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Biology of Reproduction 59, 298-308 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Rat Testicular Extracellular Superoxide Dismutase: Its Purification, Cellular Distribution, and Regulation1

Dolores Mrukc,d, Chin-Ho Chengc, Yan-Ho Chengc, Meng-yun Moc, Josephine Grimac, Bruno Silvestrinie, Will M. Leed, , and C. Yan Cheng2,c

c Population Council, Center for Biomedical Research, New York, New York 10021 d Department of Zoology, University of Hong Kong, Hong Kong, People's Republic of China e Institute of Pharmacology and Pharmacognosy, University of Rome "La Sapienza," 00185 Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using multiple HPLC steps, we have identified and purified a 68-kDa polypeptide (as estimated by gel permeation HPLC) to apparent homogeneity, from primary Sertoli cell-enriched culture medium, that consisted of two monomers of 35 ({alpha} chain) and 33 kDa (ß chain) on SDS-polyacrylamide gel running under reducing conditions. Partial N-terminal amino acid sequence analysis of these two monomers revealed sequences of NH2-DXGESGVDLADRL (SODEX-{alpha}) and NH2-XXDTGESGVDLADXL (SODEX-ß), which are identical to rat extracellular superoxide dismutase (SODEX) with the exceptions that SODEX-{alpha} and SODEX-ß are missing, respectively, four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from their N-termini, compared to rat SODEX, suggesting that the cleavage sites of the SODEX gene in the testis are different from that of other organs. Studies by sequential use of reverse transcription and polymerase chain reaction (PCR) using two SODEX primers have demonstrated the expression of SODEX in the heart, brain, lung, kidney, epididymis, testis, Sertoli, and germ cells, with low expression in the liver and ovary and no expression in the uterus, spleen, or thymus. Nucleotide sequence analysis of this 447-base pair PCR product from Sertoli cells revealed that its sequence is equivalent to the sequence of previously published rat SODEX. During testicular maturation, the SODEX steady-state mRNA level increased significantly from 20 to 60 days of age and then declined at 90 days of age. Such an increase in the testicular SODEX expression during maturation is not likely a result of an up-regulation by germ cells, since germ cells isolated from either 20- or 60-day-old rats when cocultured with Sertoli cells failed to elicit an increase in SODEX expression in the cocultures. Using primary Sertoli cell cultures in vitro, it was found that Sertoli cell SODEX expression was stimulated by interleukin-1{alpha} but not by either interferon-{gamma} or basic fibroblast growth factor. These results illustrate that Sertoli cells as well as germ cells synthesize and/or secrete a testicular variant of SODEX that may provide essential clues to understanding superoxide radical-mediated damage in the gonad.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Superoxide dismutases (SODs), catalases, and peroxidases are among the presently known antioxidant enzymes that protect cells from damage produced by various forms of reactive oxygen species such as superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-). These radicals are generated in low quantities as by-products during normal cellular metabolism. Reactive oxygen species have been implicated in numerous pathological states such as aging, inflammation, cancer, heart disease (for reviews, see [1–3]), degeneration of motor neurons in the brain and spinal cord [4], human immunodeficiency virus type 1 infection [5], and infertility [6–8]. The mechanism by which SODs protect cells from oxygen radicals is to scavenge O2- by converting it into H2O2, which in turn is broken down into H2O (for review, see [9]) in the cytoplasm by glutathione peroxidase [10] and in peroxisomes by catalases [11]. Depending on the transition metal ion found at their active site, SODs can be categorized into one of three types: copper/zinc (Cu/Zn SOD), manganese (Mn SOD), and iron (Fe SOD). Cu/Zn SOD is found primarily in the cytosol of eukaryotes, in chloroplasts, and in some species of bacteria; Mn SOD in prokaryotes and in mitochondria of eukaryotes; and Fe SOD in prokaryotes. An extracellular form of Cu/Zn SOD (SODEX), which is distinct from the cytosolic form in terms of molecular weight and amino acid composition, is also found in eukaryotes (for reviews, see [1, 2]).

