Maternal histone deacetylase is accumulated in the nuclei of Xenopus oocytes as protein complexes with potential enzyme activity.
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Reversible acetylation of core histones plays an important regulatory role in transcription and replication of chromatin. The acetylation status of chromatin is determined by the equilibrium between activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The Xenopus protein HDACm shows sequence homology to other putative histone deacetylases, but its mRNA is expressed only during early development. Both HDACm protein and acetylated non-chromosomal histones are accumulated in developing oocytes, indicating that the key components for histone deposition into new chromatin during blastula formation are in place by the end of oogenesis. Here we show that the 57 kDa HDACm protein undergoes steady accumulation in the nucleus, where it is organized in a multiprotein complex of approx. 300 kDa. A second, major component of the nuclear complex is the retinoblastoma-associated protein p48 (RbAp48/46), which may be used as an adaptor to contact acetylated histones in newly assembled chromatin. The nuclear complex has HDAC activity that is sensitive to trichostatin A, zinc ions and phosphatase treatment. The 57 kDa protein serves as a marker for total HDAC activity throughout oogenesis and early embryogenesis. The active HDACm complex and its acetylated histone substrates appear to be kept apart until after chromatin assembly has taken place. However, recombinant HDACm, injected into the cytoplasm of oocytes, not only is translocated to the nucleus, but also is free to interact with the endogenous chromatin. (+info)
Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22).
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In this study we describe the pre-clinical development and clinical application of preimplantation genetic diagnosis (PGD) by fluorescence in-situ hybridization (FISH) for two non-related carriers (one male and one female) of the most common balanced reciprocal translocation: t(11;22)(q25;q12). For the couple with the female carrier, enumeration of the sex chromosomes in the embryos was also indicated (husband: 47,XXY karyotype). Four-colour FISH analysis was performed on six blastomeres from three embryos. No embryo transfer was possible because all the embryos were unbalanced. Three PGD cycles, with two-colour FISH, were carried out for the couple with the male translocation carrier. A total of 35 embryos were biopsied and diagnosed by FISH; nine out of the 35 embryos (25. 7%) were normal and seven of them were transferred (two embryos from the first and four from the third cycle), six out of 35 embryos (17%) were unbalanced, three out of 35 embryos (5.7%) were triploid or polyploid, 10 out of 35 embryos (28.6%) were mosaic and seven out of 35 embryos (20%) were chaotic. Diagnosis failed in 2.9% of the embryos. The spermatozoa of the male carrier were also analysed using three-colour FISH. Only 29.1% of the sperm cells seemed to be balanced or normal. By choosing probes lying on both sides of the breakpoints and by using a combination of sub-telomeric or locus-specific probes and centromeric probes, the use of three-colour FISH enabled detection of all the imbalances in sperm and/or cleavage-stage embryos in the patients. This may improve risk assessment and genetic counselling in the future for translocation carriers. (+info)
Fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis for 21-hydroxylase deficiency in congenital adrenal hyperplasia.
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Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase. The disease is characterized by a range of impaired adrenal cortisol and aldosterone synthesis combined with an increased androgen synthesis. These metabolic abnormalities lead to an inability to conserve sodium and virilization of females. The most common mutation causing the severe form of CAH is a conversion of an A or C at nucleotide (nt) 656 to a G in the second intron of the steroid 21-hydroxylase gene (CYP21) causing aberrant splicing of mRNA. A couple was referred to our centre for preimplantation genetic diagnosis (PGD) for 21-hydroxylase deficiency in CAH. A PGD was set up to detect the nt656 A/C-->G mutation using fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. Using DNA or single cells from the father, the normal allele could not be amplified. Non-amplification of the normal allele has been previously described in asymptomatic carriers, therefore the PCR was further developed using heterozygous lymphoblasts from the mother. The PCR was shown to be highly efficient (96% amplification), accurate (0% contamination) and reliable (0% allelic drop-out). The couple started PGD treatment and the second PGD cycle resulted in a twin pregnancy. The genotype of the fetuses was determined in our laboratory using chorionic villus sampling material using the method described here. Both fetuses were shown to be heterozygous carriers of the mutation, and two healthy girls were born. (+info)
The transcription factor GATA6 is essential for early extraembryonic development.
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The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue. (+info)
Genetic markers to predict polygenic disease: a new problem for social genetics.
