Rat Spermatogenic Cell β-d -Galactosidase: Characterization, Biosynthesis, and Immunolocalization

Experimental Cell Research 261, 139 –149 (2000) doi:10.1006/excr.2000.5057, available online at http://www.idealibrary.com on

Rat Spermatogenic Cell ␤-D-Galactosidase: Characterization, Biosynthesis, and Immunolocalization Marjorie D. Skudlarek,* 1 Aida Abou-Haila,† ,1 and Daulat R. P. Tulsiani* ,2 *Department of Obstetrics & Gynecology and Department of Cell Biology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, Tennessee 37232-2633; and †UFR Biome´dicale, Universite Rene´ Descartes, 45 rue des Saints-Pe`res, 75270 Paris Cedex 06, France

In this study we have demonstrated that the rat sperm acrosomal ␤-D-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain ␤-galactosidase and four other glycohydrolases (␤-D-glucuronidase, ␣-D-mannosidase, ␣-Lfucosidase, and ␤-N-acetylglucosaminidase). With the exception of ␣-L-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of ␤-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t 1/2 of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive. © 2000 Academic Press 1

M.D.S. and A.A.-H. contributed equally to this work. To whom correspondence should be addressed. Fax: (615) 3224358. E-mail: [email protected]. 2

Key Words: spermatogenesis; germ cell ␤-galactosidase; sperm acrosome; acrosomal glycohydrolases; mammalian fertilization.

INTRODUCTION

␤-D-Galactosidase (EC 3.2.1.23) is an exoglycohydrolase which cleaves ␤-galactosyl residues from glycoproteins and glycolipids [5]. The enzyme has been reported in all mammalian tissues examined including male reproductive tissues [4] and spermatozoa [3, 25]. The enzyme is present in a latent form in the lysosomes [29] and in soluble forms in body fluids, including serum [29] and epididymal luminal fluids [26]. The soluble enzyme in the rat luminal fluid occurs in two molecular forms of 97 and 84 kDa which differ mainly in carbohydrate content [32]. The two isoforms were shown by our group to optimally cleave a synthetic substrate (p-nitrophenyl (PNP) ␤-D-galactopyranoside) at acidic pH and two glycoprotein substrates ([ 3H]galfetuin and [ 3H]gal-ovomucoid) at neutral pH [32]. The latter finding suggests that both isoforms of ␤-galactosidase would be functional at the neutral pH within the epididymal lumen. Evidence presented in earlier reports strongly suggested that the epididymal fluid ␤-galactosidases have a role in the modification of sperm plasma membrane glycoproteins during epididymal maturation of spermatozoa [31, 32]. Our group recently generated antiserum against homogenous preparations of the 97-kDa isoform of ␤-galactosidase purified from rat epididymal fluid. The monospecific antibody (IgG fraction) purified from antiserum was used to immunolocalize the enzyme in fixed testicular sections and spermatozoa. The indirect immunostaining protocol used on paraffin sections of adult rat testis localized the enzyme in round spermatids beginning in stage IV of the seminiferous epithelium cycle, but failed to reveal immunopositive reaction in late spermatocytes [3]. Since several exoglycohydrolases including ␤-D-glucuronidase [1, 2], N-acetyl ␤-D-glucosaminidase [12, 17] and ␣-L-fucosi-

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dase [11], believed to be important in mammalian fertilization [2, 28], are first expressed in spermatocytes, we wondered if ␤-galactosidase truly has a unique distribution. Therefore, a new high-titer antibody was generated for further characterization of the enzyme. The purposes of studies reported here were (i) to examine expression of the enzyme in rat testicular germ cells and (ii) to examine its synthesis and processing in the purified spermatogenic cells. Biochemical studies included in this report indicate that ␤-galactosidase, like other glycohydrolases, is first expressed in the diploid germ cells. Multiple biochemical approaches as well as biosynthetic studies in highly enriched populations of testicular germ cells indicate that spermatocytes and round spermatids contain several molecular forms of the enzyme, some of which appear to be processed in a cell-specific manner. MATERIALS AND METHODS Materials. Adult male Sprague–Dawley rats (3– 4 months old) were either from Sasco (Omaha, NE) or from Centre de Production Animale (Orle´ans, France). The animals were housed in our animal facilities under 16L:8D conditions with free access to food and water for at least 3 days before any experiment. The animals were either anesthetized (see below) or sacrificed by CO 2 asphyxiation. All procedures used were approved by the Institutional Animal Care Review Board. All PNP– glycopyranoside substrates, gold-labeled (10 nm) anti-rabbit (IgG) goat IgG fraction, bovine serum albumin (fraction V), p-aminophenyl ␤-D-thiogalactopyranoside immobilized on 4% beaded agarose, and a fluorescein-labeled affinity-purified antirabbit (IgG) goat IgG fraction (1.5 mg protein/ml) were from Sigma Chemical Co. (St. Louis, MO). Other reagents used for immunoperoxidase studies were from Vector Laboratories (Burlingame, CA). Glutaraldehyde EM grade (2%) used for electron microscopy was from Taab Laboratories (England), and Lowicryl K4M was from Chemische Werke Lowi Gmbh & Co. (Wailkraiburg, Germany). All analytical gel electrophoresis reagents, including electrotransfer and immunoblotting reagents, biotinylated standard marker proteins, and the protein assay kit, were from Bio-Rad Laboratories (Richmond, CA). [ 35S]Methionine (1160 Ci/mmol) was purchased from NEN Research Products (Boston, MA). Medium for culturing of testicular germ cells was derived from the protocol of Grootegoed et al. [10] as described previously [24], except that the germ cells were starved for 30 min in methionine-free minimum essential medium (MEM) before labeling with [ 35S]methionine. All other chemicals were obtained commercially and were of the highest purity available. Purification of ␤-galactosidase and production of antiserum. ␤-Galactosidase (97-kDa form) was purified to apparent homogeneity from rat epididymal fluid by our published procedure [32]. The enzyme preparations were used for the production of antiserum by immunizing a female virgin New Zealand White rabbit (⬃1.5 kg). Briefly, the rabbit was immunized with 200 ␮g of the enzyme protein, emulsified in Freund’s complete adjuvant as described [34]. Immunization was repeated every 8 –10 days with the same amount of purified enzyme emulsified in Freund’s incomplete adjuvant. Blood was collected by cardiac puncture after a total of five immunizations (on day 63) and serum was prepared as described [34]. Affinity-purified polyclonal antibody (rabbit IgG fraction) was prepared from the preimmune or immune serum on a column of immobilized protein G as described [33]. The monospecific IgG fraction was prepared on a column of immobilized ␤-galactosidase.