To date, the various antioxidant systems in the testis have not been extensively studied. For instance, previous reports have indicated the presence of SODs [12], glutathione [13–16], and glutathione-dependent enzymes such as glutathione-S transferase [12, 16, 17] and glutathione reductase [12, 15, 17] in the testis, but the exact cellular localizations of these enzymes are unknown. For SODEX, the epididymis is known to be a major site of expression in the rat [18]. Numerous studies have demonstrated that SODEX, a tetrameric glycoprotein found predominantly in the extracellular matrix of tissues and to a lesser degree in extracellular fluids, has a high affinity for heparin and is bound to heparin sulfate proteoglycan in the glycocalyx on cell surfaces as well as in the connective tissue matrix [19]. However, the properties and distribution of SODEX in the rat are different from those of other mammals studied thus far. In the rat, SODEX is dimeric, has a low affinity for heparin, and does not bind to heparin sulfate in vivo [19]. Furthermore, a single amino acid mutation from Asp to Val at position 28 from the N-terminus can convert the rat SODEX to a tetramer with a high affinity for heparin sulfate [20]. As such, the identification and isolation of SODEX from Sertoli cell-enriched culture medium, and the demonstration of its expression in Sertoli cells, are significant in that there have been no reports indicating that Sertoli cells indeed produce and/or express SODEX.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sertoli Cells and Sertoli Cell-Enriched Culture Medium (SCCM) Preparation

Primary cultures of Sertoli cells were prepared from 20-day-old male Sprague-Dawley rats (Charles River, Kingston, MA) by sequential enzymatic treatments as previously described [21]. The resulting Sertoli cells were plated in 100-mm Petri dishes at a density of 5 x 104 cells/cm2 in serum-free Ham's F-12 nutrient mixture and Dulbecco's Modified Eagle's medium (F-12/DMEM, 1:1, v:v) (Gibco BRL, Gaithersburg, MD) supplemented with insulin (10 µg/ml), human transferrin (5 µg/ml), epidermal growth factor (2.5 ng/ml), and bacitracin (5 µg/ml). Cells were maintained in a humidified atmosphere of 95% air:5% CO2 (v:v) at 35°C. To obtain Sertoli cells with a purity greater than 95%, cultures were hypotonically treated 48 h after plating with 20 mM Tris, pH 7.4, at 22°C for 2.5 min to lyse contaminating germ cells [22] and then were washed two times with F-12/DMEM. Sertoli cells were then cultured for an additional 8 days. Spent media collected on Days 4 and 8 were stored at -20°C until use.

Purification of SODEX from Primary SCCM

Preparation of SCCM. Batches of 6–10 L of SCCM prepared as described above were used for the purification of SODEX. Media were pooled, concentrated at 4°C, and equilibrated against 20 mM Tris, pH 7.4, at 22°C using a Millipore (Bedford, MA) Minitan tangential ultrafiltration unit equipped with eight Minitan plates with a molecular weight cutoff at 10 000. The sample was then centrifuged at 45 000 x g for 45 min to remove cellular debris and filtered through a 0.2-µm filter unit.

Anion-exchange HPLC. About 200 mg of proteins from 6–10 L of SCCM obtained from the preceding step was loaded onto a Pharmacia Biotech (Piscataway, NJ) anion-exchange HPLC preparative column (Mono Q, HR 16/10, 16 x 100 mm, i.d.). Bound proteins were eluted using a linear gradient from 0% to 80% solvent B (20 mM Tris, pH 7.4, at 22°C, containing 600 mM NaCl) at a flow rate of 4 ml/min over a period of 90 min. The eluents were monitored by UV absorbance at 280 nm, and fractions of 4 ml were collected. An aliquot from selected fractions was withdrawn for SDS-PAGE, and proteins were visualized by silver staining as detailed elsewhere [23].

C8 reverse-phase HPLC. Fractions obtained from the preceding step containing SODEX were pooled, lyophilized, resuspended in solvent A (5% acetonitrile:95% H2O containing 0.1% trifluoroacetic acid, v:v), and loaded onto a Vydac (Separations Group, Hesperia, CA) C8 reverse-phase HPLC column (4.6 x 250 mm, i.d.) at a flow rate of 1 ml/min. Bound proteins were eluted using a linear gradient from 5% to 80% solvent B (95% acetonitrile:5% H2O containing 0.1% trifluoroacetic acid, v:v) over a period of 45 min. The eluents were monitored by UV absorbance at 280 nm, and fractions of 1 ml were collected. An aliquot from selected fractions was withdrawn for SDS-PAGE. For all subsequent HPLC steps, unless otherwise noted, the compositions of solvents A and B were the same as in this HPLC step, the eluents were monitored by UV absorbance at 280 nm, fractions of 1 ml were collected, an aliquot from selected fractions was withdrawn for SDS-PAGE, and proteins were visualized by silver staining. During the initial purification steps, SODEX was monitored by its electrophoretic mobility on SDS-PAGE, and its identity was subsequently verified by direct protein sequencing.

