Essential Role of the Apolipoprotein E Receptor-2 in Sperm Development
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m302157200
ISSN1083-351X
AutoresOlav M. Andersen, Ching‐Hei Yeung, Henrik Vorum, Maren Wellner, Thomas K. Andreassen, Bettina Erdmann, Eva‐Christina Mueller, Joachim Herz, Albrecht Otto, Trevor G. Cooper, Thomas E. Willnow,
Tópico(s)Clusterin in disease pathology
ResumoThe apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility. The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility. Apolipoprotein (apo) E receptor-2 (apoER2) 1The abbreviations used are: apoER2, apolipoprotein E receptor-2; LDL, low density lipoprotein; PHGPx, phospholipid hydroperoxide glutathione peroxidase. is a member of the low density lipoprotein (LDL) receptor gene family and a prototype receptor that acts both in endocytosis and in signal transduction (1Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Google Scholar, 2Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Google Scholar, 3Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Google Scholar, 4Beffert U. Morfini G. Bock H.H. Reyna H. Brady S.T. Herz J. J. Biol. Chem. 2002; 277: 49958-49964Google Scholar). In particular, apoER2 functions as a cellular receptor for Reelin, a signaling factor that regulates neuronal migration processes in the embryonic brain. Reelin binds to apoER2 and to the related very low density lipoprotein receptor on the surface of post-mitotic neurons that migrate to their proper position in the developing brain (2Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Google Scholar, 5D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Google Scholar). Binding of Reelin results in tyrosine phosphorylation of Disabled 1, an adaptor protein bound to the receptor tails, and in activation of down-stream signaling pathways involving the Src family of tyrosine kinases and phosphatidylinositol 3-kinase (4Beffert U. Morfini G. Bock H.H. Reyna H. Brady S.T. Herz J. J. Biol. Chem. 2002; 277: 49958-49964Google Scholar, 6Howell B.W. Herrick T.M. Cooper J.A. Genes Dev. 1999; 13: 643-648Google Scholar, 7Arnaud L. Ballif B.A. Forster E. Cooper J.A. Curr. Biol. 2003; 13: 9-17Google Scholar, 8Bock H.H. Herz J. Curr. Biol. 2003; 13: 18-26Google Scholar). Defects in these signaling cascades as in apoER-2-deficient mice cause abnormal layering of neurons in the cortex, hippocampus, and cerebellum (9Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Google Scholar). Aside from post-mitotic neurons in the brain, apoer2 transcripts are also abundant in the placenta, the ovaries, and the epididymis (1Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Google Scholar, 3Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Google Scholar, 10Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Google Scholar). The function of the receptor in these tissues, however, remains unclear. In the present study, we aimed at elucidating novel roles for apoER2 in tissues other than the brain. We focused our attention on the male reproductive system because apoER2 is highly expressed in the principal cells of the epididymis (3Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Google Scholar) and because receptor-deficient mice suffer from male infertility (9Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Google Scholar). Here we have identified a crucial role for the receptor in sperm maturation, in particular in the acquisition and development of sperm motility. Materials—The generation of apoER2-deficient mice has been described before (9Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Google Scholar). Because of male infertility, the line was bred in-house by mating of homozygous-deficient (apoer2–/–) females with males heterozygous for the receptor gene defect (apoer2+/–). All experiments reported in this study were performed on apoer2+/– and apoer2–/– littermates. As a source for clusterin, we employed conditioned, serum-free medium from Madin-Darby canine kidney cells that secrete large amounts of the protein (11Graichen R. Lösch A. Appel D. Koch-Brandt C. J. Biol. Chem. 1996; 271: 15854-15857Google Scholar). A soluble fragment encompassing the extracellular domain of murine apoER2 was expressed as a hexahistidine fusion protein and purified from 293 EBNA cells (Invitrogen) by