Isolation of testicular germ cells. Mixed germ cells were prepared following enzymatic disruption of rat testes as described [19]. Highly enriched spermatogenic cells (spermatocytes, round spermatids, condensed/elongated spermatids, and residual bodies) were prepared from the mixed germ cells on a Staput sedimentation chamber by unit gravity sedimentation on 1200 ml of 2– 4% linear BSA gradient by the procedure of O’Brien [19] as stated previously [20]. Preparation of germ cell extracts. Highly enriched (⬎95% pure) spermatocytes (100 ⫻ 10 6 cells) or round spermatids (206 ⫻ 10 6 cells) were suspended in 1 ml of ice-cold 20 mM phosphate– citrate buffer, pH 4.3, containing 0.1 M NaCl, 1% Triton X-100 (v/v), and 25 mM benzamidine, and the suspension was sonicated for five 10-s bursts using a Fisher sonicator (Model 300, Fisher sonic dismembrator) set at energy level 40. The sonicated cells were centrifuged at 105,000g for 30 min in a Beckman ultracentrifuge at 4°C. The supernatant was removed by aspiration and the residue was extracted one more time by suspending in a small volume (0.5 ml) of the above buffer followed by sonication and centrifugation as above. The combined supernatant, containing ⬎95% of the initial ␤-galactosidase activity present in a given cell suspension, was designated germ cell extract and used for further studies. Affinity column chromatography. Unless otherwise stated, all purification steps were carried out at 0 – 4°C. ␤-Galactosidase activity present in the germ cell extracts was affinity purified on an immobilized p-aminophenyl ␤-D-thiogalactopyranoside column (0.5 ⫻ 6.0 cm) equilibrated with the above phosphate– citrate buffer containing 0.1 M NaCl. The germ cell extracts were separately applied to the column at a flow rate of 2 ml/h followed by washing with 25 ml of the above buffer at a flow rate of 4 –5 ml/h. The bound enzyme was eluted with the above buffer containing 7 M urea [32]. The enzymatically active fractions were pooled, concentrated to a small volume using a microconcentrator, dialyzed against 10 mM Tris–HCl buffer containing 0.1 M NaCl and 0.02% sodium azide, and assayed for PNP–␤galactosidase activity. Aliquots containing 0.05– 0.08 units/tube were dried in a Speed Vac and stored frozen at ⫺20°C. Localization of ␤-galactosidase in testicular germ cells and spermatozoa. The ␤-galactosidase was immunolocalized using monospecific IgG fraction using three different protocols. For immunostaining of testicular sections, rats were anesthetized by intraperitoneal injection of 6% chloral, and the tissues were fixed by perfusion through the left ventricle with Bouin’s fixative. The testes were removed, cut into small pieces, and immersed for 20 h in the same fixative. After fixation, the tissue pieces were dehydrated and embedded in paraffin before thin sections (⬃5 ␮m) were prepared. The sections were freed from paraffin and hydrated as described [1]. The sections were quenched for endogenous peroxidase activity followed by blocking the nonspecific binding sites with normal goat serum. The sections were incubated with the preimmune/immune IgG fraction (5 ␮g protein/ml) for 1 h at 37°C followed by diluted goat anti-rabbit IgG, avidin– biotin– horseradish complex (ABC kit, Vector Laboratories), and the immunopositive reaction was revealed by our published procedure [1]. Immunofluorescence studies. The binding of monospecific (experimental) or preimmune (negative control) IgG to the mixed germ cells and spermatozoa (before and after permeabilization with methanol) was examined by indirect immunofluorescence as described [1, 30], except that the concentration of primary antibody (monospecific/ preimmune IgG) was reduced to 5 ␮g protein/ml. Following incubation in diluted FITC-labeled secondary antibody, the cells were washed in PBS, mounted, and observed under a confocal microscope using Nomarski differential interference contrast optics [2]. Immunoelectron microscopy. An adult rat was anesthetized as above and the tissues were fixed by perfusion through the left ventricle for 10 min with 1% buffered glutaraldehyde as described [1]. Following this fixation, each testis and cauda epididymidis was removed, cut into small pieces, and immersed in the above fixative for

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TABLE 1 Glycohydrolase Activities in Rat Testicular Germ Cells a Germ cells (m units/10 6 cells) Enzyme b

SC

RS

C/ES

RB

␤-D-Galactosidase ␤-D-Glucuronidase ␣-D-Mannosidase ␣-L-Fucosidase ␤-N-Acetylglucosaminidase

12.4 ⫾ 2.9 1.6 ⫾ 0.2 15.3 ⫾ 1.1 44.8 ⫾ 2.7 31.0 ⫾ 2.7

6.6 ⫾ 0.4 0.8 ⫾ 0.1 7.5 ⫾ 0.5 51.1 ⫾ 3.0 7.4 ⫾ 0.2

3.7 ⫾ 0.6 0.5 ⫾ 0.1 4.3 ⫾ 0.2 21.5 ⫾ 0.4 4.3 ⫾ 0.6

3.9 ⫾ 0.2 0.5 ⫾ 0.1 5.2 ⫾ 0.6 36.7 ⫾ 0.7 4.3 ⫾ 0.2

a The germ cells were prepared from rat testis as described under Materials and Methods. Fractions rich in spermatocytes (SC), round spermatids (RS), condensed/elongated spermatids (C/ES), and residual bodies (RB) were separately pooled and used for the enzyme assays. The purity of pooled germ cells was examined by phase-contrast microscopy as described [20]. The cell purity was SC ⬎95% and RS ⬎90, C/ES composition was condensed 90% and elongated 10%, and RB ⬎85%. Values are average of four separate experiments in triplicate with ⫾ SD. b All enzymes were assayed using PNP– glycoside substrates as described under Materials and Methods.

50 min at room temperature. The tissues were dehydrated and embedded in Lowicryl K4M. Ultrathin sections were prepared and incubated with primary antibody (monospecific or preimmune IgG, 5 ␮g protein/ml) as described [1]. Following incubation with the goldlabeled secondary antibody, the sections were stained with uranyl acetate before electron microscopy. Radiolabeling of spermatocytes and round spermatids. Enriched populations of spermatocytes (⬎95% pure, impurities being 1.2% Sertoli cells; 2.4% round spermatids; and 1.2% condensed/elongated spermatids) or round spermatids (⬎95% pure, impurities being 4.5% condensed/elongated spermatids) were pooled and 1.5 ⫻ 10 7 cells/ml were incubated in 3 ml of methionine-free MEM. After 30 min at 34°C, the methionine-depleted cells were centrifuged at 400g for 5 min, and the pelleted cells were suspended in the above medium containing [ 35S]methionine (400 ␮Ci/ml) and incubated at 34°C under 5% CO 2 in air. After 30 min of labeling (pulse), the cells were pelleted and washed four times, by suspending in 2 ml of PBS and centrifugation (400g/5 min). The washed cells were either frozen (pulse) or suspended in 3 ml of medium containing nonradioactive methionine and cultured for pulse/chase studies as described in each experiment. Extraction, immunoprecipitation, and SDS–PAGE of 35S-labeled ␤-galactosidase. The frozen cells were suspended in ice-cold 0.1 M Tris–HCl buffer, pH 7.5, containing 0.15 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and a protease inhibitor cocktail [3]; sonicated; and centrifuged at 105,000g/30 min. Supernatant was removed by aspiration and supplemented with unlabeled enzyme carrier (2 units), and the supernatant was mixed with 60 ␮g of affinity-purified immune or preimmune IgG. Samples were incubated at 34°C for 30 min followed by overnight incubation at 4°C with gentle rocking. Following these incubations, the immunoprecipitates were collected by centrifugation [3]. The immune/preimmune precipitates were washed five times with Tris–NaCl–SDS buffer by suspension and centrifugation, dissolved in urea–SDS and electrophoresed on 7% polyacrylamide gels (SDS–PAGE) under reducing conditions [15]. Gels were exposed to Biomax MR film, and the radioactive bands were revealed by processing after 1 week. In pulse-chase studies, individual bands were cut from gels and solubilized in NCS solubilizer (24), and the radioactivity was measured by liquid scintillation spectroscopy. Electrotransfer and immunoblotting. Affinity-purified ␤-galactosidase isoforms were separated by SDS–PAGE, and the resolved polypeptides were transferred to nitrocellulose sheets by the method of Towbin et al. [27]. The immunoreactive isoforms were detected as described [23]. Enzyme and protein assays. PNP– glycosidase activities were quantified by measuring the release of p-nitrophenol in a standard

incubation mixture (0.5 ml) containing 5 mM substrate, 0.2% Triton X-100, and the desired buffer as described [29]. After incubation for 1 h at 37°C, the reaction was stopped by the addition of 1.0 ml of alkaline buffer adjusted to pH 10.7 [29]. The p-nitrophenol released was quantified by measuring the absorbance of the samples at 400 nm. One unit is the amount of enzyme that catalyzes the release of 1 ␮mol PNP/h. Protein was measured by calorimetric method of Bio-Rad according to the manufacturer’s instructions, using bovine serum albumin as the standard protein.

