Possible Function of Caudal Nuclear Pocket: Degradation of Nucleoproteins by Ubiquitin-Proteasome...

ARTICLE

Possible Function of Caudal Nuclear Pocket: Degradation ofNucleoproteins by Ubiquitin–Proteasome System in RatSpermatids and Human Sperm

Celina M. Haraguchi, Tadashi Mabuchi, Shuji Hirata, Tomoko Shoda, Toshinobu Tokumoto,Kazuhiko Hoshi, and Sadaki Yokota

Biology Laboratory (CMH,SY), Department of Biochemistry (TM), and Department of Gynecology and Obstetrics (SH,TS,KH),Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan, and Faculty ofScience, Department of Biology and Geosciences, Shizuoka University, Shizuoka, Japan (TT)

SUMMARY Many temporarily functioning proteins are generated during the replacementof nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus.This study was designed to clarify the involvement of the ubiquitin–proteasome degradationsystem in the nucleus of rat developing spermatids. Thus, we studied the nuclear distributionof polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm usingpostembedding immunoelectron microscopy. We divided the nuclear area of late spermatidsinto two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocketin the neck region. The latter was located in the caudal nuclear region andwas surrounded byredundant nuclear envelope. We demonstrated the presence of pUP in the dense area andnuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of ratspermatids. Using quantitative analysis of immunogold labeling, we found that fluctuationof pUP and proteasome levels in late spermatogenesis was mostly synchronized with dis-appearance of histones and transitional proteins reported previously. In the nuclei of humansperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuolesand clear spots. These results strongly suggest that pUP occur in the dense nuclear area ofdeveloping spermatids and that the ubiquitin–proteasome system is more actively opera-tional in the nuclear pocket than dense area. Thus, the nuclear pocket might be the deg-radation site for temporarily functioning proteins generating during condensation ofchromatin in late spermatids. (J Histochem Cytochem 55:585–595, 2007)

KEY WORDS

nucleoproteins

polyubiquitin

proteasomes

spermatogenesis

sperm

degradation sites

nuclear pocket

nuclear vacuoles

DURING SPERMIOGENESIS, a number of chromatin rear-rangements occur in the nuclei of rat spermatogeniccells and, as spermiogenesis progresses, testis-specifichistones are synthesized (Meistrich et al. 1985). In latesteps of spermiogenesis, almost all histones displace totransition proteins, and the latter are then replaced byprotamines during nuclear condensation of spermatidand spermatozoa (Grimes and Henderson 1984; Grimesand Smart 1985; Ward and Coffey 1991; Kistler et al.

1996; Wouters-Tyrou et al. 1998; Dadoune 2003). Dur-ing replacement of nucleoproteins, many temporarilyfunctioning proteins are formed andmight be degraded.Thus, efficient degradation machinery is required forthe removal of these proteins. However, there is vir-tually no information on the process of degradation ofproteins in the nuclei of developing spermatids.

Twocellular proteindegradation systemshavebeende-scribed: the lysosomal system and ubiquitin–proteasomesystem. It is likely that the latter is involved in thedegradation of nucleoproteins because degradation ofnuclear proteins might occur within the nuclei of sper-matids. In the ubiquitin–proteasome system, the targetproteins are conjugated to ubiquitin, which is a highlyconserved 76-amino acid polypeptide. Ubiquitin is cova-lently attached to the target protein by an isopeptide

Correspondence to: Dr. Sadaki Yokota, Biology Laboratory,Interdisciplinary Graduate School of Medicine and Engineering,University of Yamanashi, Chuo-shi, Yamanashi 409-3898, Japan.E-mail: [email protected]

Received for publication October 30, 2006; accepted January 25,2007 [DOI: 10.1369/jhc.6A7136.2007].

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bond between the C-terminal glycine of ubiquitin andthe e-amino group of a lysine in substrate protein (Chauet al. 1989; Ciechanover et al. 2000). The ubiquitinationprocess involves activities of three enzymes: activatingenzyme (E1), conjugating enzyme (E2), and protein ligase(E3) (Haas and Rose 1982; Hershko et al. 1983;Ciechanover 1994; Hershko and Ciechanover 1998). Asuccessive ubiquitin molecule is usually added to thelysine residues of the previous ubiquitin to produce apolyubiquitin chain. Polyubiquitinated proteins (pUP)are recognized and degraded by the 26S proteasome(Hough et al. 1987; Ganoth et al. 1988; Tanaka 1998).The 26S proteasome is a giant multimeric protease pres-ent in nuclear and cytosolic compartments and consists ofa 20S core complex and 19S regulatory complex. In thepresent study we used antibodies against pUP and pro-teasome subunits to search for degradation sites in thenuclei of spermatids.

The nucleus of rat spermatids contains a non-condensed nuclear area in the caudal region. This areaappears in step 9 and disappears in step 19, character-istically surrounded by a redundant nuclear envelopeand is called “nuclear pocket” (Lalli and Clermont1981; Clermont et al. 1993). This nuclear area has beenreported in spermatids and sperm of many mammals(Franklin 1968; Fawcett and Phillips 1969; Zamboniand Stefanini 1970). However, the physiologicalfunction of the nuclear pocket is currently not clear.In the present study we divided the nuclear area intotwo regions: the dense area composed of condensedchromatin and the nuclear pocket to compare the lo-calization of ubiquitin–proteasome system-related pro-teins in these two nuclear areas. Although the nuclearpocket is hardly observed in the rat epididymal sperm,it is found in human ejaculated sperm (Westbrook et al.2001). Furthermore, the human sperm nucleus containssmall clear spots and large electron-less dense areasknown as nuclear vacuoles (Zamboni 1987; Francavillaet al. 2001). Westbrook et al. (2001) suggested that thevacuoles are in continuity with the nuclear pocket.

The aim of the present study was to map the nucleo-protein degradation sites within the nuclei of sperma-tids and sperm and, hence, determine the involvementof the ubiquitin–proteasome degradation system in thenucleus of rat developing spermatids. Toward this goal,we localized two proteins, pUP and proteasome, in-volved with the ubiquitin–proteasome degradationsystem, in the nuclei of rat spermatids and sperm.More-over, considering the continuity of the nuclear vacuolesand clear spots to the nuclear pocket, we also localizedthese proteins in nuclei of human ejaculated sperm.Results showed the presence of pUP in the dense areaand nuclear pocket and proteasome in the nuclearpocket and clear spots of rat spermatids. In the nuclei ofhuman sperm, pUP was detected in the dense area,whereas proteasome was in the nuclear vacuoles and

clear spots. These results strongly suggested that theclear spots and nuclear pocket of rat spermatids, as wellas the nuclear vacuoles of human sperm, are degrada-tion sites for nucleoproteins in the nucleus.

Materials and Methods

Animal

Twenty four 9-week-old male Wistar albino rats weighing200–250 g were fed on appropriate standard diets and waterad libitum, until used. Five-week-old ICR mice were used.Human ejaculated sperm was obtained from two volunteers.All experiments were performed in accordance with the rulesand regulations of the Committee for Research at the Uni-versity of Yamanashi.

Antibodies

Mousemonoclonalantibody (MAb, cloneGC3a) againstgold-fish ovary proteasomes was described previously (Tokumotoet al. 2000). Mouse MAb (IgM) to polyubiquitinated proteins(clone FK1)was fromAffinityResearch Products (Exeter, UK).Rabbit anti-mouse IgM and horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgGwere fromDako (Tokyo, Japan).Alexa Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor568-labeled goat anti-mouse IgGwere fromMolecular Probes(Eugene, OR). Mouse serum was prepared from the blood ofICR mouse.

Preparation of Heads of Rat Epididymal Sperm

Rat caudal epididymides were dissected out and teased in ice-cold 50 mM Tris–HCl-buffered saline (TBS) containing pro-tease inhibitors (1 mM PMSF, 2 mg/ml leupeptin, 10 mg/mlpepstatin, 5 mg/ml antipain, and 10 mg/ml aprotinin) (all fromSigma-Aldrich; St Louis, MO) and 10 mM iodoacetamide(Sigma-Aldrich), an inhibitor of protein deubiquitination(Matsui et al. 1982; Baarends et al. 1999). Sperm suspensionwas filtered through 100-mm nylon mesh and centrifuged at3600 3 g at 4C. The pellet was suspended in a small volumeof TBS. Sperm heads were isolated by the method of Calvin(1976). Briefly, the sperm suspension was sonicated to cleaveheads from the tails. The resulting suspension was mixed with2.2 M sucrose to yield a final sucrose concentration of 1.8 M,loaded on discontinuous sucrose gradient (2.2Mand 2.05M),and centrifuged at 91,000 3 g for 60 min with a BeckmanOptima XL 80K ultracentrifuge (Beckman-Coulter; Fullerton,CA) equipped with a swinging bucket rotor (SW41 Ti) at 4C.Resulting pellets containing almost pure headswere suspendedin a small volume of TBS and stored at 270C until use.

