Hepatic selenoprotein P (SePP) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice

SePP (selenoprotein P) is central for selenium transport and distribution. Targeted inactivation of the Sepp gene in mice leads to reduced selenium content in plasma, kidney, testis and brain. Accordingly, activities of selenoenzymes are reduced in Sepp(-/-) organs. Male Sepp(-/-) mice are infertile. Unlike selenium deficiency, Sepp deficiency leads to neurological impairment with ataxia and seizures. Hepatocyte-specific inactivation of selenoprotein biosynthesis reduces plasma and kidney selenium levels similarly to Sepp(-/-) mice, but does not result in neurological impairment, suggesting a physiological role of locally expressed SePP in the brain. In an attempt to define the role of liver-derived circulating SePP in contrast with locally expressed SePP, we generated Sepp(-/-) mice with transgenic expression of human SePP under control of a hepatocyte-specific transthyretin promoter. Secreted human SePP was immunologically detectable in serum from SEPP1-transgenic mice. Selenium content and selenoenzyme activities in serum, kidney, testis and brain of Sepp(-/-;SEPP1) (SEPP1-transgenic Sepp(-/-)) mice were increased compared with Sepp(-/-) controls. When a selenium-adequate diet (0.16-0.2 mg/kg of body weight) was fed to the mice, liver-specific expression of SEPP1 rescued the neurological defects of Sepp(-/-) mice and rendered Sepp(-/-) males fertile. When fed on a low-selenium diet (0.06 mg/kg of body weight), Sepp(-/-;SEPP1) mice survived 4 weeks longer than Sepp(-/-) mice, but ultimately developed the neurodegenerative phenotype. These results indicate that plasma SePP derived from hepatocytes is the main transport form of selenium supporting the kidney, testis and brain. Nevertheless, local Sepp expression is required to maintain selenium content in selenium-privileged tissues such as brain and testis during dietary selenium restriction.     

Ecdysone agonist inducible transcription in transgenic tobacco plants

A novel chemical-induced gene regulatory system for plants consisting of two molecular components is described. The first, or regulatory, cassette comprises a chimeric receptor composed of the hinge and ligand binding domains of the Heliothis virescens ecdysone receptor and the transactivation domain of the Herpes simplex VP16 protein fused to the DNA binding domain and transactivation of a mammalian glucocorticoid receptor. The second component, a reporter cassette, contains six copies of the glucocorticoid response element (GRE) fused to the minimal 35SCaMV promoter and β-glucuronidase. The system uses a commercially available non-steroidal ecdysone agonist, RH5992 (tebufenozide), as an inducer. Activation of gene expression is shown in both tobacco transient protoplasts and transgenic plants. The response is ligand dependent and is modulated by the change in minimal promoter context. The system is capable of inducing transgene activity up to 420-fold corresponding to 150% of the activity observed with positive controls (35SCaMV:GUS).     

Comparison of different selenocompounds with respect to nutritional value vs. toxicity using liver cells in culture

The essential micronutrient selenium (Se) exerts its biological effects mainly through enzymatically active selenoproteins. Their biosynthesis depends on the 21st proteinogenic amino acid selenocysteine and thus on dietary Se supply. Hepatically derived selenoprotein P (SEPP) is the central selenoprotein in blood controlling Se transport and distribution. Kidney-derived extracellular glutathione peroxidase is another relevant serum selenoprotein depending on SEPP for biosynthesis. Therefore, secretion of SEPP by hepatocytes is crucial to convert nutritional sources into serum Se, supporting Se status and selenoprotein biosynthesis in other tissues.In order to compare the bioactivity of 10 different selenocompounds, their dose-dependent toxicities and nutritional qualities to support SEPP and glutathione peroxidase biosynthesis were determined in a murine and two human liver cell lines. Characteristic dose- and time-dependent effects on viability and SEPP production were observed. Incubations with 100 nM sodium selenite, l- or dl-selenocystine, selenodiglutathione or selenomethyl-selenocysteine increased SEPP concentrations in the culture medium up to 6.5-fold over control after 72 h. In comparison, sodium selenate, l- or dl-selenomethionine or methylseleninic acid was less effective and increased SEPP by 2.5-fold under these conditions. As expected, ebselen did not increase selenoprotein production, supporting its classification as a stable selenocompound. Methylseleninic acid, l-selenocystine, selenodiglutathione or selenite induced cell death in micromolar concentrations, whereas selenomethionine or ebselen was not toxic within the concentration range tested.Our results indicate that hepatic selenoprotein production and toxicity of selenocompounds do not correlate with and rather represent compound-specific properties. The favourable profile of selenomethylselenocysteine warrants its consideration as a promising option for supplementation purposes.     

Creation of ecdysone receptor chimeras in plants for controlled regulation of gene expression

Transformation with a chimeric receptor containing the glucocorticoid transactivation and DNA-binding domains fused to an ecdysteroid receptor ligand-binding domain permits ecdysone agonist-inducible gene expression in monocotyledonous plant cells. The inducible system is based on the specific activation of a chimeric receptor containing the ligand-binding domain of the Heliothis virescens ecdysteroid receptor and the inducer RH5992 (a 20-hydroxyecdysone agonist). RH5992 is an non-steroidal agrochemical with a high specificity for lepidopteran ecdysone receptors. Addition of RH5992 to transformed cells results in high levels of inducible expression in a ligand-specific manner, particularly when the effector receptor is coupled to the strong transactivator VP16. A chimeric construct containing the Drosophila ecdysone ligand-binding domain failed to activate reporter gene activity with RH5992, while activation was observed in the presence of muristeroneA. The system described provides the basis for an inducible gene expression system that is compatible with agricultural use. Received: 24 September 1998 / Accepted: 15 January 1999     

Selenoprotein P is required for mouse sperm development

Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10-17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1(-/-)) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1(-/-) males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1(-/-) males appear to be the same as those observed in wild-type (Sepp1(+/+)) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1(-/-) males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.