RESULTS

Glycohydrolase Activities in Rat Spermatogenic Cells Highly enriched populations of testicular germ cells (spermatocytes, round spermatids, condensed/elongated spermatids) and residual bodies were prepared by a unit gravity sedimentation protocol as described under Materials and Methods. The procedure allowed us to obtain enriched (⬎90% purity) germ cells and residual bodies. The isolated germ cells were assayed for ␤-galactosidase and four other acid glycohydrolase activities. Data presented in Table 1 demonstrate the presence of all five glycohydrolase activities in rat spermatogenic cells. With the exception of ␣-fucosidase activity which was similar in spermatocytes and round spermatids, the other four glycohydrolases demonstrated two-to threefold higher enzyme activities per cell in the diploid cells. Furthermore, the sum of the glycohydrolase activities present in condensed/elongated spermatids and residual bodies nearly equals the amount of the enzyme present in the round spermatids (Table 1). These results provided evidence suggesting that sperm-associated glycohydrolase activities are first expressed in the diploid (spermatocytes) spermatogenic cells. Characterization of Germ Cell ␤-Galactosidase Activity Since ␤-galactosidase occurs in several molecular forms [3, 32], attempts were made to identify and char-

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Synthesis and Turnover of ␤-Galactosidase Precursor Forms in Spermatocytes and Round Spermatids

FIG. 1. Immunoblot analysis of ␤-galactosidase purified from rat testicular germ cells. Approximately 0.05 and 0.08 units of affinitypurified enzyme from spermatocytes (lanes 1 and 2) and spermatids (lanes 3 and 4) were resolved on SDS–PAGE and the various molecular forms were identified by Western blot analysis using either affinity-purified immune (lanes 2 and 4) or preimmune (lanes 1 and 3) IgG fractions. Other details are described under Materials and Methods. The identity of the 103-kDa band seen in the immune lane of the round spermatids (lane 4) is not known. The positions of biotinylated standard marker proteins are shown on the left. Molecular weight ⫻ 10 ⫺3.

acterize the molecular forms of the enzyme present in the diploid and haploid germ cells. This was done by first purifying the enzyme from the germ cell extracts by affinity chromatography on a column of immobilized p-aminophenyl ␤-D-thiogalactopyranoside as described under Materials and Methods. The ␤-galactosidase activities purified from spermatocytes and round spermatids were resolved by SDS–PAGE and transferred to a nitrocellulose sheet, and the immunoreactive polypeptides were detected by Western blot analysis. Data presented in Fig. 1 demonstrate that under steady-state conditions, both diploid and haploid germ cells contain several molecular forms of ␤-galactosidase ranging in size from 103 to 62 kDa. Interestingly, the 103- and 62-kDa immunoreactive polypeptides present in the round spermatids were not detected in the spermatocytes (Fig. 1).

Cultured spermatogenic cells were incubated for 30 min with [ 35S]methionine, and the newly synthesized enzyme from the spermatocytes and round spermatids was detected by immunoprecipitation and electrophoresis in SDS–PAGE (Fig. 2). Following an initial 30-min labeling (pulse), the spermatogenic cells were pelleted, washed, and incubated for 10 h in nonradioactive methionine medium as described under Materials and Methods. ␤-Galactosidase from the diploid and haploid cells was immunoprecipitated, and the molecular forms were detected by electrophoresis and autoradiography as described under Materials and Methods. Synthesis of large molecular weight forms of ␤-galactosidase (90/88 kDa) in the spermatocytes (Fig. 2A, lanes 2 and 3) and round spermatids (Fig. 2B, lanes 2 and 3) was followed by its conversion to lower molecular weight forms (64/62 kDa) after 10 h of chase (Figs. 2A and 2B, lanes 4 and 5). Quantitative data for the time course of conversion of precursor forms to mature forms are presented in Fig. 3. No radiolabel was lost from the precursor form (90/88 kDa) in round spermatids for up to 2 h after initiation of the chase. In contrast, over 15% of the precursor forms were converted to the 64-kDa mature form in the spermatocytes within 2 h. Data presented in Fig. 3 allowed us to calculate the t 1/ 2 for turnover of the ␤-galactosidase precursor to the mature forms as 6.5 and 9 h in spermatocytes and round spermatids, respectively. Another obvious difference between these germ cells was the molecular weight of the mature forms after a 10-h chase. In spermatocytes, most of the radioactivity was incorporated into 64-kDa mature form, whereas a 62kDa radioactive band was the predominant mature form in round spermatids (Figs. 2 and 3).

TABLE 2 Apparent K m for Rat Spermatogenic Cell and Cauda Sperm ␤-Galactosidase

Kinetic Properties of ␤-Galactosidase in the Spermatogenic Cells and Spermatozoa

␤-Galactosidase activities present in the spermatogenic cells and spermatozoa were found to optimally cleave PNP– galactoside at an acidic pH of 3.5 (data not shown). The PNP–substrate concentration vs enzyme activity studies using enriched populations of germ cells generated a linear double-reciprocal plot for all cells. An apparent K m calculated from the plots is presented in Table 2.

a

Germ cell a

K m (mM) b

Spermatocytes Round spermatids Condensed/elongated spermatids Residual bodies Cauda spermatozoa

0.80 0.82 0.83 1.02 0.52 c

Rat germ cells and residual bodies were prepared as described under Materials and Methods. The purity of germ cells was similar to the values reported in Table 1. b Values reported were calculated from a linear double-reciprocal plot generated following substrate concentration vs enzyme activity using PNP– galactoside as substrate at pH 3.5. c The apparent K m reported is from a previously published report [25].

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FIG. 2. Synthesis of ␤-galactosidase and conversion of the newly synthesized precursor to mature forms. Enriched populations of spermatocytes (A) or round spermatids (B) were pooled, and 1.5 ⫻ 10 7 cells/ml were incubated in 3 ml of methionine-free MEM for 30 min, followed by incubation in methionine-free MEM supplemented with [ 35S]methionine (400 ␮Ci/ml) as described under Materials and Methods. Following 30 min of pulse labeling (lanes 2 and 3), the cells were washed and incubated (chase) in nonradioactive medium for 10 h (lanes 4 and 5). After this incubation, the cells were washed and sonicated, and the radiolabeled ␤-galactosidase was immunoprecipitated as described under Materials and Methods. Immunoprecipitates were electrophoresed on 7% polyacrylamide gels containing SDS (SDS–PAGE) at 30 mA constant current and prepared for fluorography. Lane 1, 14C-labeled standard marker proteins (Amersham); lanes 2 and 4, preimmune IgG; and lanes 3 and 5, immune IgG. Molecular weight ⫻ 10 ⫺3.

Localization of ␤-Galactosidase in Rat Spermatogenic Cells and Cauda Spermatozoa In a recent report, our group presented immunohistochemical results in the testis suggesting that ␤-galactosidase is first expressed in round spermatids beginning at stage IV of the seminiferous epithelium cycle [3]. However, the studies failed to reveal immunopositive reaction in spermatocytes. Since several acid glycohydrolases are first expressed in spermatocytes [1, 11, 12, 16] and the fact that our biochemical approaches provided evidence suggesting that the enzyme is present in the diploid cells (see above), we prepared a new polyclonal antibody with titer 2.5 to 3 times higher than the previous antibody by immunizing the rabbit with a higher amount of the purified ␤-galactosidase. In addition, since preservation of the antigenic sites during tissue fixation is an important factor which may have an influence on the immunopositive/immunonegative results, we attempted localization studies with the new antibody using multiple approaches. First, we examined the binding of preimmune/immune (monospecific) IgG fraction to the rat cauda spermatozoa by indirect immunofluores-

cence microscopy carried out before (live sperm) or after permeabilization with methanol. Since similar studies were published recently [3], they are briefly summarized as follows: (i) cauda spermatozoa treated with preimmune IgG showed immunostaining on the midpiece before or after permeabilization with methanol. (ii) The nonpermeabilized sperm treated with immune IgG showed nonspecific immunostaining on the midpiece. (iii) In contrast, permeabilized sperm treated with immune IgG revealed intense immunopositive staining mostly on the dorsal side (acrosome region) of the sperm head. (iv) Most of the cytoplasmic droplets were detached from the spermatozoa and displayed intense immunostaining; however, the very few cytoplasmic droplets still attached to sperm showed light staining only when the cells were permeabilized. These data were similar to earlier findings with sperm cells [3] and suggested that the new antibody was suitable for immunolocalization studies. The acrosomal localization of ␤-galactosidase was confirmed by electron microscopy carried out on rat cauda epididymidis sections. The immunopositive reaction was seen in the epididymal epithelial cells (data

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FIG. 3. Kinetics of loss of radiolabel from precursor forms of ␤-galactosidase and its simultaneous incorporation into mature forms. Enriched spermatocytes or round spermatids were incubated in 3 ml of MEM supplemented with [ 35S]methionine (400 ␮Ci/ml) for 30 min (pulse) as described in the legend to Fig. 2. The cells were washed and reincubated in nonradioactive medium for various time intervals (chase). At the indicated time, the cells were washed and the radiolabeled ␤-galactosidase was immunoprecipitated as described under Materials and Methods. Immunoprecipitates were electrophoresed on 7% SDS–PAGE under reducing conditions. Radioactivity was quantitated in individual band(s) as described under Materials and Methods. In this representative experiment, each point represents the mean of two samples. Apparent molecular weight of the 90/88-kDa precursor forms (F) and the 64-kDa (E) and 62-kDa (Œ) mature forms was calculated using 14C-labeled standard marker proteins. Half-lives of precursor forms were obtained from linear regression analysis.

not shown) and in the intraacrosomal region of the sperm head as evident by the presence of gold particles only when the immune IgG was used. The gold particles were present in the acrosomal head cap, but not in the equatorial region (see below in Fig. 6F).

It should be noted that ␤-galactosidase and other glycohydrolases present in the sperm acrosome are synthesized and packaged in the acrosome during sperm development in the testis [2]. It was, therefore, important to examine stage-specific localization of the enzyme during spermatogenesis. This was attempted using light and electron microscopic approaches in three different ways. First, we examined the binding of preimmune (Fig. 4A) and immune (Figs. 4B– 4D) IgG to the testicular cells present in paraffin sections of the rat testis using the immunoperoxidase approach. Data show the following: (i) No immunopositive reaction was seen in the somatic (Leydig or Sertoli) or spermatogenic cells when preimmune IgG was used as the primary antibody; and (ii) an intense immunopositive reaction was seen in Leydig cells (data not shown) and a moderate reaction in the cytoplasm of Sertoli cells extending perpendicularly throughout the epithelium (Fig. 4B). In the spermatogenic cells, immunopositive reaction was confined to granules dispersed in the cytoplasm around the nucleus of the late spermatocytes (pachytene spermatocytes) as well as the round spermatids (Figs. 4B– 4D). The Golgi apparatus and the acrosomal vesicle seen at one pole of the round spermatids show an intense immunopositive reaction (Fig. 4C). The forming acrosome in the elongated spermatids was immunopositive (Fig. 4D). In addition, an intense immunopositive reaction was seen in the residual bodies present near the lumen of the seminiferous tubule (Fig. 4B). In the second approach, we examined the binding of preimmune or immune IgG to isolated germ cell populations before and after permeabilization with methanol

FIG. 4. Distribution of ␤-galactosidase in rat testis during spermatogenesis. Testicular sections were treated with the preimmune (A) or immune (B) IgG fraction (5 ␮g protein/ml) and the immunoreaction was detected as described under Materials and Methods. Note the presence of an immunopositive reaction in the cytoplasm of Sertoli cells (arrows) and granules dispersed in the cytoplasm around the nucleus of pachytene spermatocytes (p), round spermatids (rs), and the forming acrosome in elongated spermatids (es). The formed acrosomal vesicle (arrowhead) in round spermatids (C) and the residual bodies (rb) show an intense immunopositive reaction. Original magnification ⫻1200. Stages of cycle of seminiferous epithelium in B–D are: B, stage VI; C, stage V; and D, stage VII.

