Embryological methods in ascidians: the Villefranche-sur-Mer protocols.

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells.

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc alpha 1,3(Fuc alpha 1,2)-Gal beta 1,4GlcNAc beta 1-, GalNAc alpha 1,6Gal beta 1-, Gal beta 1-, Gal beta 1,4GlcNAc- and Gal beta 1,3GalNAc alpha 1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with alpha 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and alpha 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.

Primary culture of cells from human chorion laeve: steroid metabolism and properties of cells grown in defined media supplemented with 0.1% or 10% fetal calf serum.

The purpose of this study was to develop primary cultures of human chorion laeve cells and examine certain aspects of steroid metabolism during culture. Tissues obtained by elective cesarean section at term (38-40 weeks) were dispersed with collagenase. Cells were isolated on Percoll gradients at the interface between 20% and 40% Percoll and examined in primary culture for up to 1 week. Cultures were carried out in chemically defined media supplemented with 10% or 0.1% fetal calf serum (FCS). The morphological and biochemical properties of the cells were different in the two systems. In 0.1% FCS, cells formed clumps of tissue within 16 h of plating, and there was no cell replication. In contrast, in 10% FCS, the cells formed a carpet of tissue and reached confluence after 5 days in culture, resulting in increased DNA and protein content and thymidine incorporation in the dishes. Three steroidogenic enzymes were studied during culture: alkyl steroid sulfatase, estrogen sulfatase and 3 beta-hydroxysteroid dehydrogenase. The sulfatases had higher activities in 0.1% than in 10% FCS, and their activities decreased markedly during the culture period. In contrast, 3 beta-hydroxysteroid dehydrogenase activity was higher in 10% FCS than in 0.1% FCS. Activity remained constant during the culture period in 0.1% FCS and increased in 10% FCS. In the latter system this increase resulted in the enzyme maintaining a constant specific activity during culture. These studies describe two viable systems of chorion laeve cells in primary culture, which may be valuable for studying long term and/or subtle effects on various metabolic aspects of this tissue.