conventional Ni2+-affinity chromatography. Analysis of Sperm Morphology—For in situ analysis of sperm morphology, the epididymis was prepared, and the mid/distal cauda luminal content was flushed out. The remaining epididymal tissue was immerse-fixed in 3% glutaraldehyde for 1 h and transferred to phosphate-buffered saline solution. The organ was cut into six regions representing initial segment, remaining caput, proximal corpus, mid-corpus, distal corpus, and proximal cauda, and the tangled mass of fixed luminal sperm was dissected from the center of each region. Individual sperm were released by coarse mincing and brief sonication (0.5 s, 25 W, amplitude 20, Vibracell™ Sonicator, Sonics & Materials Inc., Danbury, CT). Wet preparations of sperm were examined at 20× magnification for flagellar angulation and for the presence of abnormal mid-pieces. Analysis of Sperm Motility—For analysis of sperm motility, a tubule segment was dissected from the proximal cauda epididymidis, and the luminal content was transferred into 100 μl of Biggers Whitten Whittingham (BWW) medium (12Yeung C.H. Cooper T.G. Mol. Hum. Reprod. 2001; 7: 819-828Google Scholar) (osmolality, 400 mmol/kg) containing 12 mg/ml bovine serum albumin. Sperm were allowed to disperse for 2 min at 37 °C, further diluted, and loaded onto siliconized slides (40-μm chamber depth) for video recording. Approximately 200 motile sperm were analyzed for kinematics using a computer-assisted sperm analysis system (IVOS, Version 10.8, Hamilton-Thorne Research, Beverly, MA) by tracking each motile cell at 50 frames/s for 60 frames. Kinematic parameters included the vigor of movement (curvilinear velocity), the average swimming velocity (average path velocity), the speed of forward progression (straight line velocity), the amplitude of lateral head displacement, the beat cross frequency, and the linearity of swim path. Percentage motility was counted, and the tail morphology of the motile sperm was assessed and classified as straight, hairpin, or angulated. Electron Microscopy—Sperm were flushed from the cauda epididymidis and fixed in 2.5% glutaraldehyde, 0.1 m phosphate buffer for 24 h, post-fixed in 1% osmium tetroxide for 1 h, dehydrated, and embedded in Poly/Bed 812 (Polysciences, Inc., Eppelheim, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Philips EM 400T at an acceleration voltage of 80 kV. Endocrine Analysis—Testosterone and dihydrotestosterone levels in the epididymis were determined using specific radioimmunoassay (ICN pharmaceuticals, Costa Mesa, CA) and enzyme-linked immunosorbent assay (IBL, Hamburg, Germany) according to manufacturers' recommendations after tissue extraction of the steroids by methanol and solid phase extraction procedures. The osmotic pressure of luminal contents in the cauda epididymidis was measured as described before (12Yeung C.H. Cooper T.G. Mol. Hum. Reprod. 2001; 7: 819-828Google Scholar). Two-dimensional SDS-PAGE Analysis of Epididymal Sperm and Fluid—Total protein preparations were obtained from flushed caudal sperm or from sperm-free luminal content and dissolved in lysis buffer. Rehydration buffer was added, and the first dimension was run with immobilized pH gradient stripe (3–10L, Amersham Biosciences). The second-dimension run was performed on homemade polyacrylamide gels (12%T - (total solid content), 3%C - (ratio of cross-linker to acrylamide monomer) (13Vorum H. Liu X. Madsen P. Rasmussen H.H. Honoré B. Biochim. Biophys. Acta. 1998; 1386: 121-131Google Scholar) that were fixed in 50% (v/v) methanol, 12% (v/v) acetic acid, and 0.0185% (v/v) formaldehyde, washed in 35% (v/v) ethanol, and stained with silver nitrate. Staining was stopped in 50% (v/v) methanol, 12% (v/v) acetic acid, and the gels were dried between cellophane sheets (14Mortz E. Krogh T.N. Vorum H. Gorg A. Proteomics. 