Virginia P. Winfrey

Apolipoprotein E Receptor-2 (ApoER2) Mediates Selenium Uptake from Selenoprotein P by the Mouse Testis

Selenium is a micronutrient that is essential for the production of normal spermatozoa. The selenium-rich plasma protein selenoprotein P (Sepp1) is required for maintenance of testis selenium and for fertility of the male mouse. Sepp1 trafficking in the seminiferous epithelium was studied using conventional methods and mice with gene deletions. Immunocytochemistry demonstrated that Sepp1 is present in vesicle-like structures in the basal region of Sertoli cells, suggesting that the protein is taken up intact. Sepp1 affinity chromatography of a testicular extract followed by mass spectrometry-based identification of bound proteins identified apolipoprotein E receptor 2 (ApoER2) as a candidate testis Sepp1 receptor. In situ hybridization analysis identified Sertoli cells as the only cell type in the seminiferous epithelium with detectable ApoER2 expression. Testis selenium levels in apoER2-/- males were sharply reduced from those in apoER2+/+ males and were comparable with the depressed levels found in Sepp1-/- males. However, liver selenium levels were unchanged by deletion of apoER2. Immunocytochemistry did not detect Sepp1 in the Sertoli cells of apoER2-/- males, consistent with a defect in the receptor-mediated Sepp1 uptake pathway. Phase contrast microscopy revealed identical sperm defects in apoER2-/- and Sepp1-/- mice. Co-immunoprecipitation analysis demonstrated an interaction of testis ApoER2 with Sepp1. These data demonstrate that Sertoli cell ApoER2 is a Sepp1 receptor and a component of the selenium delivery pathway to spermatogenic cells.

Deletion of Apolipoprotein E Receptor-2 in Mice Lowers Brain Selenium and Causes Severe Neurological Dysfunction and Death When a Low-Selenium Diet Is Fed

Selenoprotein P (Sepp1) is a plasma and extracellular protein that is rich in selenium. Deletion of Sepp1 results in sharp decreases of selenium levels in the brain and testis with dysfunction of those organs. Deletion of Sepp1 also causes increased urinary selenium excretion, leading to moderate depletion of whole-body selenium. The lipoprotein receptor apolipoprotein E receptor-2 (apoER2) binds Sepp1 and facilitates its uptake by Sertoli cells, thus providing selenium for spermatogenesis. Experiments were performed to assess the effect of apoER2 on the concentration and function of selenium in the brain and on whole-body selenium. ApoER2−/− and apoER2+/+ male mice were fed a semipurified diet with selenite added as the source of selenium. ApoER2−/− mice had depressed brain and testis selenium, but normal levels in liver, kidney, muscle, and the whole body. Feeding a selenium-deficient diet to apoER2−/− mice led to neurological dysfunction and death, with some of the characteristics exhibited by Sepp1−/− mice fed the same diet. Thus, although it does not affect whole-body selenium, apoER2 is necessary for maintenance of brain selenium and for prevention of neurological dysfunction and death under conditions of selenium deficiency, suggesting an interaction of apoER2 with Sepp1 in the brain.

Megalin Mediates Selenoprotein P Uptake by Kidney Proximal Tubule Epithelial Cells

Selenoprotein P (Sepp1) contains most of the selenium in blood plasma, and it is utilized by the kidney, brain, and testis as a selenium source for selenoprotein synthesis. We recently demonstrated that apolipoprotein E receptor-2 (ApoER2) is required for Sepp1 uptake by the testis and that deletion of ApoER2 reduces testis and brain, but not kidney, selenium levels. This study examined the kidney Sepp1 uptake pathway. Immunolocalization experiments demonstrated that Sepp1 passed into the glomerular filtrate and was specifically taken up by proximal tubule epithelial cells. Neither the C terminus selenocysteine-rich domain of Sepp1 nor ApoER2 was required for Sepp1 uptake by proximal tubules. Tissue ligand binding assays using cryosections of Sepp1-/- kidneys revealed that the proximal tubule epithelium contained Sepp1-binding sites that were blocked by the receptor-associated protein, RAP, an inhibitor of lipoprotein receptor-ligand interactions. Ligand blotting assays of kidney membrane preparations fractionated by SDS-PAGE revealed that Sepp1 binds megalin, a lipoprotein receptor localized to the proximal tubule epithelium. Immunolocalization analyses confirmed the in vivo co-localization of Sepp1 and megalin in wild type kidneys and demonstrated the absence of proximal tubule Sepp1 uptake in megalin null mice. These results demonstrate that kidney selenium homeostasis is mediated by a megalin-dependent Sepp1 uptake pathway in the proximal tubule.

Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome.

Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were M(r) 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2-D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the proteins functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of acrosomal exocytosis.

Production of Selenoprotein P (Sepp1) by Hepatocytes Is Central to Selenium Homeostasis

