Chinese Hamster Oocytes Research Articles

Selective transport of long-chain fatty acids by FAT/CD36 in skeletal muscle of broilers

Fatty acid translocase (FAT/CD36) is a membrane receptor that facilitates long-chain fatty acid uptake. To investigate its role in the regulation of long-chain fatty acid composition in muscle tissue, we studied and compared FAT/CD36 gene expression in muscle tissues of commercial broiler chickens and Chinese local Silky fowls. The results from gas chromatography–mass spectrometry analysis of muscle samples demonstrated that Chinese local Silky fowls had significantly higher (P < 0.05) proportions of linoleic acid (LA) and palmitic acid, lower proportions (P < 0.05) of arachidonic acid (AA) and oleic acid than the commercial broiler chickens. The mRNA expression levels of fatty acid (FA) transporters (FA transport protein-1, membrane FA-binding protein, FAT/CD36 and caveolin-1) in the m. ipsilateral pectoralis and biceps femoris were analyzed by Q-PCR, and FAT/CD36 expression levels showed significant differences between these types of chickens (P < 0.01). Interestingly, the levels of FAT/CD36 expression are positively correlated with LA content (r = 0.567, P < 0.01) but negatively correlated with palmitic acid content (r = −0.568, P < 0.01). Further experiments in the stably transfected Chinese hamster oocytes cells with chicken FAT/CD36 cDNA demonstrated that overexpression of FAT/CD36 improves total FA uptake with a significant increase in the proportion of LA and AA, and a decreased proportion of palmitic acid. These results suggest that chicken FAT/CD36 may selectively transport LA and AA, which may lead to the higher LA deposition in muscle tissue.

Chronic renal failure, dialysis, and renal transplantation in Anderson-Fabry disease

Anderson-Fabry disease (AFd) is a rare, inherited, x-linked disease characterized by the deficiency of the lysosomal enzymatic alpha-galactosidase A activity (alpha-Gal-A). The enzyme defect leads to progressive accumulation of glycosphingolipids (GL) in all kinds of cells, tissues, organs, and body fluids. The clinical manifestations are very protean, the residual activity of alpha-Gal-A and/or different gene mutations might explain different phenotypes, but as yet these concepts have not been proven. Usually, patients with AFd show 3 clinical phases, more evident in men than in heterozygous women. The first phase (childhood and adolescence) is characterized by myalgia, arthralgia, acroparesthesia, fever, cutaneous angiokeratomas, and corneal opacities. The second phase is characterized mainly by renal involvement. In the third phase, severe renal impairment and involvement of cerebrovascular and cardiovascular systems are present. The progression to end-stage renal disease (ESRD) is common in hemizygous males (3rd-5th decade of life); usually, death occurs because of cerebral and/or cardiovascular complications in patients undergoing chronic dialysis therapies. The survival of patients with AFd in dialysis is better than in diabetic patients, but it clearly is decreased compared with uremic patients with other nephropathies, despite a lower mean age of uremia (50 versus 60 y). The outcome of kidney transplantation is similar to that found in other patients with ESRD, despite controversial issues published in the past. The use of a kidney donor with normal alpha-Gal-A activity in the control of the metabolic systemic disease is unproven. The recurrence of GL deposits in the kidney graft has been documented rarely. The definitive treatment for AFd is enzyme replacement therapy with purified alpha-Gal-A produced by a genetically engineered human cell line or Chinese hamster oocytes: relatively short-term studies have shown a significant treatment effect on clinical outcome measures.

Dithiothreitol induces sperm nuclear decondensation and protects against chromosome damage during male pronuclear formation in hybrid zygotes between Chinese hamster spermatozoa and Syrian hamster oocytes.

