Expressão de genes em sementes de lobeira (Solanum Lycocarpum St Hill) submetidas ao condicionamento fisiológico

Pós-graduação em Agronomia (Agricultura) - FCA

Seawater carbonate chemistry and trace element accumulation during experiments with common cuttlefish, Sepia officinalis, 2009

Cephalopods play a key role in many marine trophic networks and constitute alternative fisheries resources, especially given the ongoing decline in finfish stocks. Along the European coast, the eggs of the cuttlefish Sepia officinalis are characterized by an increasing permeability of the eggshell during development, which leads to selective accumulation of essential and non-essential elements in the embryo. Temperature and pH are two critical factors that affect the metabolism of marine organisms in the coastal shallow waters. In this study, we investigated the effects of pH and temperature through a crossed (3?2; pH 8.1 (pCO2, 400 ppm), 7.85 (900 ppm) and 7.6 (1400 ppm) at 16 and 19°C, respectively) laboratory experiment. Seawater pH showed a strong effect on the egg weight and non-significant impact on the weight of hatchlings at the end of development implying an egg swelling process and embryo growth disturbances. The lower the seawater pH, the more 110 mAg was accumulated in the tissues of hatchlings. The 109Cd concentration factor (CF) decreased with decreasing pH and 65Zn CF reached maximal values pH 7.85, independently of temperature. Our results suggest that pH and temperature affected both the permeability properties of the eggshell and embryonic metabolism. To the best of our knowledge, this is one of the first studies on the consequences of ocean acidification and ocean warming on metal uptake in marine organisms, and our results indicate the need to further evaluate the likely ecotoxicological impact of the global change on the early-life stages of the cuttlefish.

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice

Background: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. Methods: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. Results: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. Conclusions: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo

Morphogenesis occurs in 3D space over time and is guided by coordinated gene expression programs. Here we use postembryonic development in Arabidopsis plants to investigate the genetic control of growth. We demonstrate that gene expression driving the production of the growth-stimulating hormone gibberellic acid and downstream growth factors is first induced within the radicle tip of the embryo. The center of cell expansion is, however, spatially displaced from the center of gene expression. Because the rapidly growing cells have very different geometry from that of those at the tip, we hypothesized that mechanical factors may contribute to this growth displacement. To this end we developed 3D finite-element method models of growing custom-designed digital embryos at cellular resolution. We used this framework to conceptualize how cell size, shape, and topology influence tissue growth and to explore the interplay of geometrical and genetic inputs into growth distribution. Our simulations showed that mechanical constraints are sufficient to explain the disconnect between the experimentally observed spatiotemporal patterns of gene expression and early postembryonic growth. The center of cell expansion is the position where genetic and mechanical facilitators of growth converge. We have thus uncovered a mechanism whereby 3D cellular geometry helps direct where genetically specified growth takes place.

The vegetable system : or, a series of experiments, and observations tending to explain the internal structure, and the life of plants; their growth, and propagation; the number, proportion, and desposition of their constituent parts; with the true course of their juices; the formation of the embryo, the construction of the seed, and the encrease from that state to perfection /

Vol. 1 is dated 1759, vols. 2-3 1761, vol. 4 1762, and vol. 5 1763.

The Growth of Muscle Cell of Inbred Chicken and Indigeneous Chicken Embryo in The Medium of Rabbit Serum and Sheep Serum

An experiment on the growth embryonic muscle cell in the rabbit and sheep serum media was conducted in the Biotechnology Laboratory of Gadjah Mada University, Yogyakarta. The aim of this experiment was to observe the potency of the growth of embryonic muscle cell of the inbred chicken and indigeneous chicken in the medium of rabbit and sheep serum. Two kinds of embryo, the inbred and indigeneous chicken of eleven days old were used in the experiment. The rabbit and the sheep serum were prepared in the laboratory. The experiment was conducted by applying Nested Classification with basic Complete Randomized Design (CRD). Data collected was analyzed using analysis of variance and also using a proliferation index formula. Samples used in those research were the inner and outer cell nucleus after fourty eight hours of the growth. The result of the experiment indicated that the index of proliferation of embryonic muscle cell of the inbred chicken in the rabbit and sheep serum were 89.65 and 84.92 percent respectively. Whereas, the proliferation index of embryonic muscle cell of the indigeneous chicken in the rabbit and sheep serum were 86.20 and 84.82 percent respectively. The total of inner muscle cell nuclei of inbred chicken embryos was significantly higher (P<0.01) than those of indigeneous chicken embryos either in the rabbit or sheep serum, but there was no difference between the serum (P>0.05). inconclusion the muscle cell of inbred and indigeneous chicken embryos could growth in both serum but the growth muscle cell of inbred chicken embryo was better than that of indigeneous chicken embryo. (Animal Production 2(2): 75-82 (2000) Key words : tissue culture, chicken embryos, index proliferation, serum.

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.

On the growth and form of the gut.

The developing vertebrate gut tube forms a reproducible looped pattern as it grows into the body cavity. Here we use developmental experiments to eliminate alternative models and show that gut looping morphogenesis is driven by the homogeneous and isotropic forces that arise from the relative growth between the gut tube and the anchoring dorsal mesenteric sheet, tissues that grow at different rates. A simple physical mimic, using a differentially strained composite of a pliable rubber tube and a soft latex sheet is consistent with this mechanism and produces similar patterns. We devise a mathematical theory and a computational model for the number, size and shape of intestinal loops based solely on the measurable geometry, elasticity and relative growth of the tissues. The predictions of our theory are quantitatively consistent with observations of intestinal loops at different stages of development in the chick embryo. Our model also accounts for the qualitative and quantitative variation in the distinct gut looping patterns seen in a variety of species including quail, finch and mouse, illuminating how the simple macroscopic mechanics of differential growth drives the morphology of the developing gut.