Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality.

The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion(-1)) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = -0.42, respectively; P

Protein expression in the tissues of the cardiovascular system of the rat under selenium deficiency and adequate conditions.

We investigate the effects of selenium in tissues of the cardiovascular system. In order to examine the expression of the heart and aorta proteins (part of them) in the homogenate of selenium-deficient (Se(-)) and selenium control rats (Se(+)), two-dimensional electrophoresis was used by means of giga gels (30 x 35 cm). After electrophoresis, the protein expression pattern of the (Se(-)) gel and (Se(+)) gel was compared. The evaluation of the protein difference was implemented by means of a computer program suitable for the analysis of protein separated by the two-dimensional electrophoresis. In this way more than 2000 proteins a gel (heart) were detected and more than 1900 protein spots were detected in the aorta fraction. Ten significant differences were found between the gel of (Se(+)) and (Se(-)) heart homogenate of the rat and more than 15 significant differences between the gel of (Se(+)) and (Se(-)) of the aorta. By means of MALDI-MS-ESI-MS some of these proteins with different expression levels were not determined until now. Of those, three proteins were detected as the alpha myosin heavy chain (alpha-MHC), myosin light chain 1 and 2 (MLC 1 and 2), and the mitochondrial enzyme creatinine kinase. First results suggest that selenium deficiency affects myocardial energy metabolism and contractile proteins.

Metal-containing proteins in the apoptosis and redox processes in the rat prostate and human prostate cells.

Several trace elements, such as Se, Cu, Mn, and Zn are bound to proteins (metallo- and metalloidproteins) in the prostate gland. Currently, it is known that some of those elements play a role in the apoptosis of different cells and redox processes. For the detection of such proteins, analytical and biochemical procedures were combined. SEC and ICP-MS were used to detect some trace elements, which are bound to proteins in the prostate cytosol and/or in the human prostate cell lines. Several antibodies against specific proteins were tested. By means of some of these antibodies several trace element-containing proteins, such as selenoproteins and Cu- and Cu-Zn-proteins, could be identified in the prostate. In addition, the localization of such metal- and metalloid-containing proteins in the micro organelles and cytosol of the prostate indicates specific functions of these proteins because, as it is known, such metal- and metalloid proteins play a role in the apoptosis and especially in the redox processes.

Prevention against oxidative stress of eukaryotic cell membranes by selenium compounds of the rat.

By a combination of trace techniques and various biochemical methods, information about the characteristics of a 15-kDa selenoprotein was obtained. After labeling of rats in vivo with [(75)Se]selenite, subcellular fractionation of the homogenates of the prostate, lung, brain, thyroid gland, and large intestine, and gel electrophoretic separation of the proteins and subcellular fractionation, 15-kDa (75)Se was found in the cytosols of the tissues prostate > brain > lung > thyroid gland > large intestine after autoradiography. After coelectrophoresis of the separated 15-kDa labeled band obtained from each cytosolic fraction, the 15-kDa (75)Se band migrated in the same way as the combined bands isolated from the five tissue cytosols. After proteolytic cleavage in the gel of the 15-kDa labeled band obtained from the cytosol of each tissue and re-electrophoresis, the same labeled peptide pattern was found in each gel slice after autoradiography. By means of reversed-phase HPLC, we characterized a selenocysteine-containing protein that has enzymatic activity like that of glutathione peroxidase.

Detection and characterization of membrane-bound selenoproteins in the kidney of the rat.

The essential trace element selenium is of fundamental importance to human biology. Information on the membrane-bound selenoproteins was obtained by labeling of rats in vivo with (75)Se-selenite, subcellular fractionation of the tissue homogenates, separation of the proteins by SDS-PAGE, two-dimensional electrophoresis, and selenium detection by autoradiography. In this way, in addition to already known membrane-bound selenoproteins three novel selenoproteins in the molecular mass range of 38-40, 18, and 12-13 kDa were detected in the microsomal membranes.

Consequences of long-term selenium-deficient diet on the prostacyclin and thromboxane release from rat aorta.