Isolation of Nuclei From Homogenate of Rat Testis

The method described by Tata (1974) was used. Rat testeswere chopped in 0.32 M sucrose–3 mM MgCl2 and homog-enized in a Potter–Elvehjem homogenizer with a Teflon pestle.Homogenates were filtered through two layers of nylon bolt-ing cloth (100-mm nylon mesh), diluted with 0.6 volumes of0.32 M sucrose–3 mM MgCl2 and 0.22 volumes of distilledwater, and centrifuged at 700 3 g for 10 min. Pellets weresuspended in 2.4 M sucrose–1 mM MgCl2 and centrifugedat 50,000 3 g for 1 hr with a Beckman Optima XL 80K

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ultracentrifuge equippedwith a swinging bucket rotor (SW41Ti)at 4C. Nuclei were collected into pellets, suspended in a smallamount of 0.25M sucrose–1 mMMgCl2, and stored at270Cuntil use.

Isolation of 26S Proteasomes From Testes andLiver of Rat

Proteasomes were partially purified from testes and liver ofrats by the method of Tanaka et al. (2006). Briefly, rat testeswere homogenized in 25 mM Tris–HCl buffer (pH 7.5)containing 1 mM dithiothreitol (DTT) (Merck; Darmstadt,Germany), 2 mM ATP (Sigma; Tokyo, Japan), and 0.25 Msucrose. Homogenates were centrifuged at 70,000 3 g for1 hr with a Beckman Optima XL ultracentrifuge equippedwith an angle rotor (Type 70.1 Ti). The resulting supernatantswere recentrifuged at 70,000 3 g for 5 hr. Precipitates weredissolved in 400 ml of 25 mM Tris–HCl (pH 7.5) containing1 mMDDT, 20% glycerol, and 0.5 mM ATP and centrifugedat 20,000 3 g for 30 min to remove insoluble material. Theresulting supernatants (partially purified proteasomes) werestored at 270C.

Western Blot Analysis

pUP and proteasome subunits in rat testicular nuclei andsperm heads were analyzed by Western blotting. Protein con-centration was determined using the bicinchoninic acidmethod (Pierce Biotechnology; Rockford, IL) with BSA asthe protein standard. Protein concentration in the sampleswas adjusted to 10 mg/ml. Samples were mixed with an equalvolume of sample buffer for SDS-PAGE. SDS-PAGE wasperformed as described by Laemmli (1979), using 40 mg ofprotein (12.5% gel). Proteins were transferred to a PVDFmembrane. The membrane was soaked in PBS containing 5%(w/v) skim milk and incubated overnight with the primaryantibody, followed by 1-hr incubation with the secondaryantibody conjugated with HRP. Signals were visualized byDAB reaction.

Immunofluorescence and Immunoperoxidase Stainingof Semithin Sections

Rat testis was fixed in a fixative consisting of 4% parafor-maldehyde, 1% glutaraldehyde, and 0.15 M Hepes–KOH(pH 7.4) for 1 hr at 4C and cut into small tissue blocks. Afterwashing in PBS, tissue blocks were dehydrated and embeddedin Epon. Semithin sections (250-nm thick) were cut with ahistodiamond knife (Diatome; Biel, Switzerland) andmountedon clean glass slides. After removal of epoxy resin (Litwinet al. 1984), sections were treated with trypsin (0.5 mg/ml) for1–3 min and incubated with primary antibodies overnight.After washing in PBS, sections were incubated with HRP-labeled secondary antibodies corresponding to the primaryantibodies for 30 min. HRP activity was stained with DABreaction. For immunohistological control, sections were in-cubated with mouse serum instead of the primary antibody,followed by incubation with HRP-labeled secondary antibod-ies. For immunofluorescence staining of pUP, semithin sec-tions were stained in three steps: first with anti-pUP (mouseIgM), secondwith rabbit anti-mouse IgM,and thirdwithAlexaFluor 568-labeled goat anti-rabbit IgG. For immunohisto-

chemical controls, mouse serum was used instead of the pri-mary antibody. Sections were examined with a Nikon EclipseE600 fluorescence microscope (Nikon; Tokyo, Japan).

