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

Blastocytst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na-K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, beta-catenin, and the tight junction associated protein, zonula occludens protein 1, in pre-attachment bovine embryos, in vitro. Immunocytochemistry and gene specific reverse transcription--polymerase chain reaction methods were used. Transcripts for E-cadherin and beta-catenin were detected in embryos of all stages throughout pre-attachment development. Immunocytochemistry revealed E-cadherin and beta-catenin polypeptides evenly distributed around the cell margins of one-cell zygotes and cleavage stage embryos. In the morula, detection of these proteins diminished in the free apical surface of outer blastomeres. E-cadherin and beta-catenin became restricted to the basolateral membranes of trophectoderm cells of the blastocyst, while maintaining apolar distributions in the inner cell mass. Zonula occludens protein 1 immunoreactivity was undetectable until the morula stage and first appeared as punctate points between the outer cells. In the blastocyst, zonula occludens protein 1 was localized as a continuous ring at the apical points of trophectoderm cell contact and was undetectable in the inner cell mass. These results illustrate that the gene products encoding E-cadherin, beta-catenin and zonula occludens protein 1 are expressed and maintain cellular distribution patterns consistent with their predicted roles in mediating trophectoderm differentiation in in vitro produced bovine embryos

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 mug/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) 11 cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CRA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts

The dentition of the narrownose smooth-hound shark, mustelus schmitti

The present study provides a quantitative and qualitative analysis on the dentition of Mustelus schmitti, and estimates the tooth-replacement rate. In total, 47 males and 56 females of M. schmitti were collected on scientific trawl surveys conducted by the National Institute of Fisheries Research and Development (INIDEP) in Argentina during the months of November 2007, November 2008 and December 2008. The dental laminas were extracted from the jaw cartilage and attached to onionskin paper for dehydration treatment, maintaining the original jaw position. Tooth replacement rate was estimated following established methods used for fossil sharks, instead of the established technique of clipping teeth, based on the premise that tooth length within each row decreases from the lingual to the labial side of the jaw as a consequence of wear. The length difference between consecutive teeth in four representative rows should be proportional to the tooth-replacement rate. Mustelus schmitti exhibited homodont dentition, where teeth were similar in shape or design, and are arranged in a semi-pavement-like dentition. The dental formula was 47-63/50-63 for juveniles and 50-77/50-69 for adult specimens. The estimated mean replacement rate was 4 days series-1.Fil: Belleggia, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Ciencias Marinas. Laboratorio de Ictiología; Argentina. Instituto Nacional de Investigaciones y Desarrollo Pesquero; ArgentinaFil: Figueroa, Daniel Enrique. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Ciencias Marinas. Laboratorio de Ictiología; ArgentinaFil: Bremec, Claudia Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Investigaciones y Desarrollo Pesquero; Argentin