) or 1,2-propanediol (PROH) before culture, but without cryopreservation. The effect on survival was evaluated by the development in vitro and in vivo after ultrarapid freezing in 3.5 M DMSO and after slow programmed freezing in 1.5 M PROH. In vitro, the development of control ova to blastocysts was 39, 54 and 77% per treated ovum at 18, 23 and 28 h after HCG, respectively. The survival rate was 78, 92 and 96% after ultrarapid freezing and 69, 90 and 81% after slow freezing, respectively. The subsequent rate of development to blastocysts per survived ovum was 21%, 35% and 62% after ultrarapid freezing and 38, 54 and 63% after slow freezing. In vivo, however, the pronuclear age did not influence the implantation rate or the number of living fetuses per transferred ovum, for neither the control, nor the frozen/thawed ova. The implantation rate and the numbers of living fetuses were not significantly different for frozen/thawed and control ova. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than pronucleate mouse ova just before cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The use of cryopreserved aged human oocytes in a test of the fertilizing capacity of human spermatozoa.

The use of cryopreserved aged human oocytes in a diagnostic test of sperm fertilizing ability was evaluated. Oocytes arising from assisted conception cycles and showing no signs of fertilization 48 h post-insemination were cryopreserved by one of two methods. An ultrarapid method using dimethyl sulphoxide gave poor post-thaw results, with only 5/69 (7.2%) oocytes surviving. Oocytes frozen by a slow method using propanediol as the cryoprotectant gave better survival rates (359/594; 60%). Fertilization by donor spermatozoa of these thawed oocytes was poor (15/63; 24%) when the zona pellucida was left intact. To improve this, the zona was enzymatically removed using pronase. These zona-free oocytes were then inseminated with spermatozoa from a fertile donor or from men previously exhibiting fertilization failure in an in-vitro fertilization treatment cycle. The fertilization rate in the patient group (41/91; 45%) was significantly lower than in the donor group (16/18; 89%) (P less than 0.02). There was also a significant (P less than 0.03) reduction in the median number of pronuclei per oocyte (2.9 versus 4.5). These results show that aged oocytes can be effectively cryopreserved to establish a bank for use in a test to identify men with impaired sperm fertilizing capacity.

Cell Survival↗
PubMed1993

Cumulative pregnancy rate from one gamete intra-fallopian transfer (GIFT) cycle with cryopreservation of embryos: a practical mathematical calculation.

We evaluated the cumulative pregnancy rate from one gamete intra-Fallopian transfer (GIFT) cycle plus subsequent cycles in which embryos cryopreserved at the time of the original GIFT cycle were transferred. All patients who had their first GIFT cycle in our centre between January, 1989 and March, 1991 were included. Ovarian stimulation was accomplished with leuprolide acetate (luteal phase protocol) and a combination of follicle stimulating hormone and human menopausal gonadotrophin. GIFT was performed with three to five oocytes. Excess oocytes were inseminated and good quality embryos were cryopreserved at the 2- to 4-cell stage with 1-2 propanediol. When the GIFT cycle did not result in a pregnancy, uterine transfer of cryopreserved embryos was carried out in subsequent unstimulated cycles. A total of 97 patients had GIFT and 46 pregnancies were achieved (47.4%). A total of 51 patients (52.5%) had embryos frozen; of these, 21 were from the non-pregnant group (41.1%) and 30 from the pregnant group (65.2%) (P < 0.05). Up to now, 22 of them have undergone a frozen-thawed embryo transfer cycle, and two of them achieved a pregnancy. Based on these data, patients having a GIFT could theoretically expect a cumulative pregnancy rate of 52.2%.

Cryopreservation↗
PubMed1993

Assessment of ultrarapid and slow freezing procedures for 1-cell and 4-cell mouse embryos.

