Ubiquitin Signals in the Developing Acrosome during Spermatogenesis of Rat Testis : An Immunoelectron Microscopic Study
Biology Laboratory (CMH,SY), Department of Biochemistry (TM), and Department of Obstetrics and Gynecology (SH,TS,KH), University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Tamaho-cho, Yamanashi 409-3898, Japan
Correspondence to: Dr. Sadaki Yokota, Biology Laboratory, University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Tamaho-cho, Yamanashi 409-3898, Japan. E-mail: syokota{at}yamanashi.ac.jp
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Summary |
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(J Histochem Cytochem 52:13931403, 2004)
Key Words: monoubiquitin polyubiquitin acrosomal membrane Golgi vesicles immunoelectron microscopy rat spermatogenesis
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Introduction |
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The formation of the acrosome is thought to involve many proteins, which, having completed their roles, are degraded via the intracellular proteolysis system. Intracellular proteolysis involves two systems, a lysosomal system and a proteasome system. In the former, proteolysis occurs in membrane-bound compartments, called lysosomes, into which cytosolic proteins are incorporated through a process using the KFERQ sequence and binding to chaperon protein (Dice et al. 1986) or through microautophagy (de Duve and Wattiaux 1966
). In the latter proteolytic system, target proteins are tagged with a ubiquitin (UB) chain in a series of ATP-dependent enzyme reactions involving E1, E2, and E3 (Hershko et al. 1983
). The polyubiquitin-conjugated proteins are recognized and degraded by the 26S proteasome (Hershko and Ciechanover 1992
), a barrel-shaped structure consisting of one or two regulatory particles and a core particle (Ikai et al. 1991
; Loewe et al. 1995
). In the UB proteasome system, target proteins are conjugated with polyubiquitin chains and recognized and cleaved by the regulatory particles (Thrower et al. 2000
). Thus, UB has a very important role in the tagging of proteins to be degraded.
Recently, other functions of UB have been discovered: a single molecule of UB was found to modify proteins (Dupre et al. 2001; Hicke 2001
; Raiborg et al. 2003
). This monoubiquitination is involved in at least three distinct cellular functions: histone regulation, endocytosis, and vesicular transport between the trans-Golgi network (TGN) and endosomes, and in the budding of retroviruses from the plasma membrane.
During the formation of the acrosome, acrosomal proteins are thought to be transported to acrosomal vesicles from the TGN, where they are sorted from other lysosomal proteins and membrane proteins. It is not known whether UB is used for this sorting.
In this paper, we investigated the localization of UB signals in the acrosome during the spermiogenesis of rat testis using two anti-UB antibodies.
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Materials and Methods |
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Antibodies
Anti-UB antibody against bovine erythrocyte UB was prepared by the method of Hershko et al. (1982). Briefly, 10 mg of bovine red cell UB (Sigma-Aldrich; St Louis, MO) and 16 mg of keyhole limpet hemocyanin (Sigma-Aldrich) were dissolved in 1.2 ml of 0.14 M potassium phosphate (pH 7.0). To this solution, 40 µl of 3% glutaraldehyde was added by stirring at room temperature. The same amount of glutaraldehyde was added three more times at 10-min intervals. The mixture was allowed to stand for 90 min and was dialyzed overnight in the cold against 2 liters of PBS. SDS was added at a final concentration of 2% to the mixture, which was then boiled for 5 min. After cooling, AlCl3 was added to the mixture at a concentration of 1% (w/v) and slowly neutralized with 1 M NaOH (Lazarides and Hubbard 1976
). The resulting Al/SDS/protein precipitate was emulsified with the same volume of Freund's complete adjuvant. The emulsion containing 0.1 mg of UB was injected four times every 2 weeks into rabbits of 3.03.5 kg. Blood was collected 2 weeks after the last injection. The reactivity of antisera to UB was tested by dot blot analysis. All antisera reacted with 0.02 µg of UB. Specific antibody was purified on an affinity column of UB-coupled Sepharose 4B (Pharmacia; Uppsala, Sweden) and named UB1. In addition to our antibody, we used another anti-UB antibody, mouse monoclonal antibody, recognizing only polyubiquitinated proteins (FK1; Affiniti Research Products Ltd., Exeter, UK). Horseradish peroxidase (HRP) -conjugated swine anti-rabbit IgG and HRP-conjugated rabbit anti-mouse IgG and IgM were obtained from DAKO Japan (Tokyo, Japan). Cy3-conjugated goat anti-rabbit IgG was acquired from Chemicon International (Temecula, CA). Alexa 546-conjugated goat anti-rabbit IgG was purchased from Molecular Probes (Eugene, OR).
