(1/856) oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet.
Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina. (+info)
(2/856) Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa.
Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo. (+info)
(3/856) Cross-coupling between voltage-dependent Ca2+ channels and ryanodine receptors in developing ascidian muscle blastomeres.
1. Ascidian blastomeres of muscle lineage express voltage-dependent calcium channels (VDCCs) despite isolation and cleavage arrest. Taking advantage of these large developing cells, developmental changes in functional relations between VDCC currents and intracellular Ca2+ stores were studied. 2. Inactivation of ascidian VDCCs is Ca2+ dependent, as demonstrated by two pieces of evidence: (1) a bell-shaped relationship between prepulse voltage and amplitude during the test pulse in Ca2+, but not in Ba2+, and (2) the decay kinetics of Ca2+ currents (ICa) obtained as the size of tail currents. 3. During replacement in the external solution of Ca2+ with Ba2+, the inward current appeared biphasic: it showed rapid decay followed by recovery and slow decay. This current profile was most evident in the mixed bath solution (2 % Ca2+ and 98 % Ba2+, abbreviated to '2Ca/98Ba'). 4. The biphasic profile of I2Ca/98Ba was significantly attenuated in caffeine and in ryanodine, indicating that Ca2+ release is involved in shaping the current kinetics of VDCCs. After washing out the caffeine, the biphasic pattern was reproducibly restored by depolarizing the membrane in calcium-rich solution, which is expected to refill the internal Ca2+ stores. 5. The inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase (SERCAs) cyclopiazonic acid (CPA) and thapsigargin facilitated elimination of the biphasic profile with repetitive depolarization. 6. At a stage earlier than 36 h after fertilization, the biphasic profile of I2Ca/98Ba was not observed. However, caffeine induced a remarkable decrease in the amplitude of I2Ca/98Ba and this suppression was blocked by microinjection of the Ca2+ chelator BAPTA, showing the presence of caffeine-sensitive Ca2+ stores at this stage. 7. Electron microscopic observation shows that sarcoplasmic membranes (SR) arrange closer to the sarcolemma with maturation, suggesting that the formation of the ultrastructural machinery underlies development of the cross-coupling between VDCCs and Ca2+ stores. (+info)
(4/856) Preimplantation diagnosis by fluorescence in situ hybridization using 13-, 16-, 18-, 21-, 22-, X-, and Y-chromosome probes.
PURPOSE: Our purpose was to select the proper chromosomes for preimplantation diagnosis based on aneuploidy distribution in abortuses and to carry out a feasibility study of preimplantation diagnosis for embryos using multiple-probe fluorescence in situ hybridization (FISH) on the selected chromosomes of biopsied blastomeres. METHODS: After determining the frequency distribution of aneuploidy found in abortuses, seven chromosomes were selected for FISH probes. Blastomeres were obtained from 33 abnormal or excess embryos. The chromosome complements of both the biopsied blastomeres and the remaining sibling blastomeres in each embryo were determined by FISH and compared to evaluate their preimplantation diagnostic potential. RESULTS: Chromosomes (16, 22, X, Y) and (13, 18, 21) were selected on the basis of the high aneuploid prevalence in abortuses for the former group and the presence of trisomy in the newborn for the latter. Thirty-six (72%) of 50 blastomeres gave signals to permit a diagnosis. Diagnoses made from biopsied blastomeres were consistent with the diagnoses made from the remaining sibling blastomeres in 18 embryos. In only 2 of 20 cases did the biopsied blastomere diagnosis and the embryo diagnosis not match. CONCLUSIONS: If FISH of biopsied blastomere was successful, a preimplantation diagnosis could be made with 10% error. When a combination of chromosome-13, -16, -18, -21, -22, -X, and -Y probes was used, up to 65% of the embryos destined to be aborted could be detected. (+info)
(5/856) Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells.
Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest. (+info)
(6/856) Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.
Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality. (+info)
(7/856) Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs.
The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient's embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources. (+info)
(8/856) Impact of blastomere biopsy and cryopreservation techniques on human embryo viability.
The purpose of the present study was to evaluate the effect of cryopreservation on 55 embryos which had one blastomere biopsied for preimplantation genetic diagnosis of aneuploidy before freezing. The thawing outcome was compared to that obtained in 94 embryos which derived from our conventional freezing programme in patients with comparable characteristics who were treated in the same period. Their embryos were morphologically similar but the incidence of aneuploidy was 100% in the biopsy group and unknown in the controls. The percentage of embryos which survived intact after thawing was significantly lower in the biopsied group compared to the controls (9 versus 25% respectively; P < 0.025), whereas the rate of lysis was superior among biopsied embryos (34 versus 13% in the controls; P < 0.001). Similarly, the survival index was higher in the frozen-intact embryos than in the embryos which were frozen after biopsy (61 versus 38%; P < 0.001). No empty zonae resulted in the control group, while six were found after thawing biopsied embryos. In the second part of the study, blastomere biopsy was implemented on 102 thawed embryos generated by 16 patients. The chromosomal analyses revealed that 49 were normal, leading to the transfer of 2.5 +/- 0.8 embryos per patient. Only three clinical pregnancies were obtained, and are presently ongoing. In conclusion, the present findings discourage the use of conventional cryopreservation protocols in strategies involving preimplantation genetic diagnosis in human reproductive medicine. Adequate protocols are required for freezing and thawing embryos which have been subjected to biopsy procedures. (+info)