Diphenyl reverse-phase HPLC. Fractions obtained from the preceding step containing SODEX were pooled, lyophilized, resuspended in solvent A, and loaded onto a Vydac diphenyl reverse-phase HPLC column (4.6 x 250 mm, i.d.) at a flow rate of 1 ml/min. Bound proteins were eluted using a linear gradient from 5% to 80% solvent B over a period of 45 min.

C18 reverse-phase HPLC. Fractions obtained from the preceding step containing SODEX were pooled, lyophilized, resuspended in solvent A, and loaded onto a Vydac C18 reverse-phase HPLC column (4.6 x 250 mm, i.d.) at a flow rate of 1 ml/min. Bound proteins were eluted using a linear gradient of 20–70% solvent B over a period of 40 min. Fractions of 0.5 ml were collected.

Diphenyl reverse-phase HPLC. Fractions obtained from the preceding step containing SODEX were pooled, lyophilized, resuspended in solvent A, and loaded onto another Vydac diphenyl reverse-phase HPLC column (4.6 x 250 mm, i.d.) at a flow rate of 1 ml/min. Bound proteins were eluted using a linear gradient of 20–50% solvent B over a period of 30 min. Fractions of 0.5 ml were collected.

Gel permeation HPLC. Estimation of the apparent molecular weight of SODEX under native conditions by gel permeation HPLC was performed as previously described [24]. Briefly, about 200 µg each of rat {alpha}2-macroglobulin (720 kDa), human transferrin (75 kDa), BSA (68 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and cytochrome C (12.4 kDa) were fractionated onto a Bio-Rad (Richmond, CA) Bio-Sil SEC-250 (7.5 x 300 mm, i.d.) column equipped with a Bio-Rad Bio-Sil guard column (7.5 x 75 mm, i.d.) at a flow rate of 0.5 ml/min using 10 mM sodium phosphate, 0.15 M NaCl (pH 6.8), at 22°C, containing 10% ethanol (v:v). These proteins were fractionated individually as well as in a mixture by HPLC to obtain the corresponding retention times. About 50 µg of highly purified SODEX isolated from SCCM was also resolved by this column under the same conditions. The retention times of marker proteins were plotted against their molecular weights, and the resultant plot was used to estimate the apparent molecular weight of native SODEX.

Protein Microsequencing

About 100 pmol of SODEX was resolved onto a 10% T SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Applied Biosystems, Foster City, CA) using a buffer system consisting of 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11.0), at 22°C, containing 10% methanol (v:v). SODEX electroblotted onto the polyvinylidene difluoride membrane was stained by Coomassie blue R-250 and sequenced as previously described [25, 26]. Phenylthiohydantoin (PTH)-amino acids were identified and quantified by HPLC utilizing a Brownlee (Applied Biosystems) PTH-C18 (2.1 x 220 mm, i.d.) column. Protein sequencing was repeated twice using two different batches of purified protein. The repetitive yield was about 96%.

Isolation of Germ Cells

Germ cells were isolated from 20-day-old male Sprague-Dawley rats by a mechanical procedure without the use of trypsin, as previously described, since trypsinization was shown to affect the functional and cell adhesion properties of these cells [27]. These germ cells, which were more than 95% pure, consisted primarily of spermatogonia and spermatocytes with a relative percentage of about 50:50 when assessed by DNA flow cytometry as described previously [27]. This result is consistent with earlier findings based on microscopic morphological analysis [28]. These cells were used for coculture experiments within 1 h of their isolation. The somatic cell contamination was virtually negligible when assessed by various criteria as detailed elsewhere [27]. Germ cells were also isolated from 60-day-old rats without the use of trypsin as previously described [27]; these cells consisted largely of spermatogonia, spermatocytes, and round spermatids (elongate spermatids were removed by a glass wool filtration step) at a relative percentage of 16:18:65 when assessed by DNA flow cytometry [27].