2001; 1: 1359-1363Google Scholar). The Melanie software package (Geneva Bioinformatics) was used after scanning the gels for analysis of differences in the protein patterns between genotypes. For identification, protein spots of interest were cut out of the dried gels, hydrated, destained with Farmer's reagent (15Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Google Scholar), reduced, and alkylated before tryptic digestion. Chromatographic separation of the peptide mixture was performed on a LC Packings 75-μm PepMap C18 column (Dionex, Idstein, Germany) using a capillary liquid chromatography system. The eluting peptides were ionized by electrospray ionization on a quadrupole time-of-flight hybrid mass spectrometer (Micromass, Manchester, UK) fitted with a Z-spray source. The instrument selects precursor ions based on intensity for peptide sequencing by collision-induced fragmentation tandem mass spectrometry. The mass spectral data were processed into peak lists and correlated with protein databases using Mascot software (16Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Google Scholar). Quantitative Reverse Transcription-PCR—Quantitative reverse transcription-PCR was performed on total RNA samples from heterozygous and apoER2-deficient testis and entire epididymis using Taqman® technology (Applied Biosystems). The quantification was evaluated using glyceraldehyde-3-phosphate dehydrogenase mRNA as reference. The following primers and TaqMan probe were used for amplification of the PHGPx mRNA: forward primer, 5′-CCC GAT ATG CTG AGT GTG GTT-3′; reverse primer, 5′-TCC TGC CTC CCA AAC TGG T-3′; probe Fam 5′-ACG AAT CCT GGC CTT CCC CTG C-3′ Tamra. The results were imported into an Excel spreadsheet and analyzed according to the standard curve method. Clusterin Binding—Binding of clusterin to apoER2 was detected by surface plasmon resonance analysis (BIAcore) using the extracellular domain of murine apoER2 coupled to CM-5 sensor chips (23 fmol/mm2). Analysis of the interaction between clusterin and apoER2 was performed as described for other receptor ligands (17Andersen O.M. Christensen L.L. Christensen P.A. Sørensen E.S. Jacobsen C. Moestrup S.K. Etzerodt M. Thøgersen H.C. J. Biol. Chem. 2000; 275: 21017-21024Google Scholar, 18Nykjær A. Fyfe J.C. Kozyraki R. Leheste J.R. Jacobsen C. Nielsen M.S. Verroust P.J. Aminoff M. de la Chapelle A. Moestrup S.K. Ray R. Gliemann J. Willnow T.E. Christensen E.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13895-13900Google Scholar). Although male infertility had been noted in the initial characterization of the apoer2 knockout mouse (9Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Google Scholar), little was known about the mechanism causing this defect and about the role of apoER2 in male reproduction. To address this problem, we performed a detailed analysis of the male reproductive tract in receptor-deficient mice. No differences in weight or size of testis and epididymis or in tissue morphology were observed between apoer2–/– animals and control littermates. We analyzed 1000 tubule sections from 12 blocks cut from each face of halved testes from apoer2+/– and apoer2–/– animals. For heterozygotes, there were 91.2 ± 7.3% normal tubules, 2.8 ± 2.2% sections where only Sertoli cells were present, 5.8 ± 5.7% with spermatid arrest, and 0.2 ± 0.3% not characterized. For the homozygotes, the corresponding numbers were 95.8 ± 3.4, 2.2 ± 2.0, 1.7 ± 1.9, and 0.3 ± 0.9%, respectively. No significant differences were found in any category. No alterations were detected by light microscopic analysis of testicular and epididymal structures (data not shown), indicating normal development of these male reproductive organs in knockout mice. However, a striking phenotype was observed when spermatozoa (mature sperm) from the cauda epididymidis were inspected for morphology and motility (Fig. 1 and Table I). A majority (>51%) of spermatozoa from apoer2–/– animals showed coiling of the tail with the degree of bending ranging from slight angulations (<90°, 8.0% of sperm) to hairpin structures (180°, 43.0% of sperm). In comparison, 98% of spermatozoa from control males showed straight tail morphology (Table I). In addition, some sperm mid-pieces displayed abnormal kinking and fraying of axonemal structures (not shown). Spermatozoa from apoER2-deficient mice also suffered from impaired motility; the percentage of motile sperm was reduced to half the control value. All computer-assisted sperm analysis parameters were significantly reduced in the receptor-deficient mice except the vigor of movement (curvilinear velocity) and the amplitude of lateral head displacement, indicating no difference in flagellation per se but a decrease in the effectiveness of forward progression (Table I). This defect