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Many genetic markers that relate to common multifactorial disease in adults have been identified during the past 15 years. Their use as adjuncts for the diagnosis, prognosis, prediction of disease or targeting therapy for these disorders has begun, good examples being the Factor V Leiden mutation for venous-thromboembolism, lipoprotein lipase mutations for hypertriglyceridaemia and the apolipoprotein E4 variant for Alzheimer's dementia. However, extensive gene-gene and gene-environment interactions make their use more complex than markers for the simpler monogenic disorders (such as cystic fibrosis, or Duchenne's muscular dystrophy). Possible misapplication of the genetic markers for multifactorial disease in the fields of risk prediction, direct sales to the public, life assurance, employment rights, and legislation for regulation of their use are discussed. (+info)
Variability in the expression of trophectodermal markers beta-human chorionic gonadotrophin, human leukocyte antigen-G and pregnancy specific beta-1 glycoprotein by the human blastocyst.
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Improved culture conditions that support the development of human embryos to the blastocyst stage in vitro led to the prospect of blastocyst transfer to increase pregnancy rates. Thus, there is a need for characterization of possible biochemical markers able to predict the implantation potential of human blastocysts. In this study, the expression of three placental markers that are expressed prior to implantation, beta-human chorionic gonadotrophin (HCG), human leukocyte antigen (HLA)-G and pregnancy specific beta-1 glycoprotein (SP-1), was investigated. beta-HCG transcript could be detected as early as the two-cell stage, which is one to two cleavage divisions earlier than previously reported. Both beta-HCG and HLA-G transcripts could be detected in the majority of blastocysts, but their levels were highly variable. No association could be found between the amount of transcript for these genes, total cell number or cell death rate. Interestingly, there was a highly positive correlation between accumulation of beta-HCG and HLA-G transcripts. SP-1 protein concentrations were assessed in the culture medium of blastocysts using enzyme-linked immunosorbent assay. There was a significant positive correlation between SP-1 concentrations and blastocyst cell numbers. Moreover, synthetic oviductal medium enriched with potassium resulted in an SP-1 concentration twice as high as that observed using human tubal fluid medium. These data suggest that SP-1 may be used to select blastocysts with higher cell number, possibly resulting in higher pregnancy rates. (+info)
Pregnancy and birth resulting from transfer of a blastocyst observed to have one pronucleus at the time of examination for fertilization.
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This case report describes a successful full-term pregnancy and birth after the transfer of a zona-free blastocyst derived from an oocyte observed at fertilization check as having only one distinct pronucleus (PN). The patient had previously undergone four in-vitro fertilization (IVF) cycles and three frozen embryo transfer cycles, with one pregnancy resulting. In this IVF cycle, 7/19 oocytes were fertilized exhibiting two distinct PN; however, all these oocyctes failed to develop in culture and had arrested or totally fragmented by day 6 after insemination. One oocyte (1/19) displayed only one PN 18 h after insemination, but culture of this oocyte led to development of an early cavitating blastocyst by day 6. Since no other embryos were available for transfer to the patient, this embryo was transferred, resulting in a full-term pregnancy with delivery of a normal healthy boy. Observation of a single PN at the normal time of fertilization assessment would not appear to be an absolute indicator of developmental incompetence. In-vitro culture to 6 days post-insemination provides the opportunity to assess embryological development after activation of the embryonic genome. Formation of a morphologically normal blastocyst may be an indicator of a fertilized embryo with normal developmental capacity. (+info)
Migration defects of cdk5(-/-) neurons in the developing cerebellum is cell autonomous.
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Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cell cycle-related kinases. Previous neuropathological analysis of cdk5(-/-) mice showed significant changes in CNS development in regions from cerebral cortex to brainstem. Among the defects in these animals, a disruption of the normal pattern of cell migrations in cerebellum was particularly apparent, including a pronounced abnormality in the location of cerebellar Purkinje cells. Complete analysis of this brain region is hampered in the mutant because most of cerebellar morphogenesis occurs after birth and the cdk5(-/-) mice die in the perinatal period. To overcome this disadvantage, we have generated chimeric mice by injection of cdk5(-/-) embryonic stem cells into host blastocysts. Analysis of the cerebellum from the resulting cdk5(-/-) left arrow over right arrow cdk5(+/+) chimeric mice shows that the abnormal location of the mutant Purkinje cells is a cell-autonomous defect. In addition, significant numbers of granule cells remain located in the molecular layer, suggesting a failure to complete migration from the external to the internal granule cell layer. In contrast to the Purkinje and granule cell populations, all three of the deep cerebellar nuclear cell groupings form correctly and are composed of cells of both mutant and wild-type genotypes. Despite similarities of the cdk5(-/-) phenotype to that reported in reeler and mdab-1(-/-) (scrambler/yotari) mutant brains, reelin and disabled-1 mRNA were found to be normal in cdk5(-/-) brain. Together, the data further support the hypothesis that Cdk5 activity is required for specific components of neuronal migration that are differentially required by different neuronal cell types and by even a single neuronal cell type at different developmental stages. (+info)