Transcriptome Profiling of Human Pre-Implantation Development

BACKGROUND: Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. METHODOLOGY: By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. PRINCIPAL FINDINGS: We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. SIGNIFICANCE: Our database and findings provide fundamental resources for understandin

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

Apoptose e maturação placentária bovina: um estudo imunohistoquímico e morfométrico

Resumo: A liberação da placenta após o parto envolve a perda da adesão materno-fetal e ocorre somente após a maturação completa do placentoma, que está relacionada com a diminuição da celularidade dos tecidos fetal e materno. A apoptose é requerida tanto para a maturação quanto para a liberação normal da placenta após o parto. O objetivo do presente estudo foi avaliar a ocorrência de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomas de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imunoistoquímica e histoquímica. As lâminas obtidas foram coradas em HE, Picrosirius Red e submetidas à análise imunoistoquímica das proteínas Caspase 3, Caspase 8, Bax e Bid. O aumento no número de vasos não necessariamente se associou ao aumento do calibre destes durante a evolução da gestação. Os resultados de histomorfometria revelaram aumento da marcação para Bax e Caspases 3 e 8 em células trofoblásticas binucleadas no final da gestação, enquanto o Bid se manteve sem alteração significativa. A histomorfometria das células trofoblásticas mononucleadas revelou expressão alta para Bax no início de gestação, com diminuição aos 6 meses de gestação e aumento das imunomarcações para Caspases 3 e 8, e Bid com o avanço gestacional. Os colágenos tipo I e III não aumentaram do terço médio ao final da gestação, o que é importante para a diminuição da adesão materno-fetal. Esses resultados confirmam que as Caspases 3 e 8, e o Bax estão envolvidos nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial e/ou extrínseca ao longo da gestação em células trofoblásticas binucleadas, e que nas células trofoblásticas mononucleadas o Bax deixa de ser importante, enquanto o Bid e as Caspases 3 e 8 se tornam os mais significativos

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations.

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies