"In vitro" capacitation and further progesterone-induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

Diabetic Retinopathy Is Associated with Decreased Tyrosine Nitrosylation of Vitreous Interleukins IL-1α, IL-1ß, and IL-7.

OBJECTIVE: To simultaneously evaluate tyrosine nitrosylation and phosphorylation levels of vitreous interleukins of patients with diabetic retinopathy, in which abnormal tyrosine phosphorylation has been previously described. RESEARCH DESIGN AND METHODS: Specific immunoprecipitation of interleukins IL-1α, IL-1ß, IL-2 and IL-7 was carried out in samples obtained during vitrectomy performed for proliferative diabetic retinopathy in patients (n=12) and for macular hole in controls (n=12). Tyrosine nitrosylation and phosphorylation levels of the immunoprecipitated interleukins were analysed by Western blot with the respective specific antibodies and correlated. The results were also correlated with the total amount of immunoprecipitated interleukin protein. The mean phosphorylation/nitrosylation ratios of these proteins in vitreous humour of both the control group and diabetic patients were determined. RESULTS: Diabetes was associated with decreased tyrosine nitrosylation of IL-1α, IL-1ß and IL-7 and an increased tyrosine phosphorylation/nitrosylation ratio with respect to controls in IL-1α (1.58±0.22 vs. 2.74±0.39, respectively; p<0.05) and IL-7 (2.15±0.01 vs. 3.26±0.57, respectively; p<0.05). No significant changes were observed in nitrotyrosine or in the tyrosine phosphorylation/nitrosylation ratio of IL-2. CONCLUSIONS: Proliferative diabetic retinopathy is associated with concomitant and simultaneous changes in both tyrosine phosphorylation and tyrosine nitrosylation status of specific pro-inflammatory interleukins present in the vitreous fluid such as IL-1α, IL-1ß and IL-7. These changes could be related to the increase in pro-inflammatory activity detected in diabetes-induced retinopathy.

Specific expression pattern of tissue cytokines analyzed through the Surface Acoustic Wave technique is associated with age-related spontaneous benign prostatic hyperplasia in rats.

The aim of the study reported herein was to evaluate the suitability of the Surface Acoustic Wave (SAW) technique as a possible diagnostic tool in benign prostatic hyperplasia (BPH). Moreover, for the first time, the BPH model was a totally physiological using naturally aged rats with spontaneous, age-related BPH instead of the pharmacologically induced models usually used. Eighteen male Wistar rats were distributed according to their age: 6 weeks (young), 12 weeks (adult) and 12 months (old) old. Prostate gland was removed and analyzed by mini-arrays, Western blotting (WB) and SAW techniques. Mini-arrays indicated that there were significant differences in the expression of 29/34 inflammation-related cytokines. WB was carried out to confirm the results after selection of 4 cytokines from which one showed no changes, namely PDGF-AA, and the other three, which significantly increase in older animals, were CD86, ß-NGF and VEGF. Notwithstanding, WB of old rats yielded confusing results due to an anomalous migration of proteins, dismissing this technique as an useful tool in these animals. Accurate results in old rats were uniquely obtained by using the SAW technique. Thus, SAW analysis showed that there were not differences among groups in the amount of PDGF-AA. On the contrary, SAW analysis showed that amounts of CD86, ß-NGF and VEGF in old rats were 2.0, 1.9 and 5.7-fold higher than that from young ones, respectively. These results indicate that SAW is a highly accurate technique for determining changes in the cytokines expression in BPH.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels.

This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone-induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation-associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5 µm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4 h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on in vitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1 µm melatonin and used for in vitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of in vitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix.

Zonal distribution of glycogen synthesis in isolated rat hepatocytes.

Incubation of hepatocytes isolated from fasted rats with [14C]glucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with [14C]glucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of [14C]glucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.

The presence of a high-Km hexokinase activity in dog, but not in boar, sperm.

The presence of a high-Km hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog-sperm glucokinase-like protein, DSGLP), in the range of glucose concentrations of 4-10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog-sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat-liver anti-glucokinase antibody. This protein also had a molecular weight equal to that observed in rat-liver extracts, suggesting a close similarity between both the proteins. This glucokinase-like protein was distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high-Km hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog-sperm function along its life-time.

Control of glycogen deposition.

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.

Glucose and fructose have sugar-specific effects in both liver and skeletal muscle in vivo: a role for liver fructokinase.

We examined glucose and fructose effects on serine phosphorylation levels of a range of proteins in rat liver and muscle cells. For this, healthy adult rats were subjected to either oral glucose or fructose loads. A mini-array system was utilized to determine serine phosphorylation levels of liver and skeletal muscle proteins. A glucose oral load of 125 mg/100 g body weight (G 1/2) did not induce changes in phosphorylated serines of the proteins studied. Loading with 250 mg/100 g body weight of fructose (Fr), which induced similar glycemia levels as G 1/2, significantly increased serine phosphorylation of liver cyclin D3, PI3 kinase/p85, ERK-2, PTP2 and clusterin. The G 1/2 increased serine levels of the skeletal muscle proteins cyclin H, Cdk2, IRAK, total PKC, PTP1B, c-Raf 1, Ras and the ß-subunit of the insulin receptor. The Fr induced a significant increase only in muscle serine phosphorylation of PI3 kinase/p85. The incubation of isolated rat hepatocytes with 10 mM glucose for 5 min significantly increased serine phosphorylation of 31 proteins. In contrast, incubation with 10 mM fructose produced less intense effects. Incubation with 10 mM glucose plus 75 µM fructose counteracted the effects of the incubation with glucose alone, except those on Raf-1 and Ras. Less marked effects were detected in cultured muscle cells incubated with 10 mM glucose or 10 mM glucose plus 75 µM fructose. Our results suggest that glucose and fructose act as specific functional modulators through a general mechanism that involves liver-generated signals, like micromolar fructosemia, which would inform peripheral tissues of the presence of either glucose- or fructose-derived metabolites.

