Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Cardiac Neural Crest Cells and Their Role in Murine Purkinje Fiber Development

The heart beat is regulated by the cardiac conduction system (CCS), a specialized group of cells that transmit electrical impulses around the heart chambers. During development, ventricular CCS cells originate from embryonic cardiomyocytes and not from the neural crest. Nonetheless, discoveries in chick implied that the cardiac neural crest (CNC) cells contribute to proper development of the ventricular CCS. In this report, the Splotch mouse mutant (Pax3sp), in which the CNC cells do not migrate to the heart, was used to investigate whether these cells also affect proper CCS development in mammals. Homozygote mutants (Pax3Sp!Sp) are lethal on 111 Embryonic Day 13 (E13), and can be phenotyped by spina bifida and exencephaly. Pax3Spi+ mice were crossed to obtain wild type, Pax3 Spi+ and Pax3 Sp!Sp embryos. Comparison of hematoxylin and eosin stained histological sections showed less trabeculation in El2.5 cardiac ventricles of Pax3Sp!Sp. Furthermore, immunofluorescence analysis with the Purkinje fiber marker Cx40 showed a qualitative difference between wild type and mutant hearts. Quantitative analysis indicated that Pax3 Sp!Sp ventricles had fewer Cx40 expressing cells, as well as less Cx40 being expressed per cell when compared to wild type ventricles. Immunofluorescence with the H3 histome mitosis antibody showed fewer proliferating cells in the ventricles of mutant embryos when compared to controls. These results suggest that CNCC affect the morphogenesis of cardiac ventricles and the development of the ventricular CCS by contributing cellular proliferation.