The present study was undertaken to investigate whether a time lag in sperm nuclear decondensation and male pronuclear formation in the course of development of eggs is associated with any occurrence of structural chromosome aberrations in male genomes of hybrid zygotes between Chinese hamster spermatozoa and zona-free Syrian hamster oocytes. Shortly after insemination, hybrid zygotes were treated with dithiothreitol (DTT) at different concentrations (0.1-10.0 mM) for 30 min to reduce protamine disulphide (S-S) bonds and thereby accelerate sperm nuclear decondensation and male pronuclear formation. The incidence of sperm nuclear decondensation and male pronuclear formation increased with increasing DTT concentrations, indicating that a reduction in S-S bonds effectively induces these cytological events. Chromosomes of male genomes in hybrid zygotes generated by treatment with 1.0 mM, 2.5 mM and 10.0 mM DTT were analysed at the first cleavage metaphase. Incidence of structural chromosome aberrations in each treatment was 34.5%, 27.1% and 24.7%, respectively. There was a significant difference between the incidences with 1.0 mM and 10.0 mM DTT treatment. As the time lag in nuclear decondensation and male pronuclear formation was greatest in the 1.0 mM treatment condition, followed in order by 2.5 mM and 10.0 mM, it is suggested that the lag in sperm nuclear development behind egg development is responsible for structural chromosome aberrations in male genomes of hybrid zygotes.

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation.

This study was undertaken parthenogenetically to activate Chinese hamster oocytes in vitro by chemical stimuli. Oocytes were exposed to five different chemical agents, ethanol (EtOH), strontium chloride (SrCl2), cycloheximide (CHX), phorbol ester (PMA), and ionophore A23187 (IA23). No parthenogenetic activation was observed in the oocytes treated with 8% EtOH for 8-11 min, 1.7 mM and 5.0 mM SrCl2 for 1 hr, 100 microM and 400 microM CHX for 2 hr, and 81 nM and 162 nM PMA for 5 min. In contrast, 89.7% of oocytes parthenogenetically extruded the second polar body in treatment with 3 microM IA23 for 5 min, but only 22.6% of them formed a pronucleus and developed to 2-cell embryos. The remaining ova stopped their cell cycle immediately after completion of the second meiotic division. They had unichromatid chromosomes (monads), which are called MIII chromosomes. Treatment with 5 microM IA23 for 5 min was so deleterious that > 90% of oocytes were degenerated. However, oocyte activation was significantly improved when the treatment with 3 microM IA23 for 5 min was followed by treatment with 8% EtOH for 10 min, 100 microM CHX for 2 hr, 81 nM PMA for 5 min or 3 microM IA23 for 5 min: rates of pronuclear formation were 54.4%, 84.3%, 34.2%, and 54.6%, respectively. More than 80% of pronucleate ova successfully developed into 2-cell stage. Additive treatment with 5 mM SrCl2 for 1 hr had no positive effect on pronuclear formation. Incidences of aneuploidy (4.6%) and structural chromosome aberrations (1.0%) in parthenogenons produced by combined stimuli of IA23 and CHX were not significantly different from those (3.8% and 1.6%, respectively) in female pronuclei of ova fertilized in vitro, showing that combined treatments with IA23 and CHX cause neither nondisjunction at the second meiotic division nor structural aberrations in MII chromosomes. The present technique for parthenogenetic activation of Chinese hamster oocytes may be useful as an assessment system to detect aneugenic and clastogenic effects of mutagens on mammalian oocytes.