can be attributed to the abnormal tail structure.Table IEffect of apoER2 deficiency on sperm morphology and motility (mean ± S.E.)apoer2+/-apoer2-/-Sperm morphologyStraight98.0 ± 0.649.0 ± 6.5aSignificantly different (p < 0.05).Hairpin1.57 ± 0.5743.0 ± 5.9aSignificantly different (p < 0.05).Angulated0.4 ± 0.28.0 ± 3.5aSignificantly different (p < 0.05).Sperm motility% Motile70.1 ± 4.438.3 ± 4.4aSignificantly different (p < 0.05).Curvilinear velocity, μm/s142.8 ± 3.5126.4 ± 9.0Average path velocity, μm/s108.9 ± 2878.8 ± 7.2aSignificantly different (p < 0.05).Straight line velocity, μm/s94.3 ± 2.767.2 ± 6.6aSignificantly different (p < 0.05).Amplitude of lateral head displacement, μm9.4 ± 0.318.63 ± 0.68Beat cross frequency, Hz21.2 ± 0.527.3 ± 1.1aSignificantly different (p < 0.05).Linearity, %66.6 ± 1.059.3 ± 2.2aSignificantly different (p < 0.05).a Significantly different (p < 0.05). Open table in a new tab Male germ cells mature to fertilizing sperm as they move from the testis through the various segments of the epididymal duct. To test which step in sperm maturation may be defective in apoer2 knockout mice, we performed regional specific analyses of the spermatozoa and correlated our findings with the expression pattern of the receptor. The presence of apoER2 in epithelial cells of the epididymis had been reported before (3Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Google Scholar). However, no information on the specific epididymal region expressing the protein was available. Immunohistochemistry localized the receptor exclusively to the principal cells in the initial segment of the epididymis (Fig. 2). No significant apoER2 expression was detected in the adjacent caput, corpus, or cauda or in the testis (data not shown). Interestingly, the regional specific appearance of sperm tail abnormalities in apoER2-deficient males paralleled the distinct expression pattern of the receptor. Spermatozoa descending from the seminiferous tubules of the testis into the initial segment of the epididymis were normal and exhibited straight tail morphology (region 1, Fig. 3). After exit from the initial segment, distinct morphological abnormalities became apparent and progressively increased in percentage during transit of the sperm through the epididymal duct (regions 2–6). This observation suggested normal spermatogenesis in the testis but abnormal sperm maturation in the proximal epididymis of animals lacking apoER2.Fig. 3Regional sperm tail morphology in heterozygous (+/–) and apoER2-deficient mice (–/–). Sperm were isolated from consecutive epididymal segments of mice of the indicated genotypes and analyzed for straight (above) and hairpin tail morphology (middle) or for the presence of other abnormalities (e.g. defective mid-piece) (below). Values are given as percent of total sperm count. 1, initial segment; 2, caput; 3, proximal corpus; 4, mid-corpus; 5, distal corpus; 6, proximal cauda.View Large Image Figure ViewerDownload (PPT) Acquisition of sperm motility in the epididymis requires extensive structural remodeling of the spermatozoa, and an inability to adopt these changes results in structural abnormalities and in immotility (19Jones R. J. Reprod. Fertil. 1998; 53: 73-84Google Scholar). Intriguingly, the hairpin morphology of sperm from apoer2–/– males could be reverted completely upon incubation in buffer containing mild detergents (such as 0.1% Triton X-100) (Fig. 4). This phenomenon has been observed in other models of epididymal dysfunction. It indicates a failure of the sperm to regulate the intracellular osmotic pressure, termed "cell volume decrease." Sperm with defective cell volume decrease fail to counteract water influx when released into hypo-osmotic conditions, causing cell swelling and coiling of the tail. The swelling is relieved upon perforation of the sperm membrane with detergents, resulting in tail straightness (20Yeung C.H. Sonnenberg-Riethmacher E. Cooper T.G. Biol. Reprod. 1999; 61: 1062-1069Google Scholar, 21Butler A. He X. Gordon R.E. Wu H.S. Gatt S. Schuchman E.H. Am. J. Pathol. 2002; 161: 1061-1075Google Scholar). To uncover the molecular mechanism underlying this defect, we compared the protein expression pattern in sperm from apoer2–/– and apoer2+/– epididymis using two-dimensional SDS-PAGE. As exemplified in Fig. 5, a single protein spot with an apparent molecular mass of 19-kDa was significantly reduced in knockout compared with control sperm. By densitometric scanning, a 3-fold decrease in the amount of the protein was measured. Using mass spectrometry, four peptide sequences were derived from this protein spot corresponding to residues Thr76-Arg89, Ile97-Arg107, Gln108-Lys126, and Tyr180-Lys191 of murine phospholipid hydroperoxide glutathione peroxidase (PHGPx) (SwissProt data base entry O70325). PHGPx is a selenoperoxidase that plays a crucial role in spermatogenesis both in the testis and in the epididymis (22Godeas C. Tramer F. Micali F. Soranzo M. Sandri G. Panfili E. Biol. Reprod. 1997; 57: 1502-1508Google Scholar). The protein can change its physical properties and act either as a soluble enzyme or as an insoluble structural protein (23Roveri A. Ursini F. Flohe L. Maiorino M. Biofactors. 2001; 14: 213-222Google Scholar, 24Ursini F. Heim S. Kiess M. Maiorino M. Roveri A. Wissing J. Flohe L. Science. 1999; 285: 1393-1396Google Scholar). In immature spermatids in the testis, PHGPx functions as a peroxidase, controlling the oxidative milieu of the germ cell (25Roveri A. Casasco A. Maiorino M. Dalan P. Calligaro A. Ursini F. J. Biol. Chem. 1992; 267: 6142-6146Google Scholar). In the epididymis, PHGPx is catalytically inactive and covalently linked to thiol-proteins to form the sperm mitochondrial capsule (24Ursini F. Heim S. Kiess M. Maiorino M. Roveri A. Wissing J. Flohe L. Science. 1999; 285: 1393-1396Google Scholar). This capsule is a keratin-like matrix that surrounds the mitochondria wrapped in a helical arrangement around the flagellum in the sperm mid-piece. The capsule stabilizes the mitochondrial helix, and impaired PHGPx expression results in mid-piece irregularities and in mitochondria irregularly wrapped around the flagellum (26Imai H. Suzuki K. Ishizaka K. Ichinose S. Oshima H. Okayasu I. Emoto K. Umeda M. Nakagawa Y. Biol. Reprod. 2001; 64: 674-683Google Scholar). As demonstrated by two-dimensional SDS-PAGE (Fig. 5) and confirmed by Western blot analysis (data not shown), reduced expression of PHGPx was seen in epididymis- but not in testis-derived sperm of apoer2 knockout mice, indicating a defect in the structural rather than the catalytic function of the protein. Consistent with this hypothesis, a highly irregular mitochondrial helix was observed in mid-piece sections of most receptor-deficient spermatozoa (Fig. 6). Insufficient PHGPx expression was caused by translational or post-translational mechanisms, because transcriptional levels of the PHGPx gene were unchanged in knockout compared with control tissue (p = 0.16 and p = 0.60, Student's t test for epididymis and testis, respectively) (Fig. 7).Fig. 7Quantitative reverse transcription-PCR analysis of PHGPx expression in testis and epididymis. The expression levels of PHGPx in testis and epididymis of apoer2+/– (open bars) and apoer2–/– animals (closed bars) were determined by quantitative reverse transcription-PCR and indicated as relative expression intensities (mean ± S.E.).View Large Image Figure ViewerDownload (PPT) To this point, our studies had traced the likely reason for the male infertility in the apoer2–/– mice to an inability to sustain normal sperm levels of PHGPx expression in the epididymis-derived sperm. As a consequence, the spermatozoa suffer from severe structural defects of the mitochondria and from cell volume dysregulation. It is well established that the epithelial cells of the epididymis provide the proper microenvironment for structural remodeling of the spermatozoa and for acquisition of motility (27Aitken R.J. Vernet P. J. Reprod. Fertil. Suppl. 1998; 53: 109-118Google Scholar, 28Christova Y. James P.S. Cooper T.G. Jones R. J. Androl. 2002; 23: 384-392Google Scholar, 29Turner T.T. Miller D.W. Avery E.A. Biol. Reprod. 1995; 52: 1012-1019Google Scholar, 30Cooper T.G. J. Reprod. Fertil. 