Immunofluorescence Staining of SpermSmear Preparations

Rat epididymal sperm and human ejaculated sperm weresmeared on clean coverglasses, air dried, and stored at 220Cuntil used. Some smear preparations were fixed in a solutioncontaining three parts methanol and one part acetic acid for10 min at room temperature and treated with 30 mM DTTfor 1 hr at room temperature. Other smear preparationswere fixed in 0.1% glutaraldehyde and 0.1 M Hepes–KOH(pH 7.4) for 30 min at room temperature. After washing with0.1 M Tris–HCl (pH 7.4), the preparations were treated with0.05% SDS for 30 min (Robinson and Vandre 1997). After-wards, smear preparations were immunostained and exam-ined as described above.

Postembedding Immunoelectron Microscopy

Rat testes were sliced in ice-cold fixative consisting of 4%paraformaldehyde, 0.25% glutaraldehyde, and 0.15 MHepes–KOH buffer (pH 7.4); fixed for 1 hr at 4C; and cutinto small tissue blocks. Tissue blocks were dehydrated ingraded ethanol series and embedded in LR White resin at220C. Human ejaculated sperm from two volunteers wasprepared in the same manner. Thin sections were cut with a

Figure 1 (A) Western blot analysis of proteins in rat testicular nuclei(Lanes 1 and 3) and sperm heads (Lanes 2 and 4). Lanes 1 and 2: pUPsignals with mouse monoclonal antibody FK1. Lanes 3 and 4: 20Sproteasome subunits detected by mouse monoclonal antibodyGC3a. Numbers, 31, 30, and 25 show molecular mass of each sub-unit, respectively. MM, molecular mass markers. (B) Western blotanalysis of proteasomes partially purified from rat testes (Lane T)and liver (Lane L) with mouse monoclonal antibody to gold fishproteasome subunits (GC3a).

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diamond knife and mounted on nickel grids. Sections werethen incubated overnight with the primary antibodies at 4C.Reacted antibodies were visualized by protein A–gold (15 nmin diameter). For immunocytochemical control experiments,sections were incubated with mouse serum instead of theprimary antibody and then with rabbit anti-mouse IgM orIgG, followed by incubation with the protein A–gold probe.All sections were contrasted with 2% uranyl acetate for5–10 min and lead citrate for 30 sec. Sections were examinedwith a Hitachi H7000 electron microscope (Hitachi; Tokyo,Japan) at an accelerating voltage of 80 kV.

Quantitative Analysis of Gold Labeling

For quantitative analysis, 10 electron micrographs for eachselected step of spermatogenic cells were taken and enlargedto 330,000. Each step was identified by the morphologicalcriteria described by Russell et al. (1990). The nuclear area oflate spermatids was divided into two: the dense area andnuclear pocket located in the neck region and surrounded byredundant nuclear envelope (Lalli and Clermont 1981). Thenuclear areas of spermatogonia and pachytene spermatocytes,dense area, and nuclear pocket of late spermatids were esti-mated by a digitizer equipped with a computer using mor-phometry software (SigmaScan; Jandel Scientific, San Rafael,CA). Gold particles in each compartment were counted and

the labeling density (gold particle number/1 mm2) was cal-culated. Background labeling was determined to measure thearea without tissue and counting of gold particles in this area.Labeling density values of each compartment reported hererepresent values after subtraction of the background label-ing density.

Results

Western Blot Analysis of Ubiquitin–ProteasomeSystem-related Proteins in Rat Testicular Nuclei andSperm Heads

Mouse MAb (FK1) specific for polyubiquitinated pro-teins developedmany bands of variousmolecular massesin the nuclear fraction of rat testis and epididymal spermheads (Figure 1A), showing that many polyubiquinatedproteins with relatively low molecular masses were con-tained in testis nuclear fraction and sperm heads. MouseMAb (GC3a) to proteasome subunits detected three ma-jor bands with molecular masses of 31, 30, and 25 kDain the nuclear fraction (Figure 1A). In sperm head ex-tract, the 31-kDa subunitwas not observed but two othersubunits were recognized (Figure 1A). These three majorbands were commonly observed in proteasomes partially

Figure 2 Immunohistochemical stainingof rat testis. (A–C)Frozen sections of rat testis were incubated with FK1 anti-body, then with rabbit anti-mouse IgM, and finally withAlexa Fluor 568-labeled goat anti-rabbit IgG. Nuclei of step10 spermatids are partially stained (A), whereas those ofstep 15 spermatids are strongly stained (B). Residual bodiesand cross-sectioned step19 spermatids are stained (C). (D–F)Semithin sections of Epon-embedded tissue were stainedby immunoenzyme technique after removal of epoxy resin.Staining for proteasome subunits. Arrowheads indicatecondensed chromosomes in dividing spermatocytes (D).Arrows indicate nuclear staining of elongated spermatids(E). Staining is noted in residual bodies and longitudinallysectioned flagella (F). Bar5 25 mm.

