Long-term reproductive cycles in rams after pinealectomy or superior cervical ganglionectomy

Long-term cycles in diameter of the testes, colouration of the sexual skin and plasma concentrations of testosterone, FSH and prolactin were monitored in groups of pinealectomized (PINX), superior cervical ganglionectomized (SCGX), and control Soay rams living near Edinburgh (56 degrees N). In Exp. 1, PINX, SCGX and control rams were kept outside for 4 years, and well defined seasonal cycles in each of the reproductive parameters were evident in all 3 groups (e.g. testosterone cycle length assessed by sine-wave analysis: 12.08 +/- 0.17, 12.39 +/- 0.14 and 12.15 +/- 0.10 months for PINX, SCGX and control rams respectively). Qualitative differences, however, were apparent between the groups in the timing and amplitude of the reproductive cycle. The seasonal peak in reproductive function occurred from July to September in the PINX and SCGX rams, some 2 months earlier in the year than in controls, while the amplitude of the cycle was less marked in the PINX and SCGX rams. There were no significant differences between the experimental groups in the seasonal cycle in the plasma concentrations of prolactin. In Exp. 2, SCGX and control rams were kept indoors under an artificial environment with a 32-week light cycle and constant nutrition for 4 years. Compared to the controls, in which the reproductive changes were synchronized to the driving light cycle, the SCGX rams showed poorly defined reproductive cycles of lower amplitude and longer period (e.g. testosterone cycle length: 57.8 +/- 6.1 and 32.1 +/- 0.2 weeks for SCGX and control rams, respectively). There was evidence of a cycle in some of the reproductive parameters in the SCGX rams with a period close to 32 weeks during the second half of the study (e.g. testosterone cycle 32.4 +/- 0.8 weeks), which was taken to indicate social induction from the neighbouring control rams. In two further short-term experiments, SCGX rams showed a decline in testicular activity in response to receiving a restricted diet (60% of controls) and an increase in testosterone secretion in response to exposure to oestrous ewes. The overall results illustrate that PINX and SCGX rams can generate long-term synchronized cycles in pituitary and testicular activity. The animals are apparently unable to respond to changes in daylength due to the loss of the functional pineal gland but they remain competent to respond to other environmental cues such as changes in nutrition, temperature and social factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translational capacity of sheep oocytes microinjected with messenger RNA

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

In vitro pluripotency of epiblasts derived from bovine blastocysts

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Synthesis of intraspecific somatic hybrids of Solanum tuberosum: assessments of morphological, biochemical and nematode (Globodera pallida) resistance characteristics

In an attempt to produce novel agronomic traits, a number ofintraspecific somatic hybrid plants have been produced followingleaf mesophyll protoplast fusion between S. tuberosum dihaploidclones PDH 40 (possessing good tuber shape and yield) and PDH417 (possessing resistance to potato cyst nematode, G. pallida).PDH 417 protoplast-derived calli failed to regenerate plantsusing the described culture conditions preventing this parentaltype amongst the mass of regenerated fusion products. Initially,somatic hybrid plants were selected based on differential pigmentationin tuber sprouts and where possible on petal colour. Differentialmobility of patatin bands in electrophoresed tuber extractsfurther confinned hybridity. The intraspecific somatic hybridsalso showed different levels of resistance to G. pallida pathotypesPa2 and Pa3 in the somatic hybrid plants examined. Key words: Somatic hybridization, dihaploids, patatin, nematode resistance, Solanum tuberosum, potato     

Production and modifications of extracellular structures during development of chytridiomycetes

Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC Nomarski-differential contrast optics - TEM transmission electron microscopy     

Seed storage protein variation in Arachis species.

Seventy-two accessions, representing 22 species from sections Arachis, Erectoides, Extranervosae, and Triseminalae of the genus Arachis, were screened for seed storage protein polymorphism. Variation was detected between sections, between genome types, between species, and in some cases between different accessions of the same species or different seeds of the same accession. Arachis duranensis and one accession of A. cardenasii were found to have identical protein patterns. The greatest dissimilarity was found between species of the section Extranervosae and species of the section Triseminalae. Those of section Erectoides showed much similarity with some species of section Arachis. Protein polymorphism was shown to distinguish the two subspecies of A. hypogaea (fastigiata and hypogaea) in 27 of 28 cases. The seed protein profile of A. monticola was a combination of seed protein profiles from the two A. hypogaea subspecies. The relatedness between the various species was calculated and those that had the greatest similarity with A. hypogaea were A. spegazzinii and A. batizocoi.     

Acetate inhibition on growth of recombinant E. coli and expression of fusion protein TGFaα-PE40

Summary Acetate was inhibitory to the growth of early induced E. coli cells and their expression of fusion protein, transforming growth factor--Pseudomonas exotoxin 40 (TGF-PE40), but the inhibitory level was strain dependent For E. coli JM109 (pTAC-TGF57-PE40), 2 g/L of added acetate (3 g/L of total acetate in the medium) decreased TGFa-PE40 production by 38.0%. Acetate was less inhibitory to E. coli RR1, and RR1 was not affected by adding 2 g/L of acetate. However, 5 g/L of added acetate (6.7 g/L of total acetate in the medium) decreased TGF-PE40 production by 21.2%. These results indicate that higher acetate concentration was associated with inhibition of TGF-PE40 expression of E. coli JM109 during late induction.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.