Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst

Mammalian pre-implantation development culminates in the formation of the blastocyst consisting of two distinct cell lineages, approximately a third of the cells comprise the pluripotent inner cell mass (ICM) and the remainder the differentiated trophectoderm (TE). However, the contribution made by these two cell types to the overall energy metabolism of the intact blastocyst has received relatively little attention. In this study, the metabolism of the intact mouse blastocyst and isolated ICMs were determined in terms of total ATP formation (calculated from oxygen consumption and lactate formation), mitochondrial distribution and amino acid turnover to provide an indication of protein synthesis. The TE consumed significantly more oxygen, produced more ATP and contained a greater number of mitochondria than the ICM. Amino acid turnover was significantly greater (p<0.001) in the TE compared with the ICM. Specifically, there was a significant difference in the utilization of aspartate (p=0.020), glutamate (p=0.024), methionine (p=0.037), and serine (p=0.041) between the cells of the ICM and TE. These data suggest that the TE produces approximately 80% of the ATP generated and is responsible for 90% of amino acid turnover compared with the ICM. The major fate of the energy produced by the TE is likely to be the Na(+), K(+)ATPase (sodium pump enzyme) located on the TE basolateral membrane. In conclusion, the pluripotent cells of the ICM display a relatively quiescent metabolism in comparison with that of the TE.     

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis)

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco© (ES, INVE) or Aquagrow Gold© (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparβ), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.     

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20β-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 μg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.     

Substantial loss of genetic variation in a single generation of Senegalese sole (Solea senegalensis) culture: implications in the domestication process

The flatfish Solea senegalensis represents an important resource in modern mariculture and is largely raised in South Spain and Portugal. Substantial progress has been achieved in its domestication, though suitable reproduction and zootechnical conditions still remain unknown. Difficulties to obtain breeders from wild lead many companies to set up broodstocks with first generation (G1) progeny without genetic guidelines. To study the genetic processes underlying the early stage of domestication of this species, the genetic structure of four representative broodstocks from Southern Spain has been assessed by means of eight microsatellite loci . Data revealed a substantial reduction in levels of genetic variability on just one generation in stocks totally or partially composed of G1 individuals when compared with those solely integrated by individuals from wild. The genetic relatedness within the four stocks has been established, having detected close relationships between individuals from cultured origin, thus suggesting that the loss of variability is apparently due to setting up broodstocks by blind selecting of family related G1 individuals. The high proportion of siblings in these stocks can have negative consequences in future generations due to inbreeding effects. The relationships between the four broodstocks were traced, having found a common origin between two of them, which come from the same donor hatchery, thus enhancing the homogenization of the existent resources for S. senegalensis among the various stocks. This finding alerts about the risks in exchanging fish between hatcheries without knowing their genetic origin. From this study, the use of microsatellites is strongly recommended to control the genetic composition of S. senegalensis broodstocks, aimed to maintain standards of genetic health and improve their reproduction capacity, which is a key issue in the domestication process.     

Melatonin Binding Sites in Senegal Sole: Day/Night Changes in Density and Location in Different Regions of the Brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B_max) at mid‐light (ZT06) and mid‐dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2‐[¹²⁵I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid‐light (189.5±46 pg/ml) than mid‐dark (455.5±163 pg/ml). Values of B_max were statistically significantly higher in the optic tectum (5.6±0.6 and 12.3±1 fmol/mg prot, at mid‐light and mid‐dark, respectively) and in the cerebellum (7.7±1.1 and 10.6±1.3 fmol/mg prot, at mid‐light and mid‐dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down‐regulation.     

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.     

Influence of Constant Light and Darkness,Light Intensity,and Light Spectrum on Plasma Melatonin Rhythms in Senegal Sole