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purified from rat testes and liver (Figure 1B), showing thereactivity and specificity of the antibody (GC3a) for thesubunits of 20S proteasomes.

Immunofluorescence and Immunoperoxidase Stainingfor pUP and Proteasome Subunits in Rat Testis

Immunofluorescence staining for pUP identified nucleiof spermatids at steps 10–19 (Figures 2A–2C). In step10 spermatids, nuclei were weakly and partially stainedfor pUP (Figure 2A). In step 15 spermatids, nuclei werestrongly stained for pUP (Figure 2B). In stage VII, nucleiof step 19 spermatids and residual bodies were heavilystained for pUP (Figure 2C). Proteasome was stainedin the nuclei of spermatogonia, spermatocytes, andelongating spermatids (Figures 2D–2F). In addition,stained chromosomes of dividing spermatocytes werealso noted (Figure 2D). Residual bodies and flagellawere also stained for proteasomes (Figure 2F). No stain-ing was noted in sections treated with only the second-ary antibodies.

Immunofluorescence Staining for pUP andProteasome Subunits in Rat Epididymal Sperm andHuman Sperm

Diffuse stainingwasobserved forpUP (Figure3A). Stain-ing of proteasomewas evident in the small dots scatteredthroughout the nucleus of rat sperm (Figure 3B). Thenucleus of human sperm was also diffusely stained forpUP (Figure 3C). The proteasome was stained in dotsof various sizes (Figure 3D). No staining was noted insections treated only with the secondary antibodies.

Immunoelectron Microscopic Localization of pUP andProteasome in Rat Developing Spermatids

Labeling for pUP and proteasome was detected in nu-merous sites of the spermiogenic cells at various labelingintensities. In this study we focused on the localizationof these proteins in the nuclei of spermatids. Positivelabelingwas completely abolished in immunocytochem-ical control sections.

Round Spermatids (Steps 1–8 Spermatids) andElongating Spermatids (Steps 9–12). The nuclei ofspermatids from step 1 to step 12 stained weakly forpUP and proteasome. Labeling for pUP started to in-crease at steps 10–12 spermatids.

Condensing Spermatids (Steps 13–15). In step13 spermatids, gold labeling for pUP was observedin the electron-dense area, which showed progressivechromatin condensation (Figure 4A, arrows). In addi-tion, the labeling was associated with a rough networkthat appeared in the spermatids of this step. Labelingfor pUP was identified in the nuclear pocket (data notshown). No gold labeling was observed in the cyto-

chemical control sections treated with a combinationof mouse serum, rabbit anti-mouse IgM, and proteinA–gold probe (Figure 4B). Gold particles for protea-some were observed in the margins of electron-denseareas where the condensation of chromatin started(Figure 4C). Proteasome signals were also detected inthe nuclear pocket (Figure 4C, surrounded by dottedline). In immunocytochemical control sections treatedwith a combination of mouse serum/rabbit anti-mouseIgG-protein A–gold probe, no gold labeling was noted(Figure 4D). In step 15 spermatids, heavy labeling forpUP was observed in the dense area as well as in thenuclear pocket (Figure 4E). In the dense area, mostof the gold labeling was detected in the dense matrixbut not in the clear spots (Figure 4E, arrows). In con-trol sections, almost no gold particles were observed(Figure 4F). Labeling for proteasome was detected inboth areas (Figure 4G). In the dense area, approximatelyhalf of the gold labeling was associated with clearspots (Figure 4G, arrows). Labeling for proteasome

Figure 3 Immunofluorescence staining of rat epididymal sperm andhuman ejaculated sperm. (A,B) Smear preparation of rat sperm. (C,D)Human ejaculated sperm. (A,C) Staining for pUP. (B,D) Staining forproteasome. Bar 5 5 mm.

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in the nuclear pocket was stronger than in the densearea. No gold signals were observed in control sections(Figure 4H).

Condensed Spermatids (Steps 16–19). Strong labelingfor pUP was observed in the dense area of spermatids atstep 19 (Figure 5A). Most clear spots in the dense areawere devoid of gold labeling (Figure 5A, arrows). Thesize of the nuclear pocket decreased gradually andalmost disappeared in step 19 spermatids, and labelingfor pUP in this area also decreased. Moderate labeling

for proteasome was observed in the dense area of step19 spermatids, in which some gold signals were presentin the clear spots (Figure 5B, arrows). Labeling forproteasome in the nuclear pocket diminished also inthis step.