1998; 53: 119-136Google Scholar). Through regulated secretion and reabsorption, they regulate the pH, osmotic pressure, and composition of epididymal fluids and control the transfer of metabolites such as proteins, lipids, and sugars to and from the sperm surface. Given the expression of apoER2 in principal cells of the epididymis, alterations in epididymal fluid composition were considered a possible cause for the sperm maturation defect in receptor-deficient mice. Therefore, we performed a detailed analysis of the hormone, electrolyte, and protein status of the epididymal fluids in knockout and in control tissues. Testosterone and dihydrotestosterone levels in the epididymis were identical in apoer2–/– (8.9 ± 5.9 and 8.1 ± 3.2 ng/g, respectively) and apoer2+/– animals (13.3 ± 19.5 and 14.7 ± 18.4 ng/g, respectively). Furthermore, no difference in the osmotic pressure of the epididymal fluids from knockout mice and heterozygous controls was detected (484.0 ± 24.2 and 479.9 ± 18.9 mmol/kg, respectively). In contrast, by two-dimensional SDS-PAGE the accumulation of a cluster of protein spots in the 40-kDa molecular mass range was observed in the epididymal fluid of receptor-deficient animals compared with controls (Fig. 8). By tryptic digestion and mass spectrometry, these spots were identified as the α-chain of murine clusterin (derived peptide sequences corresponding to residues Val385-Arg400, Leu408-Lys424, and Phe429-Lys436 of SwissProt data base entry Q06890). Clusterin is a heterodimeric glycoprotein involved in lipid transport and in sperm maturation (31Jones S.E. Jomary C. Int. J. Biochem. Cell Biol. 2002; 34: 427-431Google Scholar, 32Rosenberg M.E. Silkensen J. Int. J. Biochem. Cell Biol. 1995; 27: 633-645Google Scholar). The protein associates with the membrane of sperm in the testis. In the epididymis, it dissociates from the sperm surface to be cleared from the ductual fluids by the principal cells. To demonstrate that apoER2 may be involved in the removal of clusterin from the epididymal fluid, we tested binding of the protein to apoER2 by surface plasmon resonance analysis (BIAcore) (Fig. 9). In these studies, clusterin bound to apoER2 in a concentration-dependent manner. The interaction was blocked by the receptor-associated protein, an inhibitor of ligand binding to members of the LDL receptor gene family. Taken together, these data identified apoER2 as a novel clusterin receptor and suggested receptor deficiency as the cause of impaired catabolism and extracellular accumulation of the protein. Human infertility affects 5–7% of all male individuals. Approximately 60% of all cases of infertility are attributed to genetic factors, and the elucidation of the underlying gene defects is an area of intensive research (33De Kretser D.M. Baker H.W.G. J. Clin. Endocrinol. Metab. 1999; 84: 3443-3450Google Scholar, 34Lilford R. Jones A.M. Bishop D.T. Thornton J. Mueller R. BMJ. 1994; 309: 570-573Google Scholar). The production of active male gametes is a complicated process that involves (i) elaboration of spermatozoa from early germ cells in the testis, (ii) development of motility and fertilizing potential in the epididymis, and (iii) the ability to undergo capacitation in the female tract. Gene defects blocking any of these steps result in impaired fertility. Consistent with the expression of apoER2 in the epididymis, this receptor plays a crucial role in the acquisition of sperm motility, and deletion of the corresponding gene in the mouse results in less motile sperm. Based on our findings we propose that apoER2 activity is required to sustain normal expression of sperm PHGPx in the epididymis. As a consequence of the receptor gene defect, expression of PHGPx is reduced, causing structural and, likely, functional defects of the sperm mitochondria, and cell volume dysregulation. Whether apoER2 directly regulates the metabolism of PHGPx or whether loss of the protein is an indirect consequence of the receptor defect still needs to be addressed. The crucial role of PHGPx in fertility has been appreciated in numerous studies (26Imai H. Suzuki K. Ishizaka K. Ichinose S. Oshima H. Okayasu I. Emoto K. Umeda M. Nakagawa Y. Biol. Reprod. 2001; 64: 674-683Google Scholar, 35Foresta C. Flohe L. Garolla A. Roveri A. Ursini F. Maiorino M. Biol. Reprod. 2002; 67: 967-971Google Scholar). Thus, PHGPx levels in human sperm are correlated positively with morphological integrity and forward motility (35Foresta C. Flohe L. Garolla A. Roveri A. Ursini F. Maiorino M. Biol. Reprod. 2002; 67: 967-971Google Scholar). When expression of the protein was assessed in spermatozoa from infertile men, more than 10% of the individuals suffered from a dramatic decrease of PHGPx expression. As a consequence, their sperm were immotile and exhibited irregularly sized mitochondria, a phenotype that closely resembles features of sperm in apoER2-deficient mice (26Imai H. Suzuki K. Ishizaka K. Ichinose S. Oshima H. Okayasu I. Emoto K. Umeda M. Nakagawa Y. Biol. Reprod. 2001; 64: 674-683Google Scholar). Loss of the sperm tail, as frequently observed in sperm from selenium-deficient mice, has also been attributed to inadequate levels of PHGPx in the mitochondrial membrane (36Behne D. Weiler H. Kyriakopoulos A. J. Reprod. Fertil. 1996; 106: 291-297Google Scholar). The correlation between PHGPx expression, mitochondrial integrity, and sperm motility is far from being clearly understood. Lack of peroxidase activity in the sperm may result in the accumulation of reactive oxygen species and in oxidative damage to the mitochondria (26Imai H. Suzuki K. Ishizaka K. Ichinose S. Oshima H. Okayasu I. Emoto K. Umeda M. Nakagawa Y. Biol. Reprod. 2001; 64: 674-683Google Scholar, 37Rao B. Soufir J.C. Martin M. David G. Gamete Res. 1989; 24: 127-134Google Scholar, 38Aitken R.J. Clarkson J.S. Hargreave T.B. Irvine D.S. Wu F.C. J. Androl. 1989; 10: 214-220Google Scholar, 39Zalata A.A. Christophe A.B. Depuydt C.E. Schoonjans F. Comhaire F.H. Mol. Hum. Reprod. 1998; 4: 111-118Google Scholar). Alternatively, absence of the protein may affect formation of the sperm mitochondrial capsule that is essential for forming the mitochondrial helix in the sperm mid-piece. Mitochondrial defects in turn cause immotility by yet unknown mechanisms (40Weinberg J.B. Doty E. Bonaventura J. Haney A.F. Fertil. Steril. 1995; 64: 408-413Google Scholar, 41Folgerø T. Bertheussen K. Lindal S. Torbergsen T. Øian P. Hum. Reprod. 1993; 8: 1863-1868Google Scholar). One simple explanation may be insufficient energy production to sustain forward motility and cell volume regulation. Several knockout mouse models of epididymal dysfunction and sperm immotility have been reported in the literature that may shed some light on a possible contribution of apoER2 to sperm maturation (42Venables J.P. Cooke H.J. J. Endocrinol. Invest. 2000; 23: 584-591Google Scholar, 43Cooke H.J. Saunders P.T. Nat. Rev. Genet. 2002; 3: 790-801Google Scholar). Spermatozoa of mice with deletion of the retinoid X receptor β gene present with hairpin tail morphology and with failure of the mitochondria to form a helix around the flagellum (44Kastner P. Mark M. Leid M. Gansmuller A. Chin W. Grondona J.M. Decimo D. Krezel W. Dierich A. Chambon P. Genes Dev. 1996; 10: 80-92Google Scholar). Accumulation of lipids in Sertoli cells and in epididymal ducts is held responsible for the sperm maturation defect in this model. Similarly, heterozygosity for the apoB gene defect (45Huang L.S. Voyiaziakis E. Chen H.L. Rubin E.M. Gordon J.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10903-10907Google Scholar) or homozygous deletion of the acid sphingomyelinase gene (21Butler A. He X. Gordon R.E. Wu H.S. Gatt S. Schuchman E.H. Am. J. Pathol. 2002; 161: 1061-1075Google Scholar) affects the lipid metabolism of the epididymis and the spermatozoa, resulting in sperm immotility. In particular, the latter model exhibits close resemblance to apoER2-deficient mice inasmuch as the sperm are characterized by detergent-reversible tail angulation (21Butler A. He X. Gordon R.E. Wu H.S. Gatt S. Schuchman E.H. Am. J. Pathol. 2002; 161: 1061-1075Google Scholar). Unfortunately, no information is available on the expression of PHGPx protein in this mouse model. Besides defects in lipid metabolism, the absence of signaling pathways in the epididymis as in c-Ros-deficient mice also results in cell volume dysregulation and in detergent-reversible tail angulation. The receptor tyrosine kinase c-Ros is an orphan receptor expressed in the initial segment and in the caput epididymis (46Sonnenberg-Riethmacher E. Walter B. Riethmacher D. Godecke S. Birchmeier C. Genes Dev. 1996; 10: 1184-1193Google Scholar). Lack of the initial segment of the epididymis in c-Ros-deficient mice coincides with cell volume dysregulation (20Yeung C.H. Sonnenberg-Riethmacher E. Cooper T.G. Biol. Reprod. 1999; 61: 1062-1069Google Scholar, 47Yeung C.H. Sonnenberg-Riethmacher E. Cooper T.G. J. Reprod. Fertil. Suppl. 