Three cryopreservation procedures were assessed for the freezing of mouse 1-cell and 4-cell embryos: the slow freezing protocols with dimethylsulphoxide (DMSO, A) and propanediol (PROH, B) and the ultrarapid procedure with DMSO (C), which was described by the Monash University group [A. Trounson et al. (1987) Fertil. Steril., 48, 843-850]. The evaluation of the different procedures included survival after freezing and thawing, further development after in-vitro culture to blastocysts and the ability to implant and to form living fetuses after transfer of early blastocysts to pseudopregnant mice. In-vitro development was lower in all frozen embryos than in the unfrozen controls. Procedures A and B induced comparable results and were significantly better than ultrarapid freezing. When unfrozen blastocysts were transferred to pseudopregnant mice, 64% of them implanted in the uterine wall and 59% developed to living fetuses. For zygotes the percentages of implantation sites and living fetuses were 47 and 33% for A, 52 and 44% for B, and 29 and 17% for C, respectively. When 4-cell embryos were cryopreserved, these results were 54 and 46% for A, 60 and 53% for B and 37 and 23% for C, respectively. In the ultrarapid procedure we also looked at the influence of the freezing solution. To set up a control, mouse zygotes and 4-cell embryos were exposed to DMSO as in the ultrarapid procedure except that they were not frozen; the survival and blastocyst formation was not different from controls, but fewer living fetuses were born (31% for zygotes and 41% for 4-cell embryos versus 67% in the controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The in-vitro and in-vivo developmental potential of frozen and non-frozen biopsied 8-cell mouse embryos.

We investigated the in-vitro and in-vivo development of cryopreserved and non-cryopreserved biopsied 8-cell mouse embryos, from which one to seven blastomeres were removed by micromanipulation. The results clearly indicate that the in-vitro and in-vivo development of biopsied 8-cell mouse embryos depended on the number of blastomeres removed: the more blastomeres removed, the greater the effect. When five, six, or seven blastomeres were removed, fewer blastocysts were formed. Furthermore, when these blastocysts were transferred to pseudopregnant females, no living young were formed, indicating the abnormality of these blastocysts. When up to three blastomeres were removed, there was no significant effect on the rate of in-vitro blastocyst formation. Living young were found even after the biopsy of four blastomeres, and after biopsy of only one or two blastomeres, the same percentage of living young was obtained as in the non-biopsied control embryos. Biopsied 8-cell mouse embryos were frozen and thawed in straws with 1,2-propanediol (1.5 M) and sucrose (0.1 M) with slow-freezing and rapid-thawing protocols. The survival after cryopreservation, defined as the percentage of embryos with the same number of blastomeres intact as the number of blastomeres before freezing, was excellent and no different from non-biopsied embryos, independent of the number of blastomeres biopsied. Furthermore, cryopreservation had no further impact on the in-vitro and in-vivo development of the biopsied embryos.

Animals↗
PubMed1994

Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes.

Survival following cryopreservation of fresh and aged human oocytes by the propanediol (PROH) procedure was observed in 51 and 73% of oocytes respectively, immediately after thawing. This survival was reduced in both types of oocytes at the time of insemination (3-4 h) to 41% in fresh and 61% in aged oocytes. Insemination of the cryopreserved and control oocytes with spermatozoa from one donor resulted in total fertilization rates similar to our in-vitro fertilization (IVF) rate for non-male factor patients. The normal fertilization rate for fresh cryopreserved oocytes was slightly lower (46%) than the rate for IVF oocytes (59%) (P < 0.05), while the abnormal fertilization rates were not significantly different (16 and 15% respectively). In contrast, a reduction in the normal fertilization rate was observed for the aged cryopreserved oocytes (13%) compared to the IVF rate (P < 0.001). Associated with this was an increase in the abnormal fertilization rate for the aged cryopreserved oocytes, which was significantly higher (47%) than the IVF rate (15%) (P < 0.001). Significant differences in the total and normal fertilization rates were observed between cryopreserved oocytes obtained from cohorts with < or = 27 (total: 84%, normal: 68%) and > 27 oocytes (total: 55%, normal: 33%) (P < 0.05). Fertilized oocytes and oocytes with abnormal or absent spindles were examined for chromosomal loss and no stray chromosomes were observed in any of these cryopreserved oocytes (n = 137). In the cryopreserved oocytes which had undergone normal fertilization, four scorable karyotypes were achieved and in all of these two sets of 23 chromosomes were observed.

Cell Survival↗
PubMed1986

Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells.

Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogenous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-3H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of [3H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experiments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoyl-2-O-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanediol-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acylation of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.

Animals↗
PubMed1978

Preferential association of newly synthesized H3 and H4 histones with newly replicated DNA.