Immunoelectron Microscopic Localization of pUP andProteasome in Rat and Human Sperm

Rat Epididymal Sperm. Sperm nuclei were stronglystained for pUP (Figure 6A) but weakly or not stained

Figure 4 Immunoelectron microscopic localization of pUP and proteasome in the nuclei of steps 13 and 15 spermatids. (A–D) Step 13spermatids. (A) Staining for pUP. Gold particles showing pUP are associated with electron-dense network and condensing chromatin (arrows).(B) Immunostaining control. Most gold particles were eliminated. (C) Staining for proteasome subunits. Gold signals are present along electron-dense network and condensing chromatin and also in the caudal nuclear pocket (area enclosed by broken line). (D) Immunostaining control.Only few gold particles are seen. (E–H) Step 15 spermatids. In this step, the nuclear pocket becomes clearer, containing electron-less densematrix and surrounded by so-called redundant nuclear envelope (areas enclosed by broken lines). (E) Staining for pUP. Gold labeling isobserved in dense nuclear matrix and nuclear pocket, but not in the clear spots (arrows). (F) Immunostaining control. No gold signals areobserved. (G) Staining for proteasome subunits. In the dense nuclear matrix, most gold particles are closely associated with clear spots (arrows)and the nuclear pocket is also heavily stained. (H) Immunostaining control. Only a few gold signals are observed. Bar 5 0.5 mm.

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for proteasome (Figure 6B). Some gold particles forproteasome were associated with small clear spots(Figure 6B, arrows).

Human Sperm. The nucleus of human ejaculatedsperm contained small clear spots that were also ob-served in the nuclei of rat sperm, as well as a large cleararea representing the vacuoles (Westbrook et al. 2001).In the next step we studied the localization of pUP andproteasome in these areas. Heavy labeling for pUP wasnoted in the dense area but not in the clear spots or inthe vacuoles (Figure 7A, arrows). Gold signals forproteasome were detected exclusively in the clear spotsand vacuoles (Figures 7B, arrows).

Quantitative Analysis of Labeling Density for pUP andProteasome in Developing Spermatogenic Cells

To verify the changes in the relative amounts of nuclearpUP and proteasome during spermatogenesis, we quan-tified the labeling density for these proteins in the densearea and nuclear pocket. The dense area consists of clearspots and dense matrix. We determined the density ofgold particles in the dense area without dividing bothregions. The nuclear pocket appeared in spermatids afterstep 9 and almost disappeared in 19 spermatids. Results

are shown in Figure 8. Labeling density for pUP in thedense area was low during early phases of spermatogen-esis but started to increase in steps 10–12 spermatids andreached the maximum at step 15. Afterwards, labelingdensity remained stable until epididymal sperm. Labelingdensity for pUP in the nuclear pocketwas at a low level insteps 10–12 spermatids, increased gradually, and reacheda maximum at step 15, decreasing thereafter. Labelingdensity for pUP in the nuclear pocket was always lowerthan in the dense area. Proteasome labeling density in thedense area remained atz40 from spermatogonia to step12 spermatids and started to increase from steps 13 to 14,reached a peak value at step 15, and then gradually de-creased. In epididymal sperm, labeling density remainedstable atz30. In the nuclear pocket, labeling density forproteasome was detected first in steps 7–9 spermatidsand then started to increase, reaching a maximum valuein step 15 spermatids, decreasing thereafter.

Discussion

In the present study, Western blot analysis of protea-somes showed several bands in the nuclear fractionisolated from rat testes, which had the same molecularmass as described for proteasomes (Tokumoto et al.2000) and were commonly observed in two 20S protea-

Figure 5 Immunoelectronmicroscopiclocalization of pUP and proteasome instep 19 spermatids. (A) Staining forpUP. Strong gold labeling is observedin the dense nuclear matrix but ishardly seen in clear spots (arrows). (B)Staining for proteasome. Nuclearstaining becomes weaker. Some goldparticles are present in clear spots(arrows). Bar5 0.5 mm.

Figure 6 Immunoelectronmicroscopiclocalization of pUP and proteasome inthe nuclei of rat epididymal sperm. (A)Labeling for pUP. Note the heavy goldlabeling in the dense nuclear matrix.(B) Labeling for proteasome. Note thegold labeling in clear spots (arrows).Bar5 0.5 mm.

