Chick embryo retina development in vitro: The effect of insulin

In this paper we study the development of chick embryo retina culturedin vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum-and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31–37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

Modification of Phospholipids with Lipases and Phospholipases

Different commercial lipases and phosphoiipases were studied in the hydrolysis and transesterification of synthetic phosphatidylcholine and soybean lecithin. Wide variations in the lipase and phospholipase activities and in the protein contents of the preparations were observed. The substrate specificity varied between different enzymes. A high degree of hydrolysis of synthetic and soybean phospholipids was achieved with both types of enzymes.

Enzymes immobilized on Celite were used in the transesterification of dimyristoyl phosphatidylcholine and oleic acid. The conversions were carried out both without solvent and in the presence of toluene. The amount of modified phosphatidylcholine was measured using HPLC. The highest amount of modified phosphatidylcholine was obtained in solvent-free transesterification. The best results were obtained with Aspergillus niyer lipase.     

Modification of Phospholipids with Lipases and Phospholipases

Different commercial lipases and phosphoiipases were studied in the hydrolysis and transesterification of synthetic phosphatidylcholine and soybean lecithin. Wide variations in the lipase and phospholipase activities and in the protein contents of the preparations were observed. The substrate specificity varied between different enzymes. A high degree of hydrolysis of synthetic and soybean phospholipids was achieved with both types of enzymes.

Enzymes immobilized on Celite were used in the transesterification of dimyristoyl phosphatidylcholine and oleic acid. The conversions were carried out both without solvent and in the presence of toluene. The amount of modified phosphatidylcholine was measured using HPLC. The highest amount of modified phosphatidylcholine was obtained in solvent-free transesterification. The best results were obtained with Aspergillus niyer lipase.     

Evaluation of reproductive barriers and realisation of interspecific hybridisations depending on genetic distances between species in the genus Helleborus

The genus Helleborus comprises 22 species, which are allocated to six sections. H. x hybridus and H. niger, which belong to different Helleborus sections, are economically important ornamentals. Several other species with minor impact exhibit interesting features, e.g. flower size, flower colour, foliage, scent and disease resistance, which should be introgressed into H. x hybridus or H. niger through interspecific hybridisation. The aims of this study were to investigate whether and which kind of hybridisation barriers occur in crosses between Helleborus species and if they differ in their manifestations, depending on the genetic distance of the respective partners. In order to obtain interspecific hybrids despite crossing barriers, a method to overcome these barriers should be developed. Crossing barriers in Helleborus were localised as predominantly post‐zygotic according to in situ pollen tube staining with aniline blue. For certain crosses, pre‐zygotic barriers could also be assumed, but pollen tube growth was not totally inhibited. Therefore, embryo rescue techniques via ovule culture were established to overcome the post‐zygotic barriers. Ovules were isolated from maternal plants 5–7 weeks after pollination in most cases and then cultured in vitro. Overall, 219 hybrids were successfully obtained, of which 16 were derived from inter‐sectional crosses. Hybrids were verified by flow cytometry and/or by molecular DNA markers.     

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1–5 mM), or NO donor (DETA NONOate 10–100 M). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 M had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 M decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.     

Citicoline: neuroprotective mechanisms in cerebral ischemia.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.     

A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P₄/E₂) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P₄/E₂ ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.