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation

Stereoselective Increase in Cholinergic Transmission by R-(+)-hyoscyamine

R-(+)-hyoscyamine, the dextro enantiomer of atropine, has been shown to amplify cholinergic transmission. R-(+)-hyoscyamine, unlike S-(—)-hyoscyamine, was able to increase acetylcholine release both in vitro and in vivo at a range of concentrations (10 −14 to 10 −12 M) and doses (5 μg/kg i.p.) which were inadequate for blocking muscarinic receptors. The increase over control values in ACh release was 15.9 ± 2.1% in in vitro experiments performed in rat phrenic nerve-hemidiaphragm preparations ( n = 6), and 63.3 + 16.3% in cortical microdialysis performed in free-moving rats ( n = 5). The maximum ACh release was reached 60 min after R-(+)-hyoscyamine administration in in vivo experiments. At the same doses and concentrations, R-(+)-hyoscyamine was also able to elicit: antinociception of a cholinergic type (55.6–112.7% depending on the test used); complete prevention of scopolamine- and dicyclomine-induced amnesia; potentiation of muscular contractions electrically evoked in isolated guinea-pig ileum (16.7 ± 3.6%) and in rat phrenic nerve-hemidiaphragm (19.9 ± 3.2%) preparations. Antinociception was performed using the hot-plate and acetic acid abdominal constriction tests in mice, and the paw pressure test in rats, while prevention of induced amnesia was evaluated in mice using the passive-avoidance test. The respective affinities (p A 2) for R-(+)- and S-(—)-hyoscyamine vs M 1 (rabbit vas deferens), M 2 (rat atrium) and M 3 (rat ileum) receptor subtypes were as follows: 7.05 ± 0.05/9.33 ± 0.03 for M 1; 7.25 ± 0.04/8.95 ± 0.01 for M 2; 6.88 ± 0.05/9.04 ± 0.03 for M 3. The respective p K i values for R-(+)- and S-(—)-hyoscyamine vs the five human muscarinic receptor subtypes expressed in Chinese hamster oocytes (CHO-K 1) were as follows: 8.21 ± 0.07/9.48 ± 0.18 for m 1; 7.89 ± 0.06/9.45 ± 0.31 for m 2; 8.06 ± 0.18/9.30 ± 0.19 for m 3; 8.35 ± 0.11/9.55 ± 0.13 for m 4; 8.17 ± 0.08/9.24 ± 0.30 for m 5. © 1997 Elsevier Science Ltd. All rights reserved.

Penetration of concanavalin-A-treated Chinese hamster oocytes by golden hamster spermatozoa in vitro, and chromosome analysis of hybrid 1-cell zygotes.

Pretreatment of zona-free Chinese hamster (CH) oocytes with three kinds of lectin--concanavalin A (Con-A), phytohaemagglutinin-P (PHA) and wheat germ agglutinin (WGA)--was attempted in order to improve penetration by golden hamster (GH) spermatozoa in vitro. Con-A had no significant effect on penetration at 2 micrograms/ml, adequately facilitated oocyte-sperm fusion at 4 micrograms/ml, and caused excessive sperm binding and resultant severe polyspermy at 10 micrograms/ml. Neither PHA nor WGA had positive effects on sperm penetration at any concentrations (2-10 micrograms/ml) examined. Using the Con-A (4 micrograms/ml) pretreatment, high rates of interspecific fertilisation and subsequent chromosome analysis of hybrid 1-cell zygotes were achieved. Among 258 CH oocytes used, 212 (82.2%) were fertilised and 153 (72.2% of fertilised ova) developed to the first cleavage metaphase. Eventually, 132 CH-derived chromosome complements and 153 GH-derived ones were successfully karyoanalysed. Incidences of aneuploidy and structural anomaly were 3.1% and 2.3% in CH complements, and 1.4% and 6.5% in GH complements, respectively. These incidences were not significantly different from those obtained by intraspecific in vivo fertilisation, suggesting that our interspecific in vitro fertilisation system does not cause chromosome aberrations.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts.

To enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37 degrees C under 5% CO2 in air. They were then treated with ionophore A23187 (20 microM) for 10 min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37 degrees C under 5% CO2 in air. In this study, 245 oocytes were used for insemination, and 198 (80.8%) were found to be penetrated by sperm; among them, 194 ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1 mM hypotaurine under the same gas phase. Within 30 h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage ('2-cell block') was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

Effect of trimethoxybenzoic compounds and their analogues on meiotic chromosome behavior of Chinese hamster oocytes

Effect of trimethoxybenzoic compounds and their analogues on meiotic chromosome behavior of Chinese hamster oocytes

Induction of aneuploidy in Chinese hamster oocytes following in vivo treatments with trimethoxybenzoic compounds and their analogues

