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Biomedical subjects

I Kola

Publications and source records attributed to I Kola.

106 records · Page 6Linked to original sources
PubMed1991

Preimplantation embryo biopsy: detection of trisomy in a single cell biopsied from a four-cell mouse embryo.

This paper describes an efficient technique for the production of metaphase spreads from single blastomeres biopsied from four-cell preimplantation mouse embryos. The karyotype obtained by chromosomal analysis of single biopsied cells is shown to be fully predictive of subsequent fetal karyotype. The data in this study also demonstrate that the entire process of embryo biopsy and karyotypic analysis of biopsied blastomeres does not adversely affect the ability of biopsied embryos to form fetuses after transfer into pseudopregnant recipients. This study has potential clinical relevance in that it demonstrates that chromosomally defective embryos can be accurately identified before implantation. In addition, the techniques developed in this study may facilitate more efficient procedures for the genesis of animal models for human disorders such as Down syndrome and Alzheimers disease.

Animals↗
PubMed1991

An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution.

This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1.5, 3.0 or 4.5 M-DMSO. Rapidly cooled straws containing 4.5 M-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4.5 M-DMSO was not influenced by straw handling as 91-92% formed blastocysts in vitro, 77-78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing less than 4.5 M-DMSO ice formation occurred throughout (1.5 M-DMSO), or in parts (3.0 M-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3.0 or 1.5 M-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3.0 M-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4.5 M-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.

Animals↗
PubMed1990

Chromosomal analysis of human oocytes fertilized by microinjection of spermatozoa into the perivitelline space.

In-vitro fertilization by sperm microinjection techniques have been shown to be effective (the birth of one child has resulted), but concerns about the chromosomal normality of embryos derived from this process have been raised. The British Interim Licensing Authority has set, as a pre-condition to the clinical application of sperm microinjection, the chromosomal evaluation of pre-embryos derived by this technique. In this study, we demonstrate that four of 18 (22%) eggs fertilized by sperm microinjection into the perivitelline space are chromosomally abnormal as compared with nine of 30 (30%) fertilized by conventional IVF procedures. These results are not significantly different. In addition, we demonstrate that nine out of 20 couples who did not previously have fertilization in multiple cycles of IVF had between one and three oocytes fertilized by sperm microinjection. These findings demonstrate that sperm microinjection does not increase the incidence of chromosomally abnormal eggs and provide reassurance and support for the clinical implementation of a technique that appears to be effective for the treatment of certain forms of male infertility.

Aneuploidy↗
PubMed1990

Localization of the murine leukemia inhibitory factor gene near the centromere on chromosome 11.

Leukemia inhibitory factor (LIF) is a glycoprotein with divergent activities: It induces the differentiation of certain myeloid leukemic cells, inhibits the differentiation of embryonic stem cells, and promotes bone remodelling in vivo and in vitro. The murine LIF gene has been assigned to the proximal region of chromosome 11 at sub-bands A1-A2, by analysis of a panel of mouse x Chinese hamster somatic cell hybrids and by in situ hybridization. Interestingly, the proximal portion of chromosome 11 has been shown, by virtue of its parental origin effects, to contain gene(s) involved in fetal growth. It is also interesting that there is a preponderance of chromosome 11 abnormalities in embryonal carcinoma cells. The localization of the murine LIF gene confirms the homology of a portion of murine chromosome 11 with human chromosome 22q, the site of the human LIF gene.

Animals↗
PubMed1989

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.

Animals↗
PubMed1989

Co-administration of ascorbic acid with cyclophosphamide (CPA) to pregnant mice inhibits the clastogenic activity of CPA in preimplantation murine blastocysts.

Cyclophosphamide (CPA) administered at a dose of 15 mg/kg body weight to pregnant inbred CBA/CaH mice 60 h after copulation significantly elevated the incidence of structural chromosomal aberrations and sister-chromatid exchanges in 96 h blastocysts. When ascorbic acid (800 mg/kg body weight) was co-administered with CPA (15 mg/kg) using different injection sites, the incidence of structural chromosomal aberrations and the number of aberrant metaphases were significantly lower as compared to the CPA-exposed embryos, but still significantly higher than untreated controls. Sister-chromatid exchanges were not significantly different in embryos exposed to CPA only when compared to those exposed to CPA and ascorbic acid. In terms of the developmental potential of embryos, pregnant mice injected with both CPA and ascorbic acid had significantly fewer resorptions per mouse, and a significantly higher number of viable fetuses retrieved on the 18th day of gestation when compared to mice injected with CPA alone. The data in this study demonstrate that the co-administration of ascorbic acid with CPA to pregnant mice ameliorates the CPA-induced clastogenicity and improves the developmental potential of these embryos. This study suggests that some of the CPA-induced genotoxicity is due to the generation of reactive oxygen molecules, but further research is necessary before this can be firmly concluded.