Western Blot Analysis of Testis Homogenate and Epididymal Sperm
Rat testis and epididymal sperm were homogenized in 0.1 M phosphate buffer containing 0.1% Triton X-100 and protease inhibitors (all from Sigma-Aldrich), 10 µM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 4 µM chymostatin, 4 µM pepstatin, and 4 µg of bestatin (Wilson and Goulding, 1986) as well as 10 mM iodoacetamide to prevent deubiquitination (Baarends et al. 1999
) and centrifuged at 100,000 x g for 1 hr. The supernatants were stored at 70C and used. Protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical; Rockford, IL) with BSA as a standard. The protein concentrations of the extracts were adjusted to 2 mg/ml, and the extracts were mixed with 1 volume of sample buffer for SDS-PAGE and heated in boiling water for 2 min. Five µg of each sample was analyzed by Western blotting.
Tissue Preparation
Testes were dissected out from rats and sliced in an ice-cold fixative consisting of 4% paraformaldehyde, 0.10.2% glutaraldehyde, 0.01% CaCl2, and 0.2 M Hepes-KOH buffer (pH 7.4). For immunofluorescence microscopy, the tissue was fixed with the same fixative containing 0.1% glutaraldehyde for 1 hr at 4C. For immunoelectron microscopy, the tissue slices were fixed in the same fixative containing 0.2% glutaraldehyde for 1 hr at 4C and then cut into small tissue blocks. The tissue blocks were dehydrated in a graded ethanol series and embedded in LR White resin at 20C. Sperm from caudal epididymis was fixed in 4% paraformaldehyde plus 0.2 M Hepes-KOH buffer (pH 7.4) for 30 min. After a brief wash in PBS, the fixed sperm was suspended in PBS containing trypsin (1 mg/ml; Difco Laboratories, Detroit, MI) and allowed to stand for 3 min at 37C. After another wash in PBS, the sperm was dehydrated and embedded in LR White resin as described above.
Smear Preparation of Sperm
Epididymal sperm was collected from the caudal epididymis and suspended in P-1 Medium (Irvine Scientific; Santa Ana, CA). After a wash with the same medium, sperm was smeared on clean glass slides coated with polylysine and fixed in 4% paraformaldehyde buffered with 0.2 M Hepes-KOH to pH 7.4 for 15 min at room temperature. After a wash with a solution consisting of 0.05 M phosphate buffer (pH 7.2) and 0.9% NaCl (PBS), the preparations were used for immunofluorescence staining.
Immunofluorescence Microscopy
The fixed tissue was embedded in Tissue-Tek (Sakura Fine Technical; Tokyo, Japan) and frozen at 20C. Frozen sections (10 µm thick) were cut with a Coldtome (Sakura Fine Technical) and mounted on clean glass slides. Sections were treated with 0.05% SDS for 30 min and then with 0.05% sodium borohydride for 30 min at room temperature (Robinson and Vandre, 2001). After a brief wash with PBS, sections were incubated with the primary antibodies, as described above, followed by Cy3- or Alexa 549-labeled secondary antibodies. For the immunocytochemical control, the incubation with the primary antibodies was omitted. Some of the smear preparations were stained in the same way but without pretreatment with SDS and sodium borohydride, whereas the others were stained after they had been treated with trypsin (1 mg/ml) for 2 min at 37C. Sections and smear preparations were examined using a Y-FL fluorescent microscope (Nikon; Tokyo, Japan) or a TCS 4D confocal laser scanning microscope (Leica Microsystems; Mannheim, Germany).