Lithium ions increase hepatic glycogen synthase stability through a proteasome-related mechanism.

Incubation of rat hepatocytes with LiCl resulted in an overall increase in the activity ratio of glycogen synthase (GS), concomitantly with a decrease in active GS kinase-3 levels. GS total activity was also increased in a dose- and time-dependent manner. This latter effect correlated with the amount of immunoreactive enzyme determined by immunoblotting. Cycloheximide and actinomycin-D did not modify LiCl action on GS activity. Lithium ions did not induce any changes in GS mRNA levels. Furthermore, the increase in the total amount of GS induced by LiCl was further augmented after addition of a specific, calpain and proteasome inhibitor. Our results indicate that LiCl increases hepatocyte GS activity through increasing both the activation state of the enzyme and its cellular content. This latter increase is mediated through a modification of the proteasome-regulated proteolytic pathway of the enzyme.

Glucose- and fructose-induced dog-sperm glycogen synthesis shows specific changes in the location of the sperm glycogen deposition.

Immunocytochemistry of glycogen deposition in dog sperm from fresh ejaculates showed that 53.7% of the population had glycogen at the midpiece, whereas 27.3% of the whole population accumulated glycogen only in the post-acrosomal region of the head. A similar distribution was observed when glycogen synthase, the enzyme which controls glycogen deposition, was studied. Incubation of spermatozoa with fructose increases the intracellular glycogen levels in a time- and concentration-dependent manner. Moreover, after incubation with 2 mM fructose for 30 min, 47.3% of the spermatozoa had glycogen located in both the midpiece and post-acrosomal zone, 25.4% of the population showed the polysaccharide only in the midpiece, and 24.7% of the spermatozoa showed a uniform distribution of glycogen all over the cell. Similar results were observed after incubation with 10-mM fructose. The distribution of glycogen synthase followed a similar pattern to that of glycogen. Incubation with glucose also induced a time- and concentration-dependent increase of glycogen content, whereas incubation with 2 mM glucose for 30 min showed that the majority of the population (81.2%) had the glycogen distributed throughout either the midpiece or the midpiece and the post-acrosomal zones together. There were practically no cells with a uniform glycogen distribution. Similar results were obtained after incubation with 10-mM glucose, whereas glycogen synthase suffered a similar glucose-induced distribution change. These results indicate that dog-sperm glycogen metabolism is modulated by changes in the activity of their controlling enzymes and also by changes in the specific location places where glycogen synthesis is produced.

Glucose 6-phosphate produced by gluconeogenesis and by glucokinase is equally effective in activating hepatic glycogen synthase.

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

Expression of a green fluorescence protein-carrier protein into mouse spermatozoa.

Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7days to, at least, 16days after inoculation. The GFP/LGS was detected in the sperm heads after an "in vitro" fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.

Evolución del estado nutricional en estudiantes de Secundaria Obligatoria a lo largo de esta etapa educativa / No disponible

Objetivos: Estudiar la evolución en el sobrepeso y/u obesidadentre estudiantes de secundaria a lo largo de todoesta etapa educativa.Métodos: Se ha calculado el IMC (Índice de masa corporal)del grupo experimental al empezar (1º) y finalizar(4º) sus estudios de enseñanza secundaria. Se ha estudiadola evolución en el porcentaje de sobrepeso y/u obesidadasí como de ciertos hábitos alimentarios.Resultados: Los resultados muestran una ligera disminuciónen el porcentaje de IMC normal a lo largo de laetapa educativa. Esta evolución dista mucho de ser aceptableporque puede comportar, por parte de nuestros jóvenes,un incremento de la obesidad cuando lleguen a laedad adulta.Conclusión: Hemos de poner al alcance de nuestros jóveneslos medios necesarios para corregir los resultadosde este estudio. Ello nos lleva a pedir a las administracioneseducativas un esfuerzo para informar y educar en elcampo de la nutrición y la alimentación a los niños yadolescentes de hoy(AU)

Background: The objective of this paper is to analyze theevolution of overweight and obesity on secondary schoolstudents among these 4 years of education.Methods: Body mass index (BMI) was calculated in apart of school children when they started (1st) and finished(4th) theirs secondary studies. Overweight and obesityevolution and some dietary habits were also studied.Results: Results showed a slow decreasing in normalBMI during secondary school. This evolution is notacceptable because it can lead to young people to increaseobesity group when they will arrive to adulthood.Conclusion: There is a clear necessity of explainingnutritional concepts to improve secondary student'sbehaviour. We demand an effort to educational authoritiesto adding food knowledge (food and nutritional sciences)to young people(AU)