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some preparations partially purified from rat testesand liver, respectively. In rat sperm heads, the 31-kDasubunit was not observed but two other subunitswere detected. It was shown that the 31-kDa subunitwas the phosphorylated form of the 30-kDa subunit(Tokumoto et al. 2000). Thus, subunits of sperm nu-clear proteasomes might be modified for their function

so that they show molecular masses slightly differentfrom those in testis nuclei. Antibody FK1 reacting withpUP, but not with free ubiquitin (Fujimuro et al. 1994),detected many bands of various molecular mass in tes-ticular nuclei and in sperm heads. The molecular massof some bands observed in the testicular nuclei was sim-ilar to those in sperm head. Results indicate the spec-ificity of the antibodies used.

Our immunofluorescence studies showed localiza-tion of pUP in various areas of spermiogenic cells in-cluding Golgi apparatus, residual bodies, flagella, andnuclei. Localization of pUP in the Golgi region was alsoreported previously (Haraguchi et al. 2004). Our studyidentified proteasome in the nuclei of spermatocytesand late spermatids and in flagella. Considered to-gether, our results suggest that pUP are produced atmultiple sites of spermiogenic cells and are degraded bythe ubiquitin–proteasome system.

Immunofluorescent staining in the present study alsoidentified pUP and proteasome in the rat sperm headand in the human sperm head. Results are consistentwith those of Western blotting. Immunofluorescent lo-calization of proteasome in the sperm of othermammalshas been reported by several groups (Wojcik et al. 2000;Sutovsky et al. 2004; Pasten et al. 2005); however, itwas detected in the neck region or acrosome region butnot within the nucleus. These results are inconsistentwith ours. Differences in the results of our study andthose of others are probably based on differences in themethods used, such as cytochemical treatments includ-

Figure 7 Immunoelectron microscopiclocalization of pUP and proteasome inthe nuclei of human ejaculated sperm.(A) Labeling for pUP. Gold particlesshowing pUP are observed in the densenuclear matrix but not in the clear spots(short arrows) and in the vacuole (longarrow). (B) Labeling for proteasome.Gold labeling is observed in the clearspots (short arrows) and in the vacuoles(long arrows) but not in the dense ma-trix. Bar5 0.5 mm.

Figure 8 Quantitative analysis of ubiquitin–proteasome system-related proteins in developing spermatogenic cells. Solid bars, label-ing density in dense area; dotted bars, labeling density in nuclearpocket. Data are expressed as mean 6 SD (n58).

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ing fixation and staining procedures, as well as theantibodies used. Immunofluorescent staining results ofhuman sperm were quite consistent with the immu-noelectron microscopic results and clearly showed thedistribution of proteasome in the clear spots and in thevacuoles and that of pUP in the dense nuclear matrix.

Relationship Between Nucleoprotein Replacementand Fluctuation of pUP and Proteasome Levels inLate Spermiogenesis

Ubiquitinated histones are detected in late spermatidsof chicken and trout (Agell et al. 1983; Nickel et al.1987). In rooster, levels of ubiquitinated H2A in-crease in late spermatids, just before replacement ofhistones by protamine (Agell et al. 1983). In the mousetestis, ubiquitinated histone H2A was detected in steps8–12 spermatids (Baarends et al. 1999), whereas poly-ubiquitinated forms of histone H2A and H3 were iden-tified in steps 9–12 spermatids of rat (Chen et al. 1998).Our immunoelectron microscopic results showed local-ization of pUP and proteasome in two distinct nuclearsites in spermiogenic cells: the dense area and nuclearpocket. The latter was recognized in steps 9–19 ratspermatids and surrounded by so-called redundant nu-clear envelope (Lalli and Clermont 1981; Clermontet al. 1993). The appearance and increase of pUP in thedense area during late spermiogenesis shown in thepresent study are similar to those of nucleoproteins de-scribed previously (Kistler et al. 1996; Oko et al. 1996;Dadoune 2003). In rats, replacement of histones bytransition proteins begins at steps 11–12 and is com-plete at steps 13–15 (Kistler et al. 1996; Oko et al.1996). In addition, replacement of transition proteinsby protamines commences at about steps 13–15 and isalmost complete at steps 16–19 (Kistler et al. 1996;Dadoune 2003). On the other hand, proteasome in thedense area increases at steps 13–14, reaches peak valuesat step 15, and then decreases to low levels at step 19.Together the increase of pUP in late spermatids suggestspolyubiquitination of histones and transition proteins.Moreover, based on the fluctuation in proteasomelevel, it is likely that these proteins are involved in thedegradation of nucleoproteins.

