Gary E. Olson

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.

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.

The present study investigates the macromolecular composition of a membrance fraction isolated from rat spermatozoa and uses specific biochemical probes to study the externally oriented plasma membrane glycoproteins of caput and cauda epididymal spermatozoa. A highly purified membrane fraction was isolated from rat cauda epididymal spermatozoa using sonication and differential centrifugation. SDS polyacrylamide gels of the isolated membrane fraction revealed a large number of Coomassie Blue staining bands (>25) and 5 PAS positive bands. The galactose oxidase-[3H]sodium borohydride technique was employed on intact spermatozoa to radioactively label externally oriented plasma membrane glycoproteins possessing terminal galactose or galactosamine residues on their oligosaccharide chains. Cauda epididymal spermatozoa possess a 37,000 dalton glycoprotein on the cell surface which is labelled by this technique, but no such component is detected on caput epididymal spermatozoa. Similarly, radioactive labelling of surface sialo-glycoproteins by sequential treatment of spermatozoa with sodium metaperiodate and [3H]sodium borohydride revealed that the 37,000 dalton glycoprotein could be labeled on cauda epididymal spermatozoa, but is not detected on caput epididymal spermatozoa. The significance of these results with respect to maturation of spermatozoa in the epididymis is discussed.

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.

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.