In vitro fertilization with normal development in the sheep

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two-to six-cell stage within 40 h (75. 8% for ovulated and 62. 6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20–22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61. 9%) and 10 (66. 6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained ( > 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress ( > 3 months).     

Plasma levels of arginine vasotocin,prolactin, aldosterone and corticosterone during prolonged dehydration in the domestic flowl: effect of dietary NaCl

Three groups of White Plymouth Rock laying hens were adapted to three levels of dietary NaCl: low-NaCl food with tap water (LOW), high-NaCl food (1% NaCl w/w added) with tap water (HT), and high-NaCl food with 0.5% NaCl for drinking (HS). The birds were subjected to water deprivation (dehydration) for 18 days. Blood sampling was done at 2-4 day intervals. Plasma concentrations of arginine vasotocin (AVT), prolactin (PRL), aldosterone (ALDO) and corticosterone (CS) were determined by radioimmunoassay. Plasma osmolality, sodium, chloride, and potassium were also determined. In the normally hydrated hens fully adapted to the diets, there was a stepwise increase from LOW to HS in plasma osmolality (305, 315, 332 mOsm, for LOW, HT and HS, respectively), [Na+] (144, 153, 161 mM) and [Cl-] (109, 119, 127 mM) as well as in [AVT] (6, 14, 18 pg/ml) and [PRL] (16, 24, 34 ng/ml). Regressing [AVT] on osmolality gave a slope of 0.30 pg . ml-1/mOsm and a threshold of 273 mOsm. The slope of [PRL] on osmolality was 0.73 ng . ml-1/mOsm. The correlation coefficient of [AVT] and [PRL] was 0.67. LOW had high [ALDO] (165 pg/ml) which was suppressed to low levels in HT and HS (5-8 pg/ml), while [CS] was the same in all groups (0.9-1.1 ng/ml). Plasma [K+] was decreased in the high-NaCl groups (5.8 mM in LOW, 4.4 and 4.7 mM in HT and HS). Dehydration resulted within 2 days generally in a sharp (5-15%) increase in osmolality, [Na+] and [Cl-], which thereafter increased more slowly during the remaining 16 days in all groups, with the slowest increase in LOW. The levels of osmolality [Na+] and [Cl-] were 5% lower in LOW than in HT and HS, which showed the same levels during the dehydration period. Plasma [AVT] and [PRL] increased 2-4 fold within 2 days of dehydration; [AVT] reached a plateau at 29 pg/ml in all groups, but [PRL] continued to rise in all groups, fastest in LOW, reaching similar levels in all groups after 14-18 days of dehydration, about 85 ng/ml. The correlation coefficient of [AVT] and [PRL] was decreased by half (to 0.32) during dehydration. Plasma [ALDO] increased in all groups with dehydration, 1.7 fold in LOW and 3-6 fold in HT and HS, but the levels reached in HT and HS were only 15-30% of that seen in LOW.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat esophageal and epidermal keratinocytes: intrinsic differences in culture and derivation of continuous lines

Serially cultivated with 3T3 feeder layer support as colonies of stratified squamous epithelium, rat epidermal and esophageal epithelial cells were readily distinguishable by three criteria. First, the epidermal colonies, exhibiting extensive piling up of squames in the centers, were more stratified than esophageal colonies. Second, in sparse culture 70 to 90% of the esophageal cells but as few as 1 to 5% of the epidermal cells were competent in cross-linked envelope formation upon treatment with the ionophore X537A. After reaching confluence, up to 90% of the cells of both types formed envelopes upon ionophore treatment. Third, epidermal cells in suspension culture reached maximal levels of spontaneously cross-linked envelopes in 1 day or less, while esophageal cells required about 4 days in suspension to reach maximal levels. A reproducible finding with both cell types was that initial colony-forming efficiencies of less than 1% increased to about 40% upon serial passage with consequent derivation of continuous lines. Sparse cultures of esophageal cells with high colony-forming ability retained a high degree of envelope competence (70 to 90%), indicating these two properties are not mutually exclusive. The derived lines exhibited reduced dependence upon feeder layer support at clonal density, but in suspension culture the cells did not grow and lost colony-forming ability with a half-time of several hours. We conclude that cells from these keratinized rat epithelia exhibit intrinsic differences in culture and become continuous lines expressing characteristic regulation of envelope competence and loss of germinative capability in suspension.     

Exceptional characteristics of amino proton exchange in guanosine compounds

Amino 1H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethyl)-aminomethane, imidazole, 2-(N-morpholino)ethanesulfonic acid, glycine, or cacodylate, all shown to be effective buffer catalysts in adenosine and cytidine proton exchange. Line broadening, seen only with phosphate and acetate, is established by intermolecular interactions, as well as by amino to water proton exchange. This absence of buffer catalysis of exchange is accounted for by the relatively small implied effect of G(N-7) protonation on amino acidity, based on similar observations with 7-methylguanosine as a model for endocyclic protonation. The requirement for diffusion-controlled proton transfer in buffer catalysis is achieved by nucleobase protonation in adenine and cytosine, but not in guanine.     

Nitrogen fixation in grassland and associated cultivated ecosystems

Summary Nitrogen fixation in the natural, Agropyron-Koeleria grassland ecosystem was studied using the C₂H₂-C₂H₄ and N¹⁵ assays. Small soil samples and also undisturbed soil cores were used for analyses. Both techniques indicated that grassland and associated cultivated soils had low fixation rates (0.6–1.8 kg/ha per 28 days in the laboratory and, 1 kg/ha per season under actual field conditions). Algal colonies (Nostoc spp.) on the soil surface were active fixers when the surface of the grassland was moist. However, their small biomass limits the extent of fixation in most areas. In native grassland, 16 legumes bore nodules. The three most common speciesVicia americana, Thermopsis rhombifolia andOxytropis sericea, all of which had active nodules, contributed 10 per cent of the total nitrogenase activity. The non-legumesElaeagnus commutata andShepherdia argentea were profusely nodulated with active nodules, but were confined to specific habitats. No nodules were found onArtemisia orOpuntia spp. The major, heterotrophic, asymbiotic bacteria in the soil were clostridia. These utilize substrates produced by aerobic cellulose and hemicellulose degrading organisms to fix N in anaerobic microsites. The C₂H₂:N₂ reduction ratio was 3 to 1 in large, aerobic core samples, but was greater under water-logged conditions where high fixation rates occurred.     

The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1. The cytosol alcohol dehydrogenase (alcohol-NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO(2) (95:5) or of O(2)+CO(2) (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO(2) (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ;ADHI-air'. 4. In addition to fraction ADHII, cells grown in the presence of O(2) have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ;ADHI-O(2)', account for about 10% of the total activity. 5. The ADHI fractions (air) and (O(2)) have similar broad pH-activity curves and similar kinetic properties, both having a lower K(m) for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Variability amongThymelaea hirsuta (L.)Endl. populations in Egypt

The present study depicts the presence of a gradient in the morphological characters ofThymelaea hirsuta (L.)Endl. leaves which correlated with the environmental gradient prevailing in the Western Mediterranean region of Egypt. The less arid and more calcareous habitats harbour individuals with obtuse and gentle curved leaf apices and gentle involute leaf margins. With the increase of aridity and decrease of CaCO₃, the leaf apices become acute and strongly curved, and the leaf margins become strongly involuted. Significant variations in seed weight, seedling emergence and viability of seed embryos inT. hirsuta, in relation to habitat types, are also shown and discussed.