Developmentally regulated markers ofin vitro-produced preimplantation bovine embroys

Abstract

Expression of various developmentally regulated markers was screened throughout the preimplantation stages of in vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as Factin, a-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine embryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells of in vitro-derived bovine embryos valid for cell line derivation.

Trophectoderm differentiation in the bovine embryo: characterization of a polarized epithelium

“…Therefore, the results obtained from in vitro derived embryos indicate that culture has little impact on embryonic E-cadherin distribution patterns. The present study reports the distribution of E-cadherin polypeptides in earlier stages than that reported by Shehu et al (1996). It Removal of E-cadherin mediated cell-cell adhesion does not prevent cell polarization (Pratt et al, 1982;Johnson et al, 1986) but rather delays and randomizes the orientation of cell polarity (Johnson et al, 1986).…”

“…Weak association of these catenin-ZO-1 complexes with E-cadherin may play a role in the shuttling of components of the tight junction to the lateral membranes mediating junction assembly (Rajasekaran et al, 1996). (Shehu et al, 1996 (Fleming et al, 1989(Fleming et al, , 1994 and tight junction formation coincide with the onset of cavitation (Ducibella and Anderson, 1975;McLaren and Smith, 1977;Pratt, 1985 …”

“…While the expression patterns of junctional complex genes are well characterized in early mouse embryos (Vestweber et al, 1987;Larue et al, 1994;Reithmacher et al, 1995), this type of analysis has only just been initiated in embryos of other mammals (Reima et al, 1993;Shehu et al, 1996). Shehu et al (1996) (Shehu et al, 1996).…”

Achievements and unmet promises of assisted reproduction technologies in large animals: a per-sonal perspective

“…Then the use of embryo co-culture with oviductal cells (Gandolfi and Moor, 1987) was the beginning of a new era. To overcome the limits in early technology and lack of scientific knowledge, in vitro produced zygotes have been cultured for a long time in the surrogate sheep oviduct that ensured cryotolerance and viability after transfer to recipients comparable to that of in vivo generated embryos (Shehu et al, 1996;Lazzari et al, 2010). For many years and still now in some laboratories, co-culture with primary cells like oviductal or cumulus cells (Galli and Moor, 1989) or with established cell lines like BRL and Vero became the routine also in the human field (Menezo et al, 2012).…”

“…It turned out however that serum (and probably some secreted factors) was detrimental for post implantation embryo development in ruminants and identified as primarily responsible for the Large Offspring Syndrome (LOS; Young et al, 1998). The presence of serum was also found detrimental for cryopreservation because it was responsible for the accumulation of lipids (Shehu et al, 1996;Lonergan and Fair, 2008). Therefore media formulations shifted towards a serum free and cell free solutions where the still undefined component remains only Bovine Serum Albumin.…”