Sertoli-Germ Cell Cocultures

Highly purified Sertoli cells isolated as outlined above were used for Sertoli-germ cell coculture experiments as previously described [29], with minor modifications. Isolated cells were plated on Matrigel (Collaborative Biochemicals, Bedford, MA) (diluted 1:7 with F-12/DMEM)-coated 24-well dishes at a density of 0.5 x 106 cells/cm2. Cultures were hypotonically treated 48 h after their isolation with 20 mM Tris, pH 7.4, at 22°C for 2.5 min to lyse contaminating germ cells [22] and then washed two times with F-12/DMEM. Media were replaced every 24 h, and cells were cultured for an additional 4 days to allow the establishment of inter-Sertoli cell tight junctions as assessed by previously published criteria [21]. These cells were then used for coculture experiments. Briefly, freshly isolated germ cells from either 20- or 60-day-old rats were cocultured with Sertoli cells for 1, 2, 3, 5, and 24 h, using a Sertoli:germ cell ratio of 1:1, in F-12/DMEM supplemented with 2 mM sodium pyruvate, 6 mM sodium DL-lactate, insulin (10 µg/ml), transferrin (5 µg/ml), and epidermal growth factor (2.5 ng/ml). Cocultures were terminated while germ cells were allowed to attach to Sertoli cells and prior to the establishment of specialized junctions with Sertoli cells, since it is known that specialized Sertoli-germ cell junctions such as desmosome-like junctions are formed by 24–48 h in vitro [30]. Cell viability of germ cells cocultured with Sertoli cells was greater than 95%, as judged by trypan blue dye exclusion test, throughout the entire culture period. Total RNA was extracted from these cells by RNA STAT-60 (Tel-test "B" Inc., Friendswood, TX) as previously described [29, 31, 32].

To determine whether germ cell influence or lack thereof on Sertoli cell SODEX was an artifact, Sertoli cells isolated as described above were cultured on Matrigel-coated 24-well dishes at a density of 0.5 x 106 cells/cm2. Cells were hypotonically treated 48 h after their isolation. Media were replaced every 24 h, and cells were cultured for an additional 4 days to allow the establishment of inter-Sertoli cell tight junctions [21]. Thereafter, Sertoli cells were cultured for 1, 2, 3, 5, and 24 h in the absence of germ cells. Total RNA was extracted from these cells by RNA STAT-60.

Treatment of Sertoli Cell Cultures with Interleukin-1{alpha} (IL-1{alpha}), Interferon-{gamma} (INF-{gamma}), and Basic Fibroblast Growth Factor (bFGF)

