Gary E. Olson

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.

A Role for Poly(ADP-ribose) Polymerase in the Transcriptional Regulation of the Melanoma Growth Stimulatory Activity (CXCL1) Gene Expression

The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription ofCXCL1 is regulated through several cis-acting elements including Sp1, NF-κB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides −94 to −78), which lies immediately upstream to the nuclear factor κB (NF-κB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds a 115-kDa protein. In this study, the IUR element has been further characterized. We show here that proximity of the IUR element to the adjacent NF-κB element is critical to its function as a positive regulatory element. Using binding site oligonucleotide affinity chromatography, we have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy and amino acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-aminobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA. The data point to the possibility that PARP may be a coactivator of CXCL1 transcription.

The Selenium-rich C-terminal Domain of Mouse Selenoprotein P Is Necessary for the Supply of Selenium to Brain and Testis but Not for the Maintenance of Whole Body Selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1–/– and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Δ240–361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Δ240–361 accounted for over 90% of the selenium in the plasma of Sepp1Δ240–361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Δ240–361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1–/– mice but they better maintained their whole body selenium. Sepp1Δ240–361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Δ240–361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1–/– mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.

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.

AKAP3 Selectively Binds PDE4A Isoforms in Bovine Spermatozoa

Abstract Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.

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.