The assembly of newly synthesized histones into chromatin during replication of MH-134SC cells was studied. Cells pulse-labeled with iododeoxyuridine and [3H]lysine were mixed with an equivalent number of normal cells labeled with [14C]lysine. Nuclease chromatin obtained from pooled cells was fractionated by buoyant-density centrifugation in a gradient containing Metrizamide and 3-iodo-1,2-propanediol. Histones extracted from heavy and normal chromatin regions of the gradient were fractionated by acid-urea gel electrophoresis, and 3H/14C ratios of individual histones were compared. The results showed highly preferential association of newly synthesized H3 and H4 with newly replicated DNA.

Cell Line↗
PubMed1980

Studies on peroxisomes. IX. Biochemical effects of simfibrate on precursor incorporation into polypeptide associated with peroxisome proliferation in rat liver.

Treatment with 1,3-propanediol-bis(2-p-chlorophenoxy-isobutyrate) (simfibrate), a hypolipidemic drug, showed little effect on liver weight and hepatic lipid level. When the effects of simfibrate on rat liver peroxisomal enzymes were examined, it was found that cyanide-insensitive fatty acyl-CoA oxidizing system and carnitine acetyltransferase activity increased from 0.38 U/g liver to 4.5 U/g liver and from 227 U/g liver to 6,450 U/g liver, respectively. The activity of D-amino acid oxidase decreased from 1.05 U/g liver to 0.39 U/g liver. Other peroxisomal enzymes including catalase and urate oxidase were not significantly changed by this drug. Of the protein components of the light mitochondrial fraction of simfibrate-treated rat liver, a polypeptide with a molecular weight of approximately 76,000, which has been suggested to be one of the peroxisomal proteins, increased in the treated rat liver (as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis) and DL-[4,5-3H]leucine-incorporation into this particular component was also stimulated to a level about 3 times that of the control.

Animals↗
PubMed1985

Inhibition of glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives.

The mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing. With an increase in pH from 4.0 to 6.0, the Ki values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slightly higher pH on inhibitor binding. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues. The modes of binding of the aminoalcohols with the two glucoamylases were very similar. Therefore, the phenomenon might be a common feature of glucoamylases in general.

Amino Alcohols↗
PubMed2006

The utility of the guppy (Poecilia reticulata) and medaka (Oryzias latipes) in evaluation of chemicals for carcinogenicity.

There has been considerable interest in the use of small fish models for detecting potential environmental carcinogens. In this study, both guppies (Poecilia reticulata) and medaka (Oryzias latipes) were exposed in the aquaria water to three known rodent carcinogens for up to 16 months. Nitromethane, which caused mammary gland tumors by inhalation exposure in female rats, harderian gland and lung tumors in male and female mice, and liver tumors in female mice by inhalation, failed to increase tumors in either guppies or medaka. Propanediol, which when given in the feed was a multisite carcinogen in both sexes of rats and mice, caused increased liver tumors in male guppies and male medaka. There was reduced survival in female guppies and no increased tumors in female medaka. 1,2,3-Trichloropropane, which when administered by oral gavage was a multisite carcinogen in both sexes of rats and mice, caused an increased incidence of tumors in the liver of both male and female guppies and medaka and in the gallbladder of male and female medaka. The results of this study demonstrate that for these three chemicals, under these specific exposure conditions, the fish appear less sensitive and have a narrower spectrum of tissues affected than rodents. These results suggest that fish models are of limited utility in screening unknown chemicals for potential carcinogenicity.

Animals↗
PubMed1989

Ultrastructural observations of human and mouse oocytes treated with cryopreservatives.

The effects of the cryopreservative agents dimethylsulfoxide (DMSO) and propanediol (PROH) on mature human and mature mouse oocytes have been examined with transmission electron microscopy. Treatment of CD-1 mouse oocytes and human preovulatory oocytes in a stepwise manner with either DMSO or PROH up to 1.5 M appears to trigger the exocytosis of 70-80% of the cortical granules in all oocytes. Successive stages in premature dehiscence, including a loss in granule electron density, fusion of the granule-limiting membrane with the oolemma, and extrusion of the cortical granule core into the perivitelline space, have been observed in all human oocytes studied. In addition, all human DMSO- and PROH-treated oocytes exhibited crypt-like invaginations and clusters of endocytic vesicles that subtend the oolemma. The presence of these crypts and pinocytotic vesicles in treated oocytes may suggest a mechanism for the retrieval of cortical granule membrane that is inserted into the original plasmalemma during exocytosis. The paucity of cortical granules in treated mouse and human oocytes as it potentially relates to an impaired ability to elicit the cortical reaction at fertilization is discussed.