It is known that peroxides, which are increased during Se deficiency because of reduced glutathione peroxidase (GSH-Px) activity, can influence the prostacyclin I2/thromboxane A2 (PGI2/TXA2) ratio. In this study we analyzed the PGI2 and TXA2 formation of aortas of long-term Se-deficient rats. Despite low GSH-Px activity in the Se-deficient group, the basal PGI2 and TXA2 formation was not different versus control animals (PGI2: 2295+/-1134 pg/mg vs 2940+/-1134 pg/mg; TXA2: 3.83+/-1.06 pg/mg vs 5.67+/-2.99 pg/mg). However, we checked the capacity of the aortas of Se-deficient rats to compensate for a suddenly increased peroxide concentration. After peroxide stimulation, the PGI2 release was significantly lower in the Se-deficient group compared to the control group (PGI2: 3507+/-1829 pg/mg vs 7986+/-2636 pg/mg). Again, the TXA2 release did not show any differences. The release ratio of PGI2/TXA2 decreased under peroxide stress in Se-deficient animals. Although long-term Se deficiency showed a relatively well-balanced metabolism under resting conditions, sudden stress, accompanied by an excessive radical production, cannot be compensated.

Mammalian selenium-containing proteins.

Mammalian selenium-containing proteins can be divided into three groups: proteins containing nonspecifically incorporated selenium, specific selenium-binding proteins, and specific selenocysteine-containing selenoproteins. Selenoproteins with known functions identified so far include five glutathione peroxidases, two deiodinases, several thioredoxin reductases, and selenophosphate synthetase 2. Alternative splicing leads to a greater variety of selenoproteins, as was shown in the cases of a specific sperm nuclei glutathione peroxidase and some thioredoxin reductases. Selenoprotein P, selenoprotein W, a 15-kDa selenoprotein, an 18-kDa selenoprotein, and several selenoproteins identified in silico from nucleotide sequence databases were found to contain selenocysteine but their functions are not known. Gel electrophoretic separation of tissue samples from rats labeled in vivo with (75)Se showed the existence of further selenium-containing proteins.

Detection of small selenium-containing proteins in tissues of the rat.

The important role of selenium in the mammalian organism has been manifested by the detection of several selenoenzymes, and there are still numerous selenium-containing proteins to be identified. After in vivo labeling of rats with [75Se]-selenite, gel electrophoretic separation of the proteins in tissue homogenates and autoradiography of the labeled bands, information on the selenium-containing proteins present in the different tissues was obtained. In the separation by SDS-PAGE and two-dimensional IEF/SDS-PAGE a large number of selenium-containing proteins or protein subunits with apparent molecular masses in the range from 116 to 8 kDa could be distinguished. This range was extended by applying a modified Tricine-SDS-PAGE, which allows the determination of smaller proteins. Using this method in the separation of the homogenates of the adrenal, brain, diaphragm, epididymis, heart, kidney, liver, lung, pituitary, prostate, skeletal muscle, spleen, thymus and thyroid, four additional selenium-containing proteins with molecular masses of approximately 7 kDa, 5kDa, 4 kDa and 3kDa were detected. The 5 kDa protein and the 7 kDa protein were identified as selenocysteine-containing selenoproteins.

Effects of selenium deficiency on the rat myocardial protein pattern-- investigation by two-dimensional gel electrophoresis.

Dietary selenium deficiency represents an etiological factor in "Keshan disease", a distinct form of an endemic cardiomyopathy. The biochemical effects of selenium depletion in the myocardium are, however, not yet known. Therefore, we investigated the changes in the myocardial protein pattern in rats after long-term selenium deficiency. The myocardial proteins were analyzed in samples from five selenium-depleted rats (Se-deficient group) and five rats supplied with adequate amounts of the element (Se-adequate group). Isoelectric focusing (IEF) with carrier ampholytes on large 2-DE gels was used for the separation of proteins in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the second dimension. The protein patterns were evaluated by means of a computer-assisted gel analysis system. The biochemical identification of the proteins of interest was achieved by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or immunoblotting. On average, 588 +/- 68 protein spots were found on the gels. No significant difference in spot numbers existed between the groups. A pattern of 270 spots with identical positions was found on every gel; 247 of these spots were not saturated and used for quantitative comparison. Thirty-five, i.e., 14 %, differed significantly in their relative intensity in the two groups. Twenty-eight protein spots were decreased in the Se-deficient group and seven were increased. Sarcomeric creatine kinase M chain, alpha-myosin heavy chain (alpha-MHC) and myosin light chain 1 and 2 (MLC 1 and 2) were largely decreased in Se-deficiency. Three protein spots were increased by more than twofold or appeared only in the Se-deficient group. A mitochondrial creatine kinase was identified in this group. The results suggest that selenium deficiency affects myocardial energy metabolism and contractile proteins. These changes probably reflect non-specific alterations in heart failure.