Light is the most important synchronizer of melatonin rhythms in fish. This paper studies the influence of the characteristics of light on plasma melatonin rhythms in sole. The results revealed that under long‐term exposure to constant light conditions (LL or DD), the total 24 h melatonin production was significantly higher than under LD, but LL and DD conditions influenced the rhythms differently. Under LL, melatonin remained at around 224 pg/ml throughout the 24 h, while under DD a significant elevation (363.6 pg/ml) was observed around the subjective evening. Exposure to 1 h light pulses at MD (mid‐dark) inhibited melatonin production depending on light intensity (3.3, 5.3, 10.3, and 51.9 µW/cm²). The light threshold required to reduce nocturnal plasma melatonin to ML (mid‐light) values was 5.3 µW/cm². Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (λ>600 nm) failed to reduce plasma melatonin significantly, far violet light (λ_max=368 nm) decreased indoleamine's concentration to ML values. These results suggest that dim light at night (e.g., moonlight) may be perceived and hence affect melatonin rhythms, encouraging synchronization to the lunar cycle. On the other hand, deep red light does not seem to inhibit nocturnal melatonin production, and so it may be used safely during sampling at night.     

Biochemical composition of cuttlefish (Sepia esculenta) eggs during embryonic development

Changes in egg volume, water content, proteins, carbohydrates, lipids, amino acids and fatty acids in cuttlefish (Sepia esculenta) were determined during embryogenesis to understand the nutritional requirements in the early life phase. The egg volume and the water content decreased significantly (P<0.05) during early embryonic development, and then increased abruptly after the beginning of organ differentiation. During embryonic development, protein was the major content in the yolk and the per cent composition varied to a large extent (55.19–78.45%) with carbohydrates (9.86–15.56%) and lipids (1.62–2.97%). Proteins and lipids decreased 23.3% and 0.4%, respectively, in dry weight, while carbohydrates increased 0.46%. Total amino acids and essential amino acids (EAAs) were stable during the early embryonic developmental stage, but decreased significantly until the eggs hatched (P<0.05). The largest utilisation of the yolk protein possibly occurred with respect to EAAs (25.7%) because of a decrease in methionine (70.3%), valine (63.0%) and phenylalanine (58.5%). The most important fatty acids were saturated fatty acid (SFA) C16:0 and polyunsaturated fatty acids (PUFAs) C22:6 and C20:5. Unsaturated fatty acids (UFAs) and SFA decreased at a similar rate during embryonic development (5.69% and 6.15%, respectively). For UFAs, monounsaturated fatty acids were consumed at a greater rate than PUFAs (29.6% and 0.05%, respectively).     

大熊猫能量和蛋白质营养需要研究进展及策略

本文在总结和分析大熊猫食性、食物选择、摄食量、组织器官和竹子中养分含量测定、养分消化代谢等研究数据的基础上,推测出大熊猫干物质摄入量、总能、消化能、粗蛋白和可消化粗蛋白需要量以及精料中赖氨酸的推荐水平。结合圈养大熊猫单位的饲喂方式和饲喂效果,该研究策略有助于确定圈养条件下大熊猫饲料的精粗比例、精料养分浓度和各养分之间的比例,为圈养大熊猫饲养标准的确立奠定基础。     

Seasonal distribution and duration of the planktonic stage of Dover sole, Solea solea, larvae in the Bay of Biscay: an hypothesis

The possibility of deriving a prediction about the effect of seasonal variations in the duration of the planktonic larval phase on the dispersal of larval Dover sole was investigated. During six cruises, from February to May 1992, the distribution of sole larvae was studied along a 100-km transect, from the offshore spawning grounds to the coastal nurseries of the Bay of Biscay (France). Samples ( n = 189) were collected with a suprabenthic sampler, and vertical profiles of water temperature and salinity were recorded simultaneously. Counts of otolith increments of larval stage 4b (onset of metamorphosis) were used to estimate the duration of the planktonic life. Duration of the larval phase decreases by about 15 days (37%) with water temperature increase (between 8° in February and 11.2° C in May). Sole larvae occur from the coastal area to 100 km offshore. Within the same cruise, no difference in the duration of the planktonic life was observed between the larvae caught in the onshore and the offshore area. In spite of seasonal differences in abundance, the extent and the shape of the larval distributions during the period of study suggest that the seasonal variations in the duration of the planktonic life did not affect the larval distribution.     

