Studies with regard to the apoptosis of testicular germ cells in rats fed a diet enriched with polyunsaturated Fatty acids.

The essential trace element selenium and polyunsaturated fatty acids (PUFA) have been used for the prevention of cancer. Both nutrients enhance the apoptosis of malignant cells and provide health benefits. However, an increased dietary intake of PUFA augments the susceptibility of lipid peroxidation and oxidative damage in many cells. So far, relatively few data are available about the interaction of selenium and PUFA in testis and thus a possible effect of both dietary components on the prevention of testicular cancer or on the apoptosis of testicular germ cells. Male germ cells in the rat contain most of the testicular phospholipid hydroperoxide glutathione peroxidase (PHGPx), mainly as the mitochondrial isoform of this selenoprotein (m-PHGPx). An experiment was therefore carried out to determine the action of fish oil, a nutrient rich in PUFA, on the testicular expression of PHGPx. Because the PHGPx formation remains nearly unchanged in the animals fed the PUFA-enriched diet, we conclude that no apoptosis of testicular germ cells is induced by an increased intake of this nutrient. The intake of fish oil in the selenium-deficient animal led to a markedly altered formation of several selenium-containing proteins, including sperm nuclei glutathione peroxidase (snGPx), also designated as the nuclear form of PHGPx (n-PHGPx), and a 10-kDa selenium-containing protein.

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

Is the distribution of selenium and zinc in the sublocations of spermatozoa regulated?

In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the insertion of zinc and selenium in sperm chromatin is regulated, the element concentrations were determined in equine spermatozoa and purified sperm nuclei. We found a significant positive correlation between the selenium concentration in equine spermatozoa and sperm nuclei. The same finding was obtained for the zinc concentration in spermatozoa and sperm nuclei. The results assume that the distribution of selenium and zinc in spermatozoa is regulated by cell signaling pathways and in this way determining the selenium and zinc amount in the chromatin of spermatozoa.

Distribution of an 18 kDa-selenoprotein in several tissues of the rat.

By combining methods for trace element analysis, tracer techniques and various biochemical and electrophoretical procedures, information on the characteristics of an 18 kDa-selenoprotein was obtained. By labeling of rats in vivo with [75Se]-selenite and gel electrophoretic separation of the proteins in tissues and subcellular fractions, a larger number of selenium-containing proteins could be distinguished. In most of the tissues investigated a labeled 18 kDa-band was present. After co-electrophoresis of the 18 kDa-bands from kidney, liver and brain we found that they all migrated in the same way. Using ultracentrifugational fractionation the 18 kDa-band was localized in the mitochondrial and microsomal membranes. Two-dimensional electrophoresis showed that it consists of a single selenium-containing protein with an isoelectric point of about 4.9-5.0. By means of proteolytic cleavage of the 18 kDa-protein and separation of its peptides by tricine-SDS-PAGE six selenium-containing peptides with molecular masses of 17, 16, 14, 12, 10, and 8 kDa were detected. After electrophoretic separation of the mitochondrial and/or microsomal proteins and acid hydrolysis of the electroeluted protein its amino acid composition was analyzed by RP-HPLC. In this way it was shown that selenium is present in the 18 kDa-protein in form of selenocysteine which is a characteristic of a genetically encoded selenoprotein.