Speciation of selenium in the mammalian organism.

As almost all of the selenium present in the mammalian organism is protein-bound, speciation is mostly concerned with the determination of the different selenium-containing proteins. Information on their distribution and their concentrations in the different tissues of the rat was obtained by means of tracer procedures which, after application of 75Se-selenite with a very high specific activity to selenium-depleted animals and electrophoretic separation of the labelled proteins, allow the determination of these compounds in the pmol to fmos range. A method was developed for the determination of selenocysteine and selenomethionine in the selenium-containing proteins. The identification of specific selenoproteins was achieved by analysis of their selenoamino acid residues and by studies on their characteristics and possible biological functions. This is being followed by the development of methods for the quantitative analysis of the selenoproteins in questions in the tissues of animals and man. In this paper the strategies and procedures used in the identification, characterization and determination of the selenium species present in the mammalian organism will be discussed.

Two new selenoproteins found in the prostatic glandular epithelium and in the spermatid nuclei.

After labeling of rats in vivo with 75Se and protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis more than 25 Se-containing bands could be distinguished. Of those proteins which were detected only in certain compartments and might therefore have tissue-specific functions, two were chosen for detailed investigation. A 15 kDa-protein was found in the prostatic epithelium where it accounted for about two thirds of the protein-bound 75Se. It was mainly present in the cytosol but was not released into the prostatic secretion. After gel chromatography it was found in the fraction which contained proteins with molecular masses of about 300 kDa. Using two-dimensional electrophoresis a pI-value of about 4.5 was determined. In the testis a specific Se-containing 34 kDa-protein was observed which appeared after the onset of puberty. It was localized in the spermatid nuclei where it contained about 80% of the Se tracer present and was found to be bound to the DNA. After extraction it partly disintegrated into a 20 kDa-protein. Both compounds contain Se in the form of selenocysteine. The fact that their formation had priority over that of glutathione peroxidase during insufficient Se intake is an indication of their biological significance. Special interest in the prostatic epithelial selenoprotein derives from a possible inverse relationship between the Se status and the incidence of prostate cancer observed in epidemiological studies, whereas with the 34 kDa-selenoprotein its appearance during the condensation phase of the spermatid nuclei might suggest its participation in some processes of sperm maturation.

Effects of a low selenium state in patients with phenylketonuria.

Eighty-seven participants of the German Collaboratory Study for Children with Phenylketonuria (PKU) presented low plasma, whole blood and hair selenium (Se) values, reduced urinary selenium excretion, and decreased plasma and erythrocyte glutathione peroxidase activity in comparison with a healthy reference group (all figures p < 0.001). Aspartate amino transferase and thyroxine (T4) concentrations in plasma were inversely correlated with the selenium blood values of the PKU children. Somatic measurements showed a negative standard deviation score of body height in the PKU children compared with reference values. Despite the different Se supply, the infants did not present any specific Se deficiency symptoms.

Effects of selenium and iodine deficiency on thyroid hormone concentrations in the central nervous system of the rat.