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FIG. 5. Confocal micrographs showing immunolocalization of ␤-galactosidase in the forming acrosome during spermiogenesis in the rat. The testicular germ cells were prepared and immunostained using anti-␤-galactosidase (monospecific IgG, 5 ␮g protein/ml) as primary antibody and FITC-labeled anti-rabbit goat IgG as the secondary antibody as described under Materials and Methods. Various phases of acrosome formation were photographed with a confocal microscope using Nomarski differential interference contrast optics (A–E) and immunofluorescence (F–J). Note the presence of intense fluorescence in the Golgi apparatus of stage 6 round spermatids (F) and the forming acrosome during progressive transformation of the elongated spermatids (G–I) and a testicular spermatozoon with fully developed acrosome (J). The elongated spermatids are: stage 8, B & G; stage 10, C & H; and stage 17, D & I. The absence of a flagellum on the elongated spermatids is due to its loss during preparation of the spermatogenic cells by enzymatic disruption of the testis.

using indirect immunofluorescence microscopy as described under Materials and Methods. The cells were observed with a confocal microscope using differential interference phase-contrast optics (Normarski). This was done in an attempt to follow the successive formation of the acrosome during the cap phase and the elongation phase of spermiogenesis. Data from this approach, presented in Fig. 5, are summarized as follows: (i) The spermatogenic cells (late spermatocytes and round spermatids) were immunostained only when the cells were permeabilized and the immune IgG was used as the primary antibody. (ii) The immunopositive reaction was diffuse over the cell cytoplasm and was more intense in vesicle-like structures present throughout the cytoplasm of the late spermatocytes and early round spermatids (data not shown). At the later stages of spermiogenesis (round spermatids in stages 5–7), an intense fluorescent cap-like structure appears at one pole of the cell (Figs. 5A and 5F). As spermiogenesis continues, several immunopositive areas were seen over an enlarging cap-like structure extending from the base of the nucleus and covering the cell cytoplasm (Figs. 5B and 5G). The immunopositive structure extends over the cell nucleus in the early elongated spermatids (Figs. 5C and 5H). In the late stage of spermatid elongation, an intense compact immunopositive reaction becomes confined to a distinct sickleshaped structure (Figs. 5D and 5I) which was also seen in the testicular spermatozoa (Figs. 5E and 5J). In the final approach, we used the postembedding immunogold procedure to localize the ␤-galactosidase in the testicular sections. An intense immunopositive

reaction was seen in the lysosomes of the somatic cells (data not shown) while in the spermatogenic cells, the enzyme was first seen in the late spermatocytes (pachytene spermatocytes) as evident by the presence of gold particles in the Golgi apparatus and lysosomelike structures (Fig. 6A). The immunolabeling was more obvious in the Golgi-associated vesicles of early round spermatids (Fig. 6B). In the round spermatids of the early Golgi phase (stages 2–3), granules present in the trans-Golgi region (presumably the proacrosomal granule) showed intensive immunolabeling in the floculent material surrounding the acrosomal granule (Figs. 6B and 6C). The round spermatids during the cap phase (stage 5) showed intense immunolabeling in the head cap extending over the nucleus and lower labeling of the acrosomal granule. In addition, gold particles were seen in small vesicles dispersed over the outer acrosomal membrane in the trans-Golgi region (Fig. 6D). The gold particles were present in the head cap of elongated spermatids (stages 8 –16) and became essentially confined to the acrosomal cap of maturing spermatids (Fig. 6E) and cauda epididymal spermatozoa (Fig. 6F). Control sections incubated in the presence of either preimmune IgG or immune IgG preadsorbed with a fivefold excess of purified ␤-galactosidase were negative (data not shown). DISCUSSION

Our group recently used an indirect immunofluorescence protocol to demonstrate that acid ␤-galactosidase

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FIG. 6. Distribution of ␤-galactosidase in the rat during spermatogenesis. Note the presence of gold particles in the Golgi stacks (arrowhead) and the Golgi vesicles as well as in lysosome-like (L) structures of the pachytene spermatocyte (A) and the Golgi stacks (arrowhead), Golgi-associated vesicles, and proacrosomal granule (pg) of early stages 2–3 of round spermatids (B, C), acrosomal vesicle (marked as A) and head cap (star) in stage 5 of round spermatids (D), and acrosomal head cap of stages 17–18 of the maturing spermatids (E) and cauda epididymal spermatozoa (F). Note the presence of some gold particles in the cytosol of Sertoli cells surrounding the spermatid in E. N, nucleus. Original magnification ⫻54,000 (A) and ⫻40,000 (B–F).