Many inhibitors of tubulin polymerization have a trimethoxybenzene ring in their molecules. Such trimethoxybenzoic compounds and their analogues may therefore have a potency to induce meiotic nondisjunction of oocytes. In this study, a single dose of reserpine (0.5 μg/g body weight), podophyllotoxin (20.0 μg/g b.w.), trimethoxybenzoic acid (500.0 μg/g b.w.) or vinblastine sulfate (3.0 μg/g b.w.) was injected intraperitoneally to mature female Chinese hamsters at the onset of the first meiotic spindle formation of oocytes. Within 6 h after spontaneous ovulation, MII oocytes were collected from the oviducts for morphological examination and cytogenetic analysis. The incidence of morphologically abnormal oocytes with unusually large first polar body or bodies increased significantly after the treatment with reserpine ( 18 202 ; 8.9%), podophyllotoxin ( 28 172 ; 16.3%) and vinblastine sulfate ( 63 197 ; 32.0%), as compared with the control ( 3 214 ; 1.4%). Chromosome analysis of oocytes revealed that podophyllotoxin and vinblastine sulfate were effective in inducing aneuploidy ( 62 154 ; 40.3% and 128 156 ; 82.1% vs. 3 198 ; 1.5% of the control) by inhibiting the formation of spindle microtubules at the first meiosis. Aneuploids were found more frequently in morphologically abnormal oocytes than in normal oocytes. No aneugenic activity of reserpine and trimethoxybenzoic acid was observed. These results indicate that trimethoxybenzoic compounds do not necessarily exhibit aneugenic activity.

CD59 costimulation of T cell activation. CD58 dependence and requirement for glycosylation.

In addition to the TCR-CD3 complex, T lymphocytes can be activated via another surface glycoprotein, the CD2 molecule. CD58 is the principal ligand for human CD2; CD59 and CD48 are two additional, low affinity ligands that have been defined for CD2. In this study, we have explored the role of CD59 in T cell activation. We have expressed human rCD58 and rCD59 molecules in Chinese hamster oocytes (CHO), and tested paraformaldehyde-treated transfectants for the ability to promote proliferation of and IL-2 secretion from PBMC and human purified T cells. We have shown that CD59 enhanced CD58-dependent T cell proliferation and IL-2 secretion in the presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, T11-2 + T11-3. CD59-dependent costimulation was dependent on several factors including the level of co-expression of CD58, the ratio of CHO cell transfectants to T cells added in culture, the concentration of mitogen, and also donor-dependent differences. As expected, CD59 costimulation of CD58-dependent T cell proliferation was inhibited by Abs directed against CD59, CD58, and CD2. In our hands, the CD59 molecule itself, in the absence of CD58, was unable to support proliferation alone even in the presence of exogenous recombinant IL-1, IL-2, or IL-6. Finally, the ability of CD59 to enhance CD58-dependent T cell responses was shown to be dependent on N-glycosylation of CD59 at amino acid Asn18.

Basic proteins in the nuage of growing Chinese hamster oocytes.

Two types of nuage aggregates, the mitochondria-associating nuage and the freely-existing nuage, appeared in the cytoplasm of the growing Chinese hamster oocytes in a growing stage-specific manner. Enzyme digestion studies on the ultrathin sections obtained from the glycol methacrylateembedded materials showed that both types of nuage contain RNA and proteins. The ethanol-phosphotungstic acid staining and also the cobalt thiocyanate staining used to examine the localization of basic proteins at the ultrastructural level revealed that the mitochondria-associating nuage contains a considerable amount of basic proteins, whereas only a few may be present in the freely-existing nuage.It was considered that the mitochondria-associating nuage may lose its basic proteins and transform into the freely-existing nuage, along with the oocyte's growth.

Characterization of paracrystalline inclusions in chinese hamster oocytes and early embryos

AbstractParacrystalline inclusions, readily visible with light microscopy, were found to be present in ovarian oocytes (beginning with unilaminar follicles), ovulated ova, and preimplantation embryos of the Chinese hamster. These inclusions appeared to be aggregates of finer filamentous structures visible only with electron microscopy. The use of various histochemical techniques suggested that the paracrystals were composed of protein with little or no lipid associated with them. Autoradiographic studies using 3H‐uridine and enzymatic studies did not support the hypothesis that the paracrystalline inclusions were composed of RNA masked by a crystalline protein lattice.