Animals↗
PubMed1989

In vitro fertilization and embryo transfer in the rhesus monkey.

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.

Animals↗
PubMed1998

Detrimental effects of sodium during mouse oocyte cryopreservation.

Cryopreservation is an established way of storing embryos, but effective methods are not available for freezing eggs. Most freezing damage is caused by high solute concentration (solution effects) and intracellular ice. Sodium salts are the major components of cryopreservation media, and the main contributor to the solution effects. The present experiments examine the effect of substituting choline for sodium as the major extracellular cation in the cryopreservation of mouse eggs. The effects of serum and various cryoprotectants were also examined. Survival, fertilization, and development were inversely related to the concentration of sodium in the freezing medium. Oocytes frozen in a choline-based medium had the highest (p < 0.001) survival and development rates. The absence of serum during thawing inhibited fertilization, whereas exposure to serum or opening the zona allowed fertilization to reach the control level. Dimethyl sulfoxide was as effective as 1,2 propanediol for obtaining high survival and fertilization rates. These results support the hypothesis that the high concentration of sodium in conventional freezing media is detrimental to cells and show that choline is a promising replacement for sodium. Reducing or eliminating sodium may allow oocytes and other cells to be frozen more efficiently.

Animals↗
PubMed1988

Buspirone, a new approach to the treatment of anxiety.

Anxiety has historically been treated by agents with a sedative component to their action. In the last decade or so it has been determined that gamma-aminobutyric acid (GABA) receptors may mediate the anxiolytic actions of the benzodiazepines, propanediol carbamates, barbiturates, and ethanol. However, inasmuch as these drugs have additional pharmacological properties (sedation, muscle relaxation, seizure control), the search for an anxioselective drug was continued. Buspirone appears to be such a drug. Clinical studies have clearly demonstrated the efficacy of buspirone in the treatment of generalized anxiety disorder without the ancillary pharmacology of earlier anxiolytics. Buspirone does not act on the GABA receptor. Rather, its most salient interaction with neurotransmitter receptors occurs at the 5-HT1A serotonin receptor. This action is supported by studies focused on receptor binding, anatomical localization, biochemistry, neurophysiology, and animal behavior. The recognition that action at 5-HT1A receptors may be a viable approach to the pharmacotherapy of anxiety is evidenced by the number of other agents of this class under development by a number of pharmaceutical companies.

Anxiety Disorders↗
PubMed1995

Eosinophilia-myalgia syndrome, toxic-oil syndrome, and diffuse fasciitis with eosinophilia.

The similarity of eosinophilia-myalgia syndrome (EMS) and toxic-oil syndrome (TOS) to systemic sclerosis and diffuse fasciitis with eosinophilia (DFE) highlights the potential for environmental agents to induce autoimmune disease. Further, a candidate etiologic agent for EMS, 3-(phenylamino)alanine, is chemically similar to the aniline derivative identified in samples of oil implicated in TOS, 3-(N-phenylamino)-1,2-propanediol, suggesting pathogenic overlap. The late-stage manifestations of EMS and TOS are muscle cramping, arthralgia, severe fatigue, and cognitive impairment. This review focuses on the divergent and parallel findings in EMS, TOS, and DFE. The formation of the Environmentally Associated Connective Tissue Disease Study Group within the American College of Rheumatology will provide a forum for the development of registries to study suspected toxin-induced disorders.

Brassica↗
PubMed1994

Felbamate pharmacology and use in epilepsy.

Felbamate, 2-phenyl-1,3-propanediol dicarbamate, is an antiepileptic drug recently approved by the United States Food and Drug Administration. It has a novel mechanism of action whereby it may decrease excitation by inhibiting glycine binding at the NMDA receptor, and it appears to have neuroprotective properties in addition to antiepileptic ones. A number of animal models have demonstrated felbamate to have a broad range of efficacy as well as a favorable safety profile. In humans it has been potentially linked to some cases of aplastic anemia. It is effective in the treatment of partial and secondarily generalized tonic-clonic seizures as well as seizures associated with the Lennox-Gastaut syndrome, especially drop attacks. It may also be effective against atypical absence as well as other seizure types. Felbamate monotherapy is generally well tolerated, with such side effects as insomnia and anorexia occurring most commonly. Felbamate shows great promise as a useful antiepileptic drug, but its role in clinical practice awaits further investigation of recently reported cases of aplastic anemia.

Animals↗