is localized within the rat sperm acrosome and cytoplasmic droplets [3]. This localization, in general, was in agreement with biochemical and morphological studies suggesting that several acid glycohydrolases, including ␤-galactosidase, are localized in the sperm acrosome [3, 25] and cytoplasmic droplets [3, 7, 8, 25]. Since acrosomal glycohydrolases [1, 2, 28], including ␤-galactosidase [18], may be important in interactions

with the vestments surrounding the ovulated egg, we have extended these studies to include localization at the light and electron microscopic level using multiple approaches. The enzyme was confined to the sperm acrosome head cap and not in the equatorial segment, as has been demonstrated for ␤-glucuronidase [1] and acrosin [9]. Although the reason for this specialized localization is not yet known, the equatorial region

MALE GERM CELL ␤-D-GALACTOSIDASE

persists even after the acrosomal contents are lost following the acrosome reaction [6]. The biochemical approaches demonstrated that the enzyme was present in purified spermatocytes, round spermatids, condensed/elongated spermatids, and residual bodies (Table 1). When the acid glycohydrolase activities were expressed per 10 6 cells, the spermatocytes showed two- to threefold higher activity for most enzymes assayed (Table 1), a result in agreement with earlier studies indicating that ␤-glucuronidase [1] and N-acetyl ␤-D-glucosaminidase [12, 16, 17], two other glycohydrolases, showed higher enzymatic activities in spermatocytes than round spermatids. Whether the high levels of glycohydrolase activity in the spermatocytes are due to a high rate of synthesis and/or slow degradation is not yet known. As discussed below, the half-life for the disappearance of precursor forms and appearance of mature forms is somewhat shorter in spermatocytes than in round spermatids (Fig. 3). Since enzymatically active mature forms of acid glycohydrolases are formed by the proteolytic processing of the enzymatically inactive precursor forms [21], it is reasonable to suggest that the observed differences in the half-life for the processing of the ␤-galactosidase precursors, and perhaps other glycohydrolases, contribute to the higher enzymatic activity in the spermatocytes. The presence of glycohydrolase activity in rat spermatocytes was not surprising since several glycohydrolases [1, 11, 12], including ␤-galactosidase [16, 17], are first expressed in diploid cells. However, this localization contrasts with a recent report from our group suggesting that ␤-galactosidase is first expressed in haploid germ cells [3]. Thus, a new high-titer antibody was produced for immunolocalization, biochemical, and synthetic studies. The monospecific antibody effectively immunoprecipitated the ␤-galactosidase activity present in the detergent solubilized spermatocytes, round spermatids, and cauda epididymal spermatozoa. These results, and the fact that ␤-galactosidase activities present within the germ cells and spermatozoa have similar pH optima, allowed us to suggest that the spermatogenic cells and spermatozoa contain immunologically and kinetically similar ␤-galactosidase activities. However, it should be noted that the K m for germ cell ␤-galactosidase is somewhat higher than that of the enzyme present in cauda spermatozoa (Table 2). A likely explanation is that whereas the germ cells possess precursor and mature forms of ␤-galactosidase (Figs. 1 and 2), the cauda spermatozoa possess only the mature form of the enzyme [3, 25]. The mature form of ␤-galactosidase present in spermatozoa is expected to have high affinity (and low K m) for the PNP–␤-D-galactopyranoside substrate. The Western blotting approaches demonstrated that under steady-state conditions, spermatocytes and round spermatids accumulate several common molecular forms

147

of ␤-gal including 88-, 70-, and 64-kDa monomers (Fig. 1). The 70-kDa monomer is likely the de-N-glycosylated form demonstrated by us to be generated following Nglycanase treatment of the newly synthesized ␤-galactosidase in round spermatids [3] and the enzyme present in the rat spermatozoa [25] and epididymal luminal fluid [32]. Interestingly, the 62-kDa form is expressed in a cell-specific manner, being present only in round spermatids (Fig. 1). We do not know at the present time whether the 103-kDa polypeptide seen in the immunoblots of the round spermatids in Fig. 1 is related to ␤-galactosidase. Since the polypeptide was not detected in biosynthetic studies (Fig. 2), we favor the possibility that this polypeptide is not related to ␤-galactosidase. A less likely possibility would be that the t 1/2 for the appearance of 103 kDa is longer than the chase time of 10 h reported in Fig. 2. Studies using longer chase times were not possible since germ cells are viable in culture for a period of 16 –18 h [19]. In order to examine synthesis and processing of ␤-galactosidase, purified spermatocytes and round spermatids were pulse labeled with [ 35S]methionine in culture, the newly synthesized enzyme was immunoprecipitated and resolved by SDS–PAGE, and the radiolabeled polypeptides were detected by autoradiography. Data presented in Fig. 2 demonstrate the presence of radioactivity in high-molecular-weight (90/88-kDa) forms in both diploid and haploid cells during an initial 30-min pulse (Figs. 2A and 2B, lanes 2 and 3), which were processed to lower molecular weight (64/62-kDa) forms during a chase period (Figs. 2A and 2B, lanes 4 and 5). The observed decreased radioactivity in higher molecular weight forms and its appearance in lower molecular weight forms during the chase indicates a precursor 3 product relationship. Data presented here confirm earlier studies from our group suggesting that ␤-galactosidase is synthesized in precursor forms of 90/88 kDa in round spermatids. In addition, we present evidence that similar precursor forms are synthesized in spermatocytes. However, the precursor forms of the enzyme in this report were processed to the 64/62-kDa rather than 56-kDa mature form seen in the earlier report [3]. We have no ready explanation for these differences. One possible explanation could be the purity of round spermatids used in the two studies. In this report, we used highly enriched round spermatids (⬎95% purity) compared to less purified cells (40% round spermatids, 50% residual bodies, and 10% elongated spermatids) used earlier. Alternatively, the new antibody used in this study may have detected different protein bands. Whatever the real reason, the fact that immunoblotting and biosynthetic approaches indicated similar mature forms in the round spermatids adds more credibility to the data presented here. The kinetics of [35S]methionine labeling into higher molecular weight proenzymes and their gradual process-