Association of Perichromatin Granules with Nuclear Pores of Growing Chinese Hamster Oocytes

Association of Perichromatin Granules with Nuclear Pores of Growing Chinese Hamster Oocytes

Primary incidences of spontaneous chromosomal anomalies and their origins and causal mechanisms in the Chinese hamster

An improved chromosomal technique was used to study primary incidences of spontaneous chromosomal aberrations classified according to their causal mechanisms in Chinese hamster oocytes and early zygotes. Chromosome slides were made from 2157 secondary oocytes, 1188 one-cell zygotes, and 756 two-cell zygotes, and karyoanalysis was successful in 1970 (91%), 1146 (96%), and 733 (97%), respectively. Paternal and maternal chromosomal complements were analyzed separately in 73% of the one-cell zygotes. In 91% of the two-cell zygotes, both blastomeres were analyzed. Incidences of abnormal chromosomal segregation taking place during first and second meiotic divisions of the oocyte, during spermatogenesis and during first somatic division were 1.6, 0.5, 0.7 and 0.5%, respectively. Ploidy anomalies included parthenogenetic haploids (0.5%) and triploids (1.5%) consisting of 0.9% of diandry, 0.3% of digyny and 0.3% of uncertain cases. Incidences of structural anomalies of male and female origins were 1.4 and 1.3%, respectively. The overall incidence of chromosomal anomalies up to the first somatic division was 8.0%. The present results are highly reliable owing to our precisely designed system of chromosome analysis. In particular, the importance of the choice of experimental animals and chromosomal technique is discussed.

Silver Staining of the Nucleolus-like Bodies in Postimplantation Rat Embryos and the Nuage in Chinese Hamster Oocytes

Silver Staining of the Nucleolus-like Bodies in Postimplantation Rat Embryos and the Nuage in Chinese Hamster Oocytes

Dose-response relationship for induction of structural chromosome aberrations in Chinese hamster oocytes after X-irradiation

Recently, we determined the precise timing of changes of meiotic chromosomal radiosensitivity in oocytes during the spontaneous estrous cycle in the Chinese hamster, by assessing MII chromosomal lesions induced after 200 rad X-irradiation at various stages prior to ovulation (Mikamo et al., 1981). It was demonstrated that early diakinesis was the most radiosensitive phase, the incidence of oocytes with structural chromosome aberrations being 15 times higher than during dictyotene stages of diestrum. A high radiosensitivity was also found in mouse oocytes at a transitional stage from late dictyotene to diakinesis, although X-irradiation was restricted only to this single stage (Reichert et al., 1975). In the present work, we investigated the dose-response relationship for induction of chromosome aberrations in Chinese hamster oocytes at the most radiosensitive stage, and compared our results with those obtained previously in the mouse.

Chromosome aberrations in metaphase II oocytes of Chinese Hamster (Cricetulus griseus) I. The sensitivity of the pre-ovulatory phase to triaziquone

In previous investigations with triaziquone, cyclophosphamide, amethopterin and X-rays we showed that the pre-ovulatory stages of mice are sensitive to the induction of numerical and structural chromosome aberrations. In the present investigation, we studied the problem whether or not this sensitivity is restricted to mice. Two doses of triaziquone were injected intraperitoneally to Chinese hamster females at different intervals before ovulation. The ovulated oocytes were collected from the ampulla and analyzed at the metaphase II stage. After treatment with triaziquone, the frequency of both numerical and structural aberrations was significantly increased over the control values. The proportion of induced structural anomalies was higher than that of the numerical ones. Chinese hamsters were less sensitive to the induction of structural and especially numerical anomalies compared with the findings with one strain of mouse.

Chromosome aberrations in metaphase II oocytes of Chinese hamster ( Cricetulus griseus) I. The sensitivity of the pre-ovulatory phase to triaziquone

In previous investigations with triaziquone, cyclophosphamide, amethopterin and X-rays we showed that the pre-ovulatory stages of mice are sensitive to the induction of numerical and structural chromosome aberrations. In the present investigation, we studied the problem whether or not this sensitivity is restricted to mice. Two doses of triaziquone were injected intraperitoneally to Chinese hamster females at different intervals before ovulation. The ovulated oocytes were collected from the ampulla and analyzed at the metaphase II stage. After treatment with triaziquone, the frequency of both numerical and structural aberrations was significantly increased over the control values. The proportion of induced structural anomalies was higher than that of the numerical ones. Chinese hamsters were less sensitive to the induction of structural and especially numerical anomalies compared with the findings with one strain of mouse.