The effects of recombinant IL-1{alpha} (specific activity, 1 x 107 U/mg protein), INF-{gamma} (specific activity, 2 x 107 U/mg protein), and bFGF (Calbiochem, San Diego, CA) on Sertoli cell SODEX mRNA expression were examined in vitro. Sertoli cells were prepared essentially as described above and cultured at a density of 0.5 x 106 cells/cm2 on Matrigel-coated 12-well dishes. After 48 h, cells were hypotonically treated to lyse contaminating germ cells [22], then given two washings with fresh F-12/DMEM and cultured for an additional 24 h. Thereafter, Sertoli cells were incubated for an additional 0–24 h in the presence of either IL-1{alpha} (10 U/ml), INF-{gamma} (100 U/ml), or bFGF (50 ng/ml) prior to termination with RNA STAT-60. To ensure that any changes detected in the mRNA expression of SODEX were not attributable to an artifact, Sertoli cells were cultured under the same conditions as described above in duplicate wells without the addition of any factors. Cells were hypotonically treated and cultured for an additional 24 h. Thereafter, Sertoli cells were terminated at various time points from 0 to 24 h with RNA STAT-60.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from cells and tissues using RNA STAT-60 as previously described [29]. RT-PCR was performed essentially as previously described [29] to examine the expression of SODEX mRNA in different tissues and cells. Briefly, 1 µg of total RNA was reverse transcribed into cDNAs using 3 µg of oligo(dT)-15 and an avian myeloblastosis virus reverse transcriptase kit (Promega, Madison, WI). Each reaction tube consisted of 5 µl of 5-strength RT buffer, 2.5 µl of 10 mM dithiothreitol, 2.5 µl of dNTP (10 mM each of dATP, dCTP, dGTP, and dTTP), 10 units of RNasin, 4 units of reverse transcriptase, and sterile double-distilled water to a final reaction volume of 25 µl. This was incubated at 42°C for 60 min. To terminate the reaction, samples were heated at 95°C for 5 min. From this reaction product, 1/5 was used; this served as a template for PCR in combination with 0.3 µg each of the sense and antisense SODEX primer pairs coamplified with either rat cytoplasmic ß-actin or ribosomal S16. Coamplification with ß-actin or S16 was included to ensure that equal amounts of RNA were reverse transcribed and amplified in each reaction tube. The primers used for the amplification of SODEX [18] and ß-actin [33] or S16 [34] were as follows: 5'-ATGGTGGCCTTCTTGTTCTGC-3' (SODEX, sense, nucleotides 109–129); 5'-GGTGTGCCGCACCCACAGCAC-3' (SODEX, antisense, nucleotides 535–555); 5'-TCACCGAGGCCCCTCTGAACCCTA-3' (ß-actin, sense, nucleotides 314–337); 5'-GGCAGTAATCTCCTTCTGCATCCT-3' (ß-actin, antisense, nucleotides 931–954); 5'-TCCGCTGCAGTCCGTTCAAGTCTT-3' (S16, sense, nucleotides 15–38); and 5'-GCCAAACTTCTTGGATTCGCAGCG-3' (S16, antisense, nucleotides 376–399). These reagents were mixed with 10 µl of the 10-strength PCR buffer, 6 µl of 25 mM MgCl2, 16 µl of dNTP (200 µM each of dATP, dCTP, dGTP, and dTTP), 5 units of Taq DNA polymerase (Promega), and sterile double-distilled water to a final reaction volume of 100 µl. The cycling parameters for the PCR reaction were denaturation at 94°C for 1 min, annealing at 61°C for 2 min, and extension at 72°C for 3 min. A total of 20–25 cycles were performed. The cycles were followed by an incubation at 72°C for 15 min. Aliquots of 5–10 µl were resolved onto 5% T polyacrylamide gels in 0.5-strength TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0, at 22°C). Under these conditions, the production of SODEX, S16, or ß-actin was in the linear phase as demonstrated in preliminary experiments when aliquots of PCR products were withdrawn from each of the PCR reaction tubes in cycles 18, 20, 22, and 25 for gel analysis. For PCR using radiolabeled primers, about 0.2 µg of the antisense primer was 5' end-labeled with [{gamma}-32P]ATP (specific activity, 2500 Ci/mmol; Amersham, Arlington Heights, IL) by using T4 polynucleotide kinase (Promega). Antisense ß-actin primer was also 5' end-labeled for coamplification as described above. PCR products were visualized by either ethidium bromide staining or autoradiography using X-OMAT AR film (Eastman Kodak, Rochester, NY).

General Methods

Protein estimation was performed by Coomassie blue dye binding assay [35] using BSA as a standard. Analytical PAGE in the presence of SDS was performed as previously described [36, 37]. All aliquots from each HPLC fraction were denatured and reduced in SDS sample buffer (0.125 M Tris, pH 6.8, at 22°C containing 1% SDS [w:v], 1.6% 2-mercaptoethanol [v:v], and 10% glycerol [v:v]). Polyacrylamide gels were silver stained as detailed elsewhere [23]. Nucleotide sequencing of SODEX was performed by the dideoxynucleotide chain termination method using Sequenase (Promega) as previously described [38]. Densitometric scanning of autoradiograms was performed using a Ultroscan XL Enhanced Laser Densitometer (Pharmacia Biotech) at 600 nm. Statistical analysis was performed by Student's t-test using the GB Statistical Analysis Software package (Version 3.0; Dynamics Microsystems, Silver Spring, MD).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and Structural Characterization of SODEX

