Expression of connexins in human preimplantation embryos in vitro

Intercellular communication via gap junctions is required to coordinate developmental processes in the mammalian embryo. We have investigated if the connexin (Cx) isoforms known to form gap junctions in rodent preimplantation embryos are also expressed in human embryos, with the aim of identifying species differences in communication patterns in early development. Using a combination of polyA PCR and immunocytochemistry we have assessed the expression of Cx26, Cx31, Cx32, Cx40, Cx43 and Cx45 which are thought to be important in early rodent embryos. The results demonstrate that Cx31 and Cx43 are the main connexin isoforms expressed in human preimplantation embryos and that these isoforms are co-expressed in the blastocyst. Cx45 protein is expressed in the blastocyst but the protein may be translated from a generally low level of transcripts: which could only be detected in the PN to 4-cell embryos. Interestingly, Cx40, which is expressed by the extravillous trophoblast in the early human placenta, was not found to be expressed in the blastocyst trophectoderm from which this tissue develops. All of the connexin isoforms in human preimplantation embryos are also found in rodents pointing to a common regulation of these connexins in development of rodent and human early embryos and perhaps other species

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS) 2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.Peer reviewe

Generation of Trophoblast Stem Cells from Rabbit Embryonic Stem Cells with BMP4

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal

Ultrastrukturuntersuchungen an der Retina des Flußkrebses Astacus Leptodactylus mit Hilfe der Gefrierbruch - und Ultradünnschnitt-Methode

Ultrastructural changes of the microvilli of the Astacus retina depending on the state of light adaptation were investigated by electron microscopy of replicas of freeze fracture faces. (1) The particles on the plasmatic fracture face (PF) of the microvillus membrane, most of which being identical with rhodopsin, are distributed statistically in the state of dark adaptation. Upon light adaptation ( $\geq$ 24 h with 70 $\mu$W cm$^{-2}$ or $\geq$ 3 h with 1-2 mW cm$^{-2}$) the particles become orientated in certain areas in a helical structure of rows (periodicity of 11 nm) around the microvillus. As shown by sprayfreezed unf ixed rhabdoms of light adapted eyes of Astacus these structures are not artef acts caused by glutaraldehyde or glycerol. As the f ormation of these rows depends on duration and intensity of the adapting light it seems to be correlated with light-induced processes in the receptor. (2) Addition of cycloheximide prevents the formation of rows of the particles during light adaptation, which might suggest a relation with protein synthesis. (3) The structured areas could possibly represent rows of metarhodopsin molecules. (4) The packing density of the particles of dark adapted retinae is found to be as great as 5000 ± 600 per $\mu$m$^{2}$ membrane area , that of lightadapted retinae as is enriched in the great as 7000 $\pm$ 1400 $\mu$m$^{-2}$ , and structured areas to 11000 $\pm$ 1000 $\mu$m$^{-2}$. The mean diameter of the particles is 80 $\mathring{A}$. (5) Under high vacuum conditions (UHV) most of the particles of the microvillus membrane are 40-50 $\mathring{A}$ in diameter,which agrees with the theoretically expected value of rhodopsin. (6) Illumination with light of higher intensity causes, in addition to the normal light-inducedstructural changes of the photoreceptor membrane, the formation of myelin-like membrane complexes, which leads to the destruction of the photoreceptor