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ing in spermatocytes and round spermatids suggest a short life span (time before the formation of mature forms) for the proenzymes (Fig. 3). In spermatocytes, ␤-galactosidase appears to have life span kinetics of 1–2 h prior to its processing to the 64/62-kDa form. However, the enzyme in round spermatids has higher life span kinetics (⬎2 h). Once started, the half-life for processing of the enzyme was 6.5 and 9 h for spermatocytes and round spermatids, respectively. To the best of our knowledge, this is the first report where an exoglycohydrolase has been demonstrated to undergo processing in a cellspecific manner during spermatogenesis. A plausible reason for the different half-lives could be that since the acrosome is formed in the early stages of spermiogenesis by fusion of proacrosomal granules [2], the acrosomal antigens synthesized in the round spermatids will be expected to have a longer half-life span. Regardless of the reasons, it does appear that newly synthesized ␤-galactosidase in round spermatids has a longer half-life span than spermatocytes. Turnover studies of mature forms (64/62 kDa) in these cells would have given us additional information; however, these studies could not be attempted owing to the limited viability of spermatogenic cells in culture [19]. It is noteworthy that although spermatocytes and round spermatids synthesize similar precursor forms of ␤-galactosidase, the major mature forms produced after a 10-h chase are different (Figs. 2 and 3). In spermatocytes, which have lysosomes but no acrosome, the major mature form had a molecular mass of 64 kDa. This form was only a minor constituent in the round spermatids where the acrosome is formed during the Golgi phase by fusion of small granules to form a large acrosomal granule [2]. These differences in the molecular mass of 64 and 62 kDa are apparently due to differential glycosylation of the polypeptide moiety as we have demonstrated for molecular forms of ␤-galactosidase in spermatozoa [25], epididymal luminal fluid [32], and the 90/88-kDa precursor forms synthesized in round spermatids [3]. Since glycan moieties are important in intracellular transport and packaging of glycosidases [14, 28, 35], it will be of interest to elucidate the structure of oligosaccharide chains of the 64- and 62kDa forms. This information may provide insight into the manner in which ␤-galactosidase and other glycohydrolases are targeted to the acrosome and lysosome. In human fibroblast [21] and rat epididymal epithelial cells [22], acid ␤-galactosidase is synthesized in an 84- to 85-kDa precursor form which is processed into a mature form of 63– 64 kDa. Similarly, the enzyme in the mouse macrophages is synthesized in precursor forms of 82– 84 kDa that are processed to a mature form of 63 kDa [24]. In lysosomes, a protective protein has been demonstrated to interact with ␤-galactosidase monomers affecting their multimerization into a high-molecular-mass aggregate of 600 – 800 kDa [13]. We have obtained no evidence for the

presence of high-molecular-mass aggregates in spermatocytes or round spermatids. The indirect immunofluorescence studies with the new antibody agree with our earlier published studies [3] and indicated that ␤-galactosidase is localized within the sperm acrosome. In addition, an immunopositive reaction was seen in the detached cytoplasmic droplets following methanol permeabilization of the sperm cells (data not included). This localization agrees with several published reports indicating the presence of glycohydrolases in the cytoplasmic droplets [3, 7, 8, 25]. The intraacrosomal localization of the enzyme in cauda spermatozoa was in agreement with immunoelectron microscopy (Fig. 6F). Next, we examined the localization of the enzyme in the spermatogenic cells during sperm development and acrosome formation. These studies were approached at the light and electron microscopic levels using multiple protocols. Data from various approaches presented in Figs. 4 – 6 allow us to conclude that ␤-D-galactosidase, like other exoglycohydrolases [1, 11, 12, 17], is first expressed in the late spermatocytes (pachytene spermatocytes). The immunoelectron microscopic studies (Fig. 6) indicate that the enzyme is present in the Golgi apparatus, Golgiassociated vesicles, and lysosome-like structures present within the spermatocytes and early round spermatids. This localization is similar to our published reports with ␤-glucuronidase [1], another exoglycohydrolase which we showed is localized in the late spermatocytes and early round spermatids. In addition, the enzyme was immunolocalized in the forming and fully formed acrosome. When we followed the immunolocalization of ␤-galactosidase during the elongation and maturation phases of spermiogenesis (Fig. 5), the immunopositive reaction was similar to the distribution of ␤-glucuronidase in our earlier studies [1, 2]. It showed the successive formation of the acrosome during the progressive transformation of the spermatids into spermatozoa with fully developed acrosome. Thus, glycohydrolases can be used as markers for acrosome formation. In summary, we report the biochemical characterization, biosynthesis, and immunolocalization of ␤-galactosidase in rat testicular germ cells. Our data indicate that diploid and haploid germ cells possess several glycohydrolases including ␤-galactosidase. Both cell types synthesize ␤-galactosidase as high-molecular-weight precursor forms which are rapidly processed to lower molecular weight mature forms in a cell-specific manner. The excellent secretarial assistance of Mrs. Loreita Little is gratefully acknowledged. We are indebted to Drs. Malika Bendahmane and Benjamin J. Danzo for critically reading the manuscript. This work was supported in part by NIH Grants HD25869 and HD34041 from the National Institute of Child Health and Human Development.

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