1998; 53: 137-147Google Scholar, 48Yeung C.H. Wagenfeld A. Nieschlag E. Cooper T.G. Biol. Reprod. 2000; 63: 612-618Google Scholar, 49Yeung C.H. Anapolski M. Sipila P. Wagenfeld A. Poutanen M. Huhtaniemi I. Nieschlag E. Cooper T.G. Biol. Reprod. 2002; 67: 269-275Google Scholar). Based on findings in the aforementioned models, a role for apoER2 in lipid metabolism or in signal transduction during sperm maturation may be considered. The function of apoER2 in cellular uptake of lipoproteins has been reported before (1Kim D.-H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.-J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Google Scholar). Although the endocytic activity of the receptor in cultured cells is low compared with other lipoprotein receptors (50Li Y. Lu W. Marzolo M.P. Bu G. J. Biol. Chem. 2001; 21: 18000-18006Google Scholar), the protein may be more active in lipoprotein uptake in the proper cellular environment of the epididymis, a phenomenon known for other members of the LDL receptor family. For example, the LDL receptor is dependent on the presence of a hepatocyte-specific adaptor to mediate LDL endocytosis in the liver but not in fibroblasts (51Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Science. 2001; 292: 1394-1398Google Scholar, 52Wilund K.R. Yi M. Campagna F. Arca M. Zuliani G. Fellin R. Ho Y.K. Garcia J.V. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Google Scholar). In analogy to its function in the brain, a role for apoER2 as signaling receptor in the epididymis is also possible. However, this function is unlikely to involve the signaling factor Reelin because reeler mice do not suffer from severe sperm tail abnormalities as seen in the apoer2 knockout animals. Some angulated tails were present in sperm samples from reelin–/– mice, but most sperm had normal tail morphology. 2O. M. Andersen, C.-H. Yeung, H. Vorum, M. Wellner, T. K. Andreassen, B. Erdmann, E.-C. Mueller, J. Herz, A. Otto, T. G. Cooper, and T. E. Willnow, unpublished information. As a first step toward identification of possible ligands that bind to apoER2 in the epididymis, we analyzed the luminal fluid for proteins accumulating in knockout samples. This approach was based on the observation that the epididymal luminal fluid contains factors that bind to the receptor in BIAcore analysis (data not shown). Using this approach we identified clusterin as an endogenous ligand for apoER2. Interestingly, this protein has also been uncovered by Schneider and coworkers as an apoER2-binding protein in independent studies. 3W. Schneider, personal communication. Although clusterin plays a role in sperm maturation, it is unlikely that loss of clusterin-mediated apoER2 function is responsible for the phenotype of the apoer2–/– mice because deletion of the clusterin gene has a rather modest effect on male fertility (53Bailey R.W. Aronow B. Harmony J.A. Griswold M.D. Biol. Reprod. 2002; 66: 1042-1053Google Scholar). Nevertheless, this finding is noteworthy because megalin, another member of the LDL receptor gene family expressed in principal cells of the epididymis, was identified earlier as an endocytic receptor for clusterin (54Morales C.R. Igdoura S.A. Wosu U.A. Boman J. Argraves W.S. Biol. Reprod. 1996; 55: 676-683Google Scholar, 55Hermo L. Lustig M. Lefrancois S. Argraves W.S. Morales C.R. Mol. Reprod. Dev. 1999; 53: 282-293Google Scholar, 56Van Praet O. Argraves W.S. Morales C.R. Mol. Reprod. Dev. 2003; 64: 129-135Google Scholar). Thus, our results demonstrate that two related receptors, apoER2 and megalin, should both be considered when studying the catabolism of clusterin in the epididymis. In summary, we have uncovered the pathophysiological basis of male infertility in apoER2-deficient mice, namely abnormal sperm morphology and subnormal motility. These findings highlight an important and previously unrecognized role of the receptor in sperm development. Further studies into the molecular pathways may yield significant new insights into the concept of sperm maturation and the cause of infertility in humans. We are indebted to H. Schulz, C. Räder, M. Eigen, S. Schütz, J. Czychi, and M. Vannauer for expert technical assistance and to C. Koch-Brandt, J. Nimpf, G. Kempermann, and R. Brigelius-Flohé for sharing reagents and unpublished data.
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