Background: Sepp1 transports selenium, but its complete role in selenium homeostasis is not known. Results: Deletion of Sepp1 in hepatocytes increases liver selenium at the expense of other tissues and decreases whole-body selenium by increasing excretion. Conclusion: Sepp1 production by hepatocytes retains selenium in the organism and distributes it from the liver to peripheral tissues. Significance: Sepp1 is central to selenium homeostasis. Sepp1 is a widely expressed extracellular protein that in humans and mice contains 10 selenocysteine residues in its primary structure. Extra-hepatic tissues take up plasma Sepp1 for its selenium via apolipoprotein E receptor-2 (apoER2)-mediated endocytosis. The role of Sepp1 in the transport of selenium from liver, a rich source of the element, to peripheral tissues was studied using mice with selective deletion of Sepp1 in hepatocytes (Sepp1c/c/alb-cre+/− mice). Deletion of Sepp1 in hepatocytes lowered plasma Sepp1 concentration to 10% of that in Sepp1c/c mice (controls) and increased urinary selenium excretion, decreasing whole-body and tissue selenium concentrations. Under selenium-deficient conditions, Sepp1c/c/alb-cre+/− mice accumulated selenium in the liver at the expense of extra-hepatic tissues, severely worsening clinical manifestations of dietary selenium deficiency. These findings are consistent with there being competition for metabolically available hepatocyte selenium between the synthesis of selenoproteins and the synthesis of selenium excretory metabolites. In addition, selenium deficiency down-regulated the mRNA of the most abundant hepatic selenoprotein, glutathione peroxidase-1 (Gpx1), to 15% of the selenium-replete value, while reducing Sepp1 mRNA, the most abundant hepatic selenoprotein mRNA, only to 61%. This strongly suggests that Sepp1 synthesis is favored in the liver over Gpx1 synthesis when selenium supply is limited, directing hepatocyte selenium to peripheral tissues in selenium deficiency. We conclude that production of Sepp1 by hepatocytes is central to selenium homeostasis in the organism because it promotes retention of selenium in the body and effects selenium distribution from the liver to extra-hepatic tissues, especially under selenium-deficient conditions.

Substructure of the postacrosomal sheath of bovine spermatozoa.

The substructure of the postacrosomal sheath and its relationship to the plasma membrane and nuclear membrane complex were examined in thin-section, negative-stain, surface-replica, and freeze-fracture preparations. The matrix of the postacrosomal sheath contains a single layer of closely associated 10- to 12-nm filamentous elements aligned parallel to the long axis of the sperm. A precise lateral interaction of the filaments is suggested from negative-stain images which reveal a second set of parallel striations extending over the surface of the sheath at 60 degrees relative to the filament long axis. Several structural differences between the posterior and anterior segments and the outer and inner surface of the postacrosomal sheath were identified. Data on structural specializations of the plasma membrane and nuclear membrane complex which relate to the asymmetric structure are presented and their potential significance in fertilization events discussed.

Mitochondria−cytoskeleton interactions in the sperm midpiece

The mitochondrial sheath of mammalian spermatozoa is adherent to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). In this manuscript we further characterize the substructure of the SMR and the outer mitochondrial membrane and provide new information on their structural interaction. The SMR resists solubilization by detergent and once partially released from the midpiece of extracted spermatozoa, it appears in negatively stained preparations as a network of longitudinally oriented ribons of fibrillar material which are laterally interconnected. In detergent-extracted specimens the SMR remains attached to the outer mitochondrial membrane thereby suggesting a firm structural interaction. Negatively stained specimens also demonstrate that the outer mitochondrial membrane possesses a paracrystalline substructure and it is suggested that ordered arrays of membrane-associated proteins are involved in the structural interaction with the SMR. The potential roles of this cytoskeletal complex during spermiogenesis and in mature sperm are discussed.

A role for the inositol kinase Ipk1 in ciliary beating and length maintenance

Cilia project from cells as membranous extensions, with microtubule structural cores assembling from basal bodies by intraflagellar transport (IFT). Here, we report a ciliary role for the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1) that generates inositol hexakisphosphate. In zebrafish embryos, reducing Ipk1 levels inhibited ciliary beating in Kupffers vesicle and decreased ciliary length in the spinal canal, pronephric ducts, and Kupffers vesicle. Electron microscopy showed that ciliary axonemal structures were not grossly altered. However, coincident knockdown of Ipk1 and IFT88 or IFT57 had synergistic perturbations. With GFP-Ipk1 enriched in centrosomes and basal bodies, we propose that Ipk1 plays a previously uncharacterized role in ciliary function.

Selenoprotein P and apolipoprotein E receptor-2 interact at the blood-brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration

Selenoprotein P (Sepp1) and its receptor, apolipoprotein E receptor 2 (apoER2), account for brain retaining selenium better than other tissues. The primary sources of Sepp1 in plasma and brain are hepatocytes and astrocytes, respectively. ApoER2 is expressed in varying amounts by tissues; within the brain it is expressed primarily by neurons. Knockout of Sepp1 or apoER2 lowers brain selenium from ~120 to ~50 ng/g and leads to severe neurodegeneration and death in mild selenium deficiency. Interactions of Sepp1 and apoER2 that protect against this injury have not been characterized. We studied Sepp1, apoER2, and brain selenium in knockout mice. Immunocytochemistry showed that apoER2 mediates Sepp1 uptake at the blood‐brain barrier. When Sepp1–/–or apoER2–/– mice developed severe neurodegeneration caused by mild selenium deficiency, brain selenium was ~35 ng/g. In extreme selenium deficiency, however, brain selenium of ~12 ng/g was tolerated when both Sepp1 and apoER2 were intact in the brain. These findings indicate that tandem Sepp1‐apoER2 interactions supply selenium for maintenance of brain neurons. One interaction is at the blood‐brain barrier, and the other is within the brain. We postulate that Sepp1 inside the blood‐brain barrier is taken up by neurons via apoER2, concentrating brain selenium in them.—Burk, R. F., Hill, K. E., Motley, A. K., Winfrey, V. P., Kurokawa, S., Mitchell, S. L., Zhang, W. Selenoprotein P and apolipoprotein E receptor‐2 interact at the blood‐brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration. FASEB J. 28, 3579–3588 (2014). www.fasebj.org