ABSTRACT
Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.
Subject(s)
Cytoplasm/transplantation , Mitochondria/physiology , Nuclear Transfer Techniques , Oocytes/cytology , Animals , Cattle , Cells, Cultured , Cytoplasmic Streaming/physiology , DNA, Mitochondrial/genetics , Embryo Culture Techniques , Embryo Transfer/veterinary , Embryo, Mammalian/metabolism , Embryo, Mammalian/physiology , Female , Fetal Development/physiology , Germ Cells/cytology , Germ Cells/ultrastructure , Nuclear Transfer Techniques/veterinary , Oocytes/ultrastructure , Pregnancy , Tissue DonorsABSTRACT
The diagnosis of bladder cancer is confirmed by histological analysis of tissue biopsies. Cytology of urine samples is a noninvasive alternative. The aim of this work was to find out whether flow cytometry of urine samples is more sensitive than cytology. For this purpose we studied 115 patients suspected of having bladder cancer. Cells isolated from urine samples were analyzed by cytometry for the expression of cytokeratin and CD 45 and for DNA measurements such as: DNA index, synthesis phase fraction and proliferative index (SPF + G2/M phase). At the same time we carried out cytological analysis. All positive cases were confirmed by histology (21/115), 18 were diagnosed by flow cytometry and 16 by cytology, with a sensitivity of 85.7% and 76.1%, respectively. Two cases were found to be positive by flow cytometry, which were not confirmed by histology, while no false positives were detected by cytology. We found that both techniques gave almost identical results for the diagnosis of bladder cancer, although there were differences in non-malignant samples. In conclusion, flow cytometry is slightly more sensitive than cytology but the combination of the two techniques improves the diagnosis.