Postembedding Immunoelectron Microscopy
Thin sections of rat testis and epididymal sperm embedded in LR White resin were cut with a diamond knife equipped with a Reichert Ultracut R (Leica; Vienna, Austria), mounted on nickel grids, and incubated overnight with the primary antibodies (5 µg/ml) at 4C, followed by protein A-gold probes 15 nm in diameter. For the immunocytochemical control experiment, the incubation of sections with the primary antibodies was omitted. Sections were stained with 2% uranyl acetate for 8 min and lead citrate for 30 sec and examined with a H7500 electron microscope (Hitachi; Tokyo, Japan) at an acceleration voltage of 80 kV.
Routine Electron Microscopy
Tissue slices of rat testis were fixed in 2% glutaraldehyde for 1 hr at 4C and cut into small tissue blocks. After a brief wash in PBS, the tissue blocks were postfixed in 1% reduced osmium tetroxide for 1 hr at room temperature. The tissue blocks were dehydrated in a graded ethanol series and embedded in Epon 812. Thin sections were stained with lead citrate and examined with an electron microscope.
Quantitative Immunoelectron Microscopy
Seven micrographs of spermatids at the steps mentioned below, which contained a Golgi region, acrosomes, and multivesicular bodies, were taken at a magnification of x15,000. The images were of spermatids at steps 13, 47, 811, 1215, and 1619. They were enlarged twofold into positive pictures. The cis-Golgi network and the trans-Golgi network were traced on the images. The areas of the cis-Golgi network, the trans-Golgi network, and the multivesicular bodies and the length of the outer membrane to which dense fibrous material was attached were estimated using a SigmaScan scientific measurement system equipped with a computer (Jandel Scientific; San Rafael, CA), and the numbers of gold particles in those areas and the length were calculated. The labeling density was expressed as the number of gold particles per square micrometer of the estimated areas or per micrometer of the estimated length.
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Results |
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Quantitative Distribution of UB Labeling in Golgi Apparatus, Fibrous Material, and Multivesicular Bodies during Spermatogenesis
To confirm the differences of specificity for UB1 and FK1 antibodies, we carried out a quantitative analysis of the distribution of UB labeling. The distribution of ubiquitinated proteins in cis- and trans-Golgi networks, the outer membrane of the head cap, and the multivesicular bodies of spermatogenic cells during spermatogenesis is summarized in Table 1. The distribution revealed using antibody UB1 was quite different from that obtained with antibody FK1. The results obtained with each antibody were just the opposite. The cis-Golgi network of early spermatids was strongly labeled with FK1 but was not labeled with UB1. The trans-Golgi network of spermatids was stained heavily with UB1 but was not stained with FK1. The fibrous material between the acrosomal granule and the outer membrane of the head cap was markedly labeled with UB1 but was not labeled with FK1. Multivesicular bodies of spermatids at steps 415 were stained heavily with UB1 but not at all with FK1.
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Discussion |
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In the present immunofluorescence analysis, acrosomes were stained with the antibody UB1. FK1 stained mainly the nuclei of spermatids, especially elongated nuclei, which were strongly stained, but not acrosomes. Both antibodies stained residual bodies with similar intensity. In epididymal sperm, a strong signal from UB1 was detected in the middle piece but only a very weak UB signal was found in the acrosome. However, when sperm was digested with trypsin, the dorsal region of the head and a small spot in the neck were strongly stained. This suggests that UB molecules are masked during passage through the epididymal tract. It is well known that the maturation of sperm occurs in the epididymal tract (Bearer and Friend 1990; Yanagimachi 1994
). A major event in this maturation is believed to be the change in plasma membrane components of sperm. Changes of immunofluorescence staining for acrosomal membrane surface antigens during the maturation of spermatozoa have been reported (Phillips et al. 1991
). It was also reported that cholesterol in the membrane of spermatozoa was dramatically modified during the maturation process (Pelletier and Friend 1983
). The present study suggests that the masking of ubiquitinated protein(s) occurs in the acrosome during maturation. Recently, it was suggested that the numbers of ubiquitinated spermatozoa decreased progressively between the caput and caudal epididymis (Sutovsky et al. 2001
). Defective spermatozoa were ubiquitinated and might be phagocytosed by epididymal epithelial cells during the epididymal passage (Sutovsky et al. 2001
). Thus, it was proposed that the ubiquitination of spermatozoa functioned as a quality-control system for sperm (Sutovsky 2003
). The role of the masking of ubiquitinated protein(s) in the head and neck during maturation is not clear. It is likely that ubiquitinated protein(s) is used in the egg after fertilization.