SODEX was purified to apparent homogeneity from a 10-liter batch of SCCM by sequential HPLC using anion-exchange (Fig. 1A) and multiple reverse-phase columns (Fig. 1, B–E). In the initial purification scheme, SODEX was monitored by its electrophoretic mobility on SDS-polyacrylamide gels by SDS-PAGE under reducing conditions (see bars in Fig. 1, A–E), which consisted of two monomeric subunits of 35 (SODEX-{alpha}) and 33 (SODEX-ß) kDa (Fig. 2A). SODEX was purified to apparent homogeneity using a combination of HPLC columns that included anion exchange (Fig. 1A), C8 reverse phase (Fig. 1B), diphenyl reverse phase (Fig. 1C), C18 reverse phase (Fig. 1D), and another diphenyl reverse phase (Fig. 1E). Figure 2A is a silver-stained SDS-polyacrylamide gel under reducing conditions showing the highly purified SODEX eluted from a diphenyl reverse-phase HPLC column in fraction 37. Partial N-terminal amino acid sequence analysis of SODEX-{alpha} (Fig. 2B) and SODEX-ß (Fig. 2C) revealed sequences of NH2-DXGESGVDLADRL and NH2-XXDTGESGVDLADXL, which are identical to rat SODEX [18, 39] with the exceptions that SODEX-{alpha} and SODEX-ß are missing four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from their N-termini, respectively, suggesting that the cleavage sites on the mature SODEX protein are different in gonadal and nongonadal tissues.



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  		FIG. 1. A–E) Purification of SODEX from SCCM. A) Results of anion-exchange HPLC. A total of 6 protein peaks were noted. The presence of SODEX in fractions 25–37 is denoted by a solid bar. B) Results of C8 reverse-phase HPLC. A total of 23 protein peaks were noted. SODEX was found in fractions under protein peaks 1–2 as denoted by a solid bar. C) Results of diphenyl reverse-phase HPLC. SODEX was found in fractions under protein peak 1. D) Results of C18 reverse-phase HPLC column. SODEX is denoted by a solid bar under protein peak 1. E) Results of a second diphenyl reverse-phase HPLC. Purified SODEX was eluted in fractions under protein peak 2. Arrow indicates where gradient began. Throughout the purification scheme, SODEX was identified by SDS-PAGE and silver staining.



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  		FIG. 2. A–E) Structural characterization of purified SODEX isolated from SCCM. A) Purified SODEX isolated from SCCM as described for Figure 1E was resolved by SDS-PAGE onto a 12.5% T SDS-polyacrylamide gel under reducing conditions and silver stained. M, protein markers with about 0.2µg protein for each marker. About 100 pmol of purified SODEX was subjected to direct protein sequencing, and 13 and 15 amino acids were identified from its NH-2 terminus for SODEX-{alpha} (B) and SODEX-ß (C), respectively. X represents an amino acid that could not be assigned unequivocally. Protein sequencing was performed as described in Materials and Methods. The abscissa represents the residue number from each Edman degradation cycle; the ordinate represents the PTH-amino acid obtained from each cycle. Two different batches of purified SODEX yielded identical results. The repetitive yield was about 96%. D) Estimation of the native molecular weight of SODEX, achieved as described in Materials and Methods (Gel permeation HPLC). Insert shows the SDS-PAGE of about 0.25 µg of purified SODEX on a 10% T SDS-polyacrylamide gel under nonreducing conditions; the gel was silver stained. M, Protein markers. E) The retention times of the marker proteins were plotted against their molecular weight, and the resultant plot was used to estimate the apparent molecular weight of native SODEX.

Subunit structural analysis of the purified SODEX under reducing (Fig. 2A) and nonreducing (Fig. 2D, insert) conditions and by gel permeation HPLC (Fig. 2, D and E) indicated that SODEX isolated from SCCM is a dimeric protein of 68 kDa consisting of two monomers of 35 (SODEX-{alpha}) and 33 (SODEX-ß) kDa.

Cellular and Tissue Distribution of SODEX

The cellular and tissue distribution of SODEX mRNA expression in various cells and tissues from male and female rats was examined by RT-PCR (Fig. 3). A 447-base pair (bp) PCR product corresponding to the expected size of SODEX mRNA was found in the heart, brain, lung, kidney, epididymis, testis, and Sertoli and germ cells with low expression in the liver and ovary, but not found in the uterus, spleen, or thymus (Fig. 3). It is apparent that the expression of SODEX is highest in the epididymis, consistent with previously published findings [18]. This 447-bp PCR product from Sertoli cells was subsequently confirmed to be authentic SODEX by direct nucleotide sequence analysis after electroelution of the PCR product and subcloning into pGem-T vector as previously described [40]. In this experiment, SODEX was coamplified with S16 to ensure that equal amounts of RNA were reverse transcribed and amplified in each reaction tube.