Daily Rhythms in the Hypothalamus-Pituitary-Interrenal Axis and Acute Stress Responses in a Teleost Flatfish,Solea senegalensis

The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p?<?.05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT]?=?14.5 and 14.4?h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT?=?1.2, 2.4, and 3.4?h, respectively), and crhbp and lactate at the end of the dark phase (ZT?=?22.3 and 23.0?h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2?±?10.7?ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6?±?11.1?ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture. (Author correspondence: jflopez@um.es)     

Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses

The effects of two metabolic inhibitors, methyl palmoxirate (MP) and amino-oxyacetate (AOA), on mouse preimplantation embryo development and cell number, and inner cell mass (ICM) cell metabolism have been examined. Two-cell embryos were cultured in media supplemented with either MP, which inhibits fatty acid oxidation, or AOA, which inhibits the transamination of glutamate into α-ketoglutarate. Embryos were scored for development daily. On day 5, expanded blastocysts were differentially labeled with fluorochromes to visualize TE and ICM cell nuclei, or the ICMs isolated by immunosurgery and their energy metabolism determined using microfluorometric methods. Embryos exposed to the two inhibitors developed into fully expanded blastocysts, although cell numbers of both the TE and ICM cells were significantly reduced compared to controls. The uptake of glucose in the presence of 1 mM MP or AOA did not differ from the controls, but less glucose was accountable for by lactate production. MP significantly reduced lactate production. In the presence of 4 mM AOA, the amount of glucose oxidized and the amount of lactate formed by ICMs were significantly reduced. The results indicate that the fuels used by isolated mouse ICMs vary in response to substrate availability and that fatty acids may be a potential energy source. © 1996 Wiley-Liss, Inc.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Is digestive capacity limiting growth at low temperatures in roach?

In roach Rutilus rutilus growth ceases below a temperature threshold of 12° C. This cessation of growth is accompanied by a reduction in feeding. Do roach decrease feeding in the cold because of reduced energy demand, caused by the decelerating effect of low temperature on metabolism and growth, or is feeding directly limited by low temperatures, leading to reduced growth rates? It was found that at low temperatures the intake and digestion of food may be limited by reduced activities of digestive enzymes. Trypsin, amylase and γ‐glutamyl transferase showed a negative compensation with respect to temperature, resulting in very low activities at acclimation temperatures of ≤12° C. Trypsin activity, falling from 400·5 ± 131·2 U g^?1 fresh mass of the gut at 27° C to 12·5 U g^?1 fresh mass at 4° C, displayed the strongest linear correlation with growth rates, suggesting that trypsin activities may set a limit to growth in the low temperature range. If protein digestion is limiting at low temperatures, this should be reflected in reduced concentrations of amino acid in the white muscle. The size of the total amino acid pool was not affected by temperature acclimation and ranged between 19·2 ± 6·2 and 25·2 ± 3·6 µmol g^?1 fresh mass of the white muscle. A decrease, however, was found of several amino acids, mainly of threonine and glutamine, in the low temperature range. Low concentrations of the essential amino acid threonine (0·14 ± 0·03 µmol g^?1 fresh mass at 12° C and 0·12 ± 0·05 µmol g^?1 fresh mass at 4° C) were probably due to nutritional or digestional limitations and may therefore have resulted from reduced trypsin activity in the cold. The non‐essential amino acid glutamine, however, can be endogenously synthesized and its low level observed at 4° C (0·16 ± 0·09 µmol g^?1 fresh mass) was not necessarily a result of low trypsin activities. It is more likely that low temperatures impair glutamine synthesis. The possibility that glutamine concentrations may be down regulated under conditions when anabolic processes are not advantageous is discussed.     

支链氨基酸对运动大鼠氨基酸代谢和运动能力的影响

观察了支链氨基酸(BCAA)对大鼠运动能力和血清游离氨基酸代谢的影响。实验用21只雄性wistar大鼠,随机分为3组:正常组、游泳对照组和游泳补充BCAA组。2个运动组每天游泳训练1h,10天后游泳6h,观察补充BCAA对大鼠游泳运动能力和血清游离氨基酸水平的影响。实验结果表明,补充BCAA可明显提高大鼠游泳存活率,抑制血清中必需氨基酸、非必需氨基酸和总氨基酸水平升高,游泳运动后大鼠的血清中乳酸和LDH的升高幅度有所降低,抑制骨骼肌LDH活力的下降。说明补充BCAA可明显提高大鼠的运动能力,减少运动造成的蛋白质分解     

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO₂ and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Summary Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and ¹H NMR analysis of peptides, synthesised by this method, were satisfactory.