Recently, a conserved UB-interacting motif (UIM), XEDEXLXXAXXXSXXEXXX, was proposed (Hofmann and Falquet, 2001), which was found in Eps15, Epsin, and HRS (Polo et al. 2002
; Raiborg et al. 2002
; Shih et al. 2002
). Thus, we carried out a cyber screening of 295 acrosomal proteins and found two candidates, sea urchin sperm receptor and pig acrosomal protein (sp32), having sequences that nearly matched the motif. It is likely that these proteins are ubiquitinated. Although we reported that cathepsin H showed the same distribution as UB in the acrosome of differentiating rat spermatids in a previous study (Haraguchi et al. 2003
), cathepsin H was not a candidate because it has no UIM motif.
In the present study, UB signals were present in the Golgi vesicles. Careful observation revealed two distribution patterns: gold particles were associated with the outer side of the vesicle membrane and were found on dense material within the vesicles. It was reported that several membrane proteins functioning as a receptor or a transporter are monoubiquitinated at the cytoplasmic tail and internalized through coated vesicles and then transported to endosomes to be degraded (Hicke 1999; Shih et al. 2000
; Katzmann et al. 2002
; Polo et al. 2002
; Raiborg et al. 2002
,2003
; Haglund et al. 2003
). Thus, evidence that UB is used for protein sorting into endosomes is accumulating. However, there has been no report so far that UB is used for the transport of proteins from the Golgi apparatus to the endoplasmic reticulum or to other compartments. In the present study, polyubiquitinated proteins revealed with the antibody FK1 were localized to the cis-Golgi network, but a few existed in the trans-Golgi network and in multivesicular bodies in which UB signals, including monoubiquitin, were detected using the UB1 antibody. In addition, no signals for polyubiquitinated proteins were detected in the fibrous material between the outer acrosomal membrane and acrosomal granules. These differences were confirmed by the quantitative analysis of ubiquitinated proteins stained with UB1 and FK1 (Table 1). Thus, the antibody FK1 recognized polyubiquitinated proteins as described (Fujimuro et al. 1994
), whereas our antibody UB1 reacted preferentially with monoubiquitin on thin sections. It is likely then that the signals in both the Golgi vesicles and the fibrous material show monoubiquitinated proteins and those in the cis-Golgi show polyubiquitinated proteins. The results suggest that monoubiquitin is involved in the selective transport of proteins between the Golgi apparatus and the acrosome. It has been shown that the sorting and delivery of proteins between the Golgi apparatus and the acrosome are less precise and that Golgi-resident proteins are present in the acrosomes of round spermatids (Moreno et al. 2000
). However, Golgi proteins mistransported to the acrosome are retrieved to the Golgi apparatus at later stages of differentiation and are completely absent in immature spermatozoa, suggesting that active anterograde and retrograde vesicular transport traffic pathways using ß-coatmer protein- and clathrin-coated vesicles operate between the Golgi apparatus and the acrosome (Moreno et al. 2000
). The present study showed that monoubiquitin-like signals in Golgi vesicles were more abundant in round than in elongating spermatids. This suggests that monoubiquitin is involved in anterograde transport between the Golgi apparatus and the acrosome. In addition, our results showed that in elongating spermatids, multivesicular bodies were heavily stained with UB1. It is likely that in the elongating spermatids, monoubiquitin is involved in the transport from the plasma membrane to multivesicular bodies. This suggests that in differentiating spermatids, monoubiquitin is involved in two different vesicular transport pathways: the transport from the Golgi apparatus to the acrosome, which occurs in early spermatids, and the transport from the plasma membrane to multivesicular bodies, which functions in late spermatids.
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Acknowledgments |
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Footnotes |
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