In addition, our data showed high levels of pUP inrat epididymal sperm. Protamine is a major nucleopro-tein found in mature sperm. It is known that protamineis replaced rapidly by oocyte-derived histones after fer-tilization, but little is known about the mechanisms ofthis replacement. The released protamine might bedegraded by the ubiquitin–proteasome system. It is thensuggested that the protamines might be polyubiqui-tinated and targeted for degradation by the ubiquitin–proteasome system present in the oocyte. Results ofpreliminary studies in our laboratories showed poly-ubiquitination of some population of protamine mole-cules in late spermiogenesis.

Function of the Nuclear PocketDuring Spermiogenesis

Our idea for the function of the nuclear pocket issummarized in Figure 9. The morphology of the nuclearpocket in mammalian spermatids has been describedin detail (Franklin 1968; Fawcett and Phillips 1969;Zamboni and Stefanini 1970; Lalli and Clermont1981), but its function is not clear. We detected pUPand proteasome in the nuclear pocket. Our quantitativeanalysis showed that labeling for proteasome appearedat steps 7–9 and then increased, whereas pUP labelingstarted at step 10 and also increased thereafter. The pro-cess of nucleoprotein replacement seems to require thedegradation of a large amount of proteins such as his-

Figure 9 Schematic diagram of degradation sites in late spermatid.Cell membrane, acrosome, and neck cytoplasm are not drawn. Nu-clear pocket is the nuclear area surrounded by redundant nuclearenvelope. Proteasome was present in the clear spots, nuclear vac-uoles, and nuclear pocket. Polyubiquitinated proteins are detectedin the dense area and nuclear pocket but not in the clear spots andnuclear vacuoles. We propose that clear spots, nuclear vacuoles, andnuclear pocket are nuclear degradation sites in late spermatids.Clear spots could move directly to the nuclear pocket (black lines) orto the periphery of the dense area and be released into the sub-acrosomal space between dense area and nuclear membrane (redline), which is open to the nuclear pocket. Nuclear vacuoles couldmove directly to the subacrosomal space and the nuclear pocket(blue line), the sites of the most extensive degradation of un-necessary proteins generated during replacement of nucleoproteins.The resultant degradation products could be transported throughnuclear pores into the neck cytoplasm (green lines) connecting toresidual body.

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tones and transition proteins, as discussed above. Wepostulate in this study that these proteins might bepolyubiquitinated in the dense area as mentioned anddegraded in the clear spots and in the nuclear pocketwhere proteasome is localized. Westbrook et al. (2001)reported the presence of SPAN-X, a sperm proteinassociated with the nucleus, in the nuclear vacuole andin the nuclear pocket of human sperm, suggesting con-tinuity between these structures. They suggested thatthis continuity facilitates transport of materials such asmRNAs or proteins targeted for degradation into thecytoplasmic droplet for disposal. In rat sperm nucleus,no nuclear vacuoles are observed but, instead, smallclear spots are present. In our study we showed thepresence of proteasome in these clear spots. It is likelythat the clear spots could move to the nuclear pocket orcontinue to the nuclear pocket as considered in humansperm (Westbrook et al. 2001). Moreover, our resultsshowed the presence of proteasome in the clear spots aswell in the vacuoles in human sperm. If pUP were trans-ported to the nuclear pocket by clear spots or vacuoles,one would expect pUP signals in both structures.However, no such pUP signal was present in the clearspots and in the vacuoles. It is possible that pUP aredegraded rapidly in the clear spots or transportedrapidly to the nuclear pocket, so that they are not ob-served in these spots. Thus, our results suggest thatthe nuclear pocket functions as a site for degradationof nucleoproteins.

In conclusion, in the present study we showed thepresence of ubiquitinated proteins and proteasome inthe clear spots, nuclear vacuoles, and nuclear pocketof spermatids and sperm. These results suggest the in-volvement of the ubiquitin–proteasome system in degra-dation of temporarily functioning proteins that appearfollowing replacement of nucleoproteins. The sites fordegradation of the proteins are the clear spots withinthe dense nuclear area and the nuclear pocket.

Acknowledgments

This work was supported in part by a Grant-in-Aid(17570158) and the Center of Excellence (21COE18999997)program from the Ministry of Education, Science, Cultureand Sport (to SY).

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