Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer

Abstract

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.

The ketone body β-hydroxybutyric acid influences agouti-related peptide expression via AMP-activated protein kinase in hypothalamic GT1-7 cells

“…An aliquot of the PCR product was electrophoresed on a 3% (w/v) agarose gel and visualised by ethidium bromide staining to confirm purity and size. To analyse the relative changes in gene expression, the 2 KDDC T method was used (Livak & Schmittgen 2001 Pöhland et al (2008).…”

Involvement of E-cadherin in early in vitro development of adult and juvenile sheep embryos

“…α-tubulin, F-actin, E-cadherin and β-catenin), could be involved in the developmental arrest normally encountered at the 8-cell stage in bovine cloned embryos (Poehland et al 2008). In agreement with this observation, we previously demonstrated that, in cows, the majority of embryos that overcome the 8-16-cell stage fastest show a correct localisation of β-catenin (Modina et al 2007a), suggesting a close relationship between embryo quality, developmental dynamics and junctional protein localisation.…”

Influence of long-term thermal stress on the in vitro maturation on embryo development and Heat Shock Protein abundance in zebu cattle

“…Western blotting analysis was performed as described by Poehland et al (2008). Matured COCs were separated mechanically (frequent pipetting with a 100 µL automatic pipette) until the denuded oocytes were obtained.…”