Animals↗
PubMed1988

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses.

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing. Malformed fetuses were obtained from oocytes that had been vitrified as well as those that had been exposed to vitrification solutions only, whereas no malformed fetuses were obtained in oocytes slow-frozen by DMSO or fresh controls--thus demonstrating that the exposure of oocytes to the vitrification chemicals was responsible for the fetal malformations. The data in this study suggest that the vitrification technique should be cautiously applied to human oocyte cryopreservation. Furthermore, the data also demonstrate that the exposure of female gametes to carcinogenic and/or teratogenic chemicals may result in malformed embryos when such oocytes are subsequently fertilized.

Aneuploidy↗
PubMed1987

Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida.

The authors report a method of micromanipulation for the insertion of a single spermatozoon under the zona pellucida of human oocytes that results in a high rate of fertilization without damage to the oocyte. Spermatozoa were exposed to calcium-depleted medium containing strontium chloride for 20 to 24 hours before resuspension in medium containing calcium to induce capacitation. Single spermatozoa treated in this way were injected under the zona pellucida of morphologically mature oocytes and fertilization was confirmed by electron-microscopy. Five of seven oocytes manipulated within 9 hours of aspiration from the follicle and 3 of 12 oocytes manipulated 23 to 28 hours after recovery from the follicle fertilized. This technique has profound implications for the possible treatment of severe male infertility.

Cell Nucleus↗
PubMed1987

Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos.

Between 1 and 4% of human oocytes fertilized in vitro are tripronuclear. It has been reported that these tripronuclear oocytes can develop to grossly normal-appearing morulae and that chromosomally, these embryos could be triploid, diploid, or severely depleted. The etiology and proportion of apparently diploid and aneuploid embryos deriving from tripronuclear human oocytes is unknown. This study provides evidence for the first time that most (18 of 29) tripronuclear human oocytes cleave directly to 3-cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4 of 29) tripronuclear human oocytes cleave to 2-cells plus an extrusion, and these embryos are diploids, whereas some (7 of 29) cleave to 2-cells, and these embryos are triploid after the first cleavage division. These findings demonstrate that most tripronuclear human oocytes have an altered cleavage pattern at the first cleavage division, that most tripronuclear human oocytes (76% in this study) do not develop into triploid embryos, and that a correlation exists between the pattern of the first cleavage division and the subsequent karyotype of these embryos. Insight into the mechanisms by which these oocytes fail to develop into triploid embryos is also provided.

Aneuploidy↗
PubMed1986

An assessment of the effects of cyclophosphamide and sodium valproate on the viability of preimplantation mouse embryos using the fluorescein diacetate test.

The fluorescein diacetate test is a measure of both esterase enzyme activity and membrane integrity and it has been shown to be useful in assessing the viability of a variety of cells cultured in vitro, mouse embryos after freezing and thawing, and mouse embryos grown under inadequate culture conditions. In this study, the effects were examined of cyclophosphamide and sodium valproate administered respectively to pregnant inbred CBA mice 60 h after fertilization, on the viability of 84-h blastocysts. Cyclophosphamide 20 and 40 mg/kg bodyweight and sodium valproate 90 mg/kg significantly increased the number of nonviable blastocysts. Cyclophosphamide 4 mg/kg and sodium valproate 23 and 45 mg/kg did not adversely affect blastocyst viability. The intensity of embryo fluorescence correlated well with the subsequent development of the embryos in culture (r = 0.863; p less than 0.001). The test had a sensitivity of 83% and a specificity of 100%. Exposure of embryos to fluorescein diacetate under the conditions of this experiment did not adversely influence the subsequent postimplantation development of 84-h blastocysts cultured in vitro for a further 120 h. These findings suggest that the fluorescein diacetate test is a simple, rapid, and nontoxic procedure that may be useful in assessing the viability of preimplantation embryos after exposure to embryotoxic drugs and chemicals.

Animals↗
PubMed1986

Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos.

The effects of cyclophosphamide (CPA), administered to pregnant inbred CBA/Ca mice 60 h after copulation, on cell number, mitotic index, chromosome structure, histone synthesis, and DNA synthesis of 84-h blastocysts, and the subsequent development of these blastocysts cultured for a further 120 h in vitro are described. Cyclophosphamide 4, 20, and 40 mg/kg significantly increased the number of chromosomally aberrant cells, chromosomal aberrations, and chromosome breaks in the blastocysts. Chromosomal rearrangements were significantly increased in the CPA 20 and 40-mg/kg treated groups, and in the 40-mg/kg group the number of cells with ring chromosomes was significantly increased. Histone synthesis and DNA synthesis were significantly inhibited in the CPA 20 and 40-mg/kg treated groups. Blastocyst cell number in each of the treated groups was less than the controls. On subsequent culture in vitro, significantly fewer embryos in the CPA 20 and 40-mg/kg groups hatched, attached, developed trophoblast outgrowths, and expanded their inner cell masses. However, the differentiation of inner cell mass into ectoderm and endoderm was impaired by all three doses of the drug. These results demonstrate that CPA administered to pregnant mice 60 h after copulation has a clastogenic effect and interferes with synthesis of DNA and histones in the preimplantation embryo, and that the drug inhibits the subsequent development and differentiation of these embryos. Cytogenetic analysis of preimplantation embryos might be a useful adjunct to the existing methods in the evaluation of the embryotoxicity of drugs and chemicals.

Animals↗
PubMed1986

Chlorpromazine inhibits the mitotic index, cell number, and formation of mouse blastocysts, and delays implantation of CBA mouse embryos.

Chlorpromazine, administered to pregnant CBA mice 56 h after copulation in single doses of 10 or 15 mg/kg bodyweight, inhibited the compaction of embryos, formation of blastocysts, and reduced the mitotic index and cell number of embryos 86 h after copulation but did not adversely influence their viability or induce structural chromosomal aberrations. Blastocyst formation was more severely affected than embryo compaction. When 86-h embryos were treated with chlorpromazine (10 or 15 mg/kg) and subsequently cultured for 120 h, there was delayed hatching from the zona pellucida, delayed attachment to the culture dish, outgrowth of the trophoblast and expansion of the inner cell mass. Mice treated identically and evaluated on the 18th day of gestation had fewer implanted embryos than did controls, and the fetuses weighed less. No resorptions, malformations or significant differences in intrauterine deaths were found. Chlorpromazine given in the same manner but at 0.5 mg/kg did not affect any of the aforementioned criteria. When 56 h embryos were cultured in vitro in the presence of 50 microM-chlorpromazine for a further 40 h, embryo compaction, blastocyst formation, the mitotic index and the total cell number were significantly reduced compared with controls. Blastocyst formation was again more severely affected than embryo compaction. The inhibition of embryo compaction, blastocyst formation, and reduction in mitotic index and cell number associated in this study with chlorpromazine in vivo and in vitro indicate that the drug inhibits the development of cleavage-stage embryos in the mouse. These effects might be mediated by antagonistic effects of calmodulin.

Animals↗
PubMedDate not supplied

Review: Pdha-2, past and present.

Pyruvate dehydrogenase (PDH) is a multiunit enzymatic complex essential for the process of generating cellular energy. One of the most important of its subunits is the E1 alpha subunit. Perturbations in the expression of this subunit lead to reduced or lost function of the PDH complex as a whole, resulting in a loss of ATP production. The consequence of such perturbations can lead to neurological abnormalities, lactic acidosis, and in males, death. Pdha-2 codes for the mouse testis isoform of the E1 alpha subunit and maps to chromosome 19 (chromosome 4 in humans). This is a fortuitous evolutionary development because the somatic isoform of the E1 alpha subunit is linked to the X-chromosome, which is not only inactivated early in spermatogenesis but is represented in only half of the haploid spermatid population. Consequently, activation of the testis-specific E1 alpha subunit is essential for the progression of spermatogenesis. Despite its importance, the molecular mechanisms governing the tight tissue- and temporal-specific regulation of Pdha-2 have, until recently, remained poorly understood. In this review, we describe our current understanding of the transcriptional regulation of Pdha-2 and propose potential mechanisms that may play a role in this process.

Adenosine Triphosphate↗