OBJECTIVE: The effects of single and combined nutritional selenium and iodine deficiency on intracellular thyroid hormone concentrations and type II 5'-iodothyronine deiodinase (5'D-II) activity were examined in different regions of the adult rat brain. DESIGN: Four groups (n = 6) of weanling female Wistar rats proceeding from a breeding line fed a selenium-deficient or a selenium-replete diet for 3 generations, were fed selenium-deficient, iodine-deficient, combined selenium- and iodine-deficient or selenium- and iodine-replete diets for 2 months before they were killed. METHODS: Tissue thyroxine (T4) and tri-iodothyronine (T3) concentrations were determined by highly sensitive RIAs after extraction of the iodothyronines from the tissue samples. The measurement of 5'D-II was based on the release of radioiodide from the 125I-labelled substrate. RESULTS: Selenium deficiency significantly decreased tissue T3 concentrations in the hippocampus, hypothalamus and striatum to 70-80% of controls, whereas no significant changes were found in the cerebellum, cerebral cortex and brain stem. Tissue T4 concentrations were only marginally affected with the exception of a 35% increase in the cerebral cortex. Iodine deficiency dramatically diminished serum T4 levels as well as intracellular T4 concentrations in all regions examined up to 10-30% of control. In spite of a threefold enhancement of 5'D-II, the iodine-deficient animals still had a significant reduction of tissue T3 concentrations (50-65% of controls) in all regions excepting the cerebellum. The combination of selenium and iodine deficiency did not significantly alter this pattern of changes. CONCLUSIONS: These findings suggest that prolonged selenium deficiency as well as iodine deficiency may compromise thyroid hormone homeostasis in the adult brain leading to tissue hypothyroidism and therefore to impaired brain function.

Identification of selenocysteine and selenomethionine in protein hydrolysates by high-performance liquid chromatography of their o-phthaldialdehyde derivatives.

A method for the identification of selenocysteine and selenomethionine in protein hydrolysates was developed. The proteins were subjected to acid hydrolysis after they had been carboxymethylated to prevent decomposition of selenocysteine during this process. After precolumn derivatization of the amino acids with o-phthaldialdehyde, the hydrolysate was chromatographed on C18 columns. The selenoamino acids were detected either by the fluorescence of their o-phthaldialdehyde derivatives (detection limit 30 pmol for selenomethionine and 170 pmol for selenocysteine) or by selenium determination in the eluate using atomic absorption spectrometry (detection limit 0.3 pmol) or, with 75Se-labelled compounds, the measurement of the tracer activity. With the latter procedure the detection limit, which depends on the specific activity of the Se tracer, could be decreased to the femtomole range. The method was successfully applied to the identification of selenocysteine in several newly found mammalian selenium-containing proteins.

Newly found selenium-containing proteins in the tissues of the rat.

The Se-containing proteins in 27 tissues of the rat were investigated by in vivo labeling with 75Se-selenite, separation of the tissue homogenate proteins by SDS-polyacrylamide gel electrophoresis, and determination of the labeled proteins by autoradiography. By using Se-depleted rats and a 75Se-tracer with a high specific activity, Se compounds present at only very low concentrations could be detected. Besides the 13 Se-containing proteins previously described, for which apparent molecular masses of 12, 15, 18, 20, 22, 25, 28, 34, 56, 60, 65, 70, and 75 kD have been found here, a further 15 75Se-labeled bands, with apparent molecular masses of 8, 10, 15.5, 16.5, 24, 32, 34.5, 38, 40, 41, 44, 45, 46.5, 53 and 116 kD could be distinguished. Two-dimensional separation of the kidney homogenate proteins showed that some of the Se-containing bands could be resolved into several labeled spots. Most of the newly found compounds were present in various tissues, but with some the enrichment in certain tissues suggested specific sites of action.

Information on the selenium status of several body compartments of rats from the selenium concentrations in blood fractions, hair and nails.

The suitability of the selenium concentrations in blood plasma, red blood cells, hair and nails to serve as indicators for the selenium status in the main body pools and for the chemical forms of the element ingested was investigated in an animal model. Selenium-deficient male rats, fed a methionine-supplemented low selenium diet, were replenished over a period of 3 months by repeated oral administration of 75Se-labeled sodium selenite or L-selenomethionine in amounts equivalent to the intake from diets with selenium concentrations of 0.3 mg/kg and 2 mg/kg. As the tracer was found to reflect the selenium distribution in the main compartments, quantitative selenium analysis in whole body, liver, skeletal muscle, heart and the monitor materials in the four groups was carried out by measuring the 75Se activity. The blood selenium level was the most suitable parameter for assessing the selenium status in the liver, while the nail selenium concentration was most suited for whole body, skeletal muscle and heart. The differences in the amount and chemical form of dietary selenium were best reflected by the ratios of the selenium levels in hair and plasma or in nails and plasma. The results suggest that with adequate or high selenium intake it might be possible to obtain information on the selenium status of the main body compartments by evaluating the selenium concentrations in several appropriate monitor materials.