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  		FIG. 3. Expression of SODEX mRNA in various tissues and cells. One microgram of total RNA was reverse transcribed into cDNA. One-fifth of this RT product was used, serving as a template for PCR using SODEX primer pairs. Coamplification was performed using S16 primer pairs. PCR products were resolved onto a 5% T polyacrylamide gel. DNA was visualized by ethidium bromide staining. A 447-bp SODEX PCR product was detected in heart, brain, lung, kidney, epididymis, testis, and Sertoli and germ cells with low expression in the liver and ovary. SODEX mRNA was not detected in the spleen, uterus, or thymus. M, DNA size markers.

Developmental Regulation of SODEX in the Testis

Since SODEX mRNA was expressed in the testis, we next investigated SODEX mRNA expression in the developing testis from 20 to 90 days of age, when there is an increase in Sertoli-germ cell interactions due to the onset of spermatogenesis. In the testis, the steady-state SODEX mRNA level increased 500-fold from 20 to 60 days of age; there was then a significant decrease in expression at 90 days of age (Fig. 4, A and B). Figure 4B shows the densitometric scanning of three autoradiograms such as the one shown in Figure 4A, but normalized against ß-actin and expressed as arbitrary units per pair of testes to assess the SODEX mRNA steady-state level per pair of testes during maturation.



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  		FIG. 4. A, B) Developmental regulation of testicular SODEX steady-state mRNA during aging. A) Autoradiogram of an RT-PCR showing a significant increase in SODEX expression between 20 and 60 days of age followed by a decrease in expression at 90 days of age. Coamplification was performed using ß-actin primer pairs. B) Densitometric scanning of three autoradiograms such as the one shown in A normalized against ß-actin; the steady-state mRNA level expressed as arbitrary units per pair of testes. *Significantly different from value for 20-day-old rats, p < 0.01; ns, not significantly different from value for 20-day-old rats.

Expression of SODEX in Sertoli Cells Cocultured with Germ Cells

Because there is an increase in the ratio of germ cells to Sertoli cells during development in the rat testis, we examined whether the increase in the SODEX mRNA level in the developing testis is attributable to an up-regulation by germ cells. Furthermore, previous studies from this laboratory have shown that when germ cells are cocultured with Sertoli cells for short periods of time up to 24 h prior to the establishment of Sertoli-germ cell junctions, there are surprising and significant changes in the mRNA expression of several genes such as cathepsin L [29], a cysteine protease that has been implicated in such pathological states as cancer [41]. Therefore, we considered it pertinent to examine whether short-term interactions between Sertoli and germ cells would influence the SODEX mRNA level in the cocultures. Sertoli cells (0.5 x 106 cells/cm2) were allowed to form intercellular junctions for 4 days in vitro. Thereafter, germ cells isolated from 20 (Fig. 5, A and B)- or 60-day-old rats (Fig. 5, C and D) were added at a ratio of 1:1 for periods of up to 24 h. Time 0 of Sertoli-germ cell cocultures represents dishes in which RNA STAT-60 was added to Sertoli cells immediately after the addition of germ cells at a Sertoli:germ cell ratio of 1:1. Cocultures were terminated at 1, 2, 3, 5, and 24 h, while germ cells were allowed to attach to Sertoli cells prior to the formation of specialized junctions with Sertoli cells. It was noted that the Sertoli cell SODEX was not affected by germ cells isolated from 20 (Fig. 5, A and B)- or 60-day-old rats (Fig. 5, C and D) from 1 to 24 h after coculture. This suggests that the increase in the steady-state SODEX mRNA level during testicular maturation is not likely to be the result of an up-regulation by germ cells. Figure 5, B and D, each show the densitometric scanning of three autoradiograms of the corresponding experiments, such as the ones shown in Figure 5, A and C, normalized against ß-actin. However, it must be noted that elongate spermatids were removed from the germ cell preparation in experiments using adult rats by the glass wool filtration step. It remains to be determined whether elongate spermatids can up-regulate Sertoli cell SODEX expression.



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  		FIG. 5. A–D) A study to assess the effect of germ cells isolated from 20 (A, B)- or 60-day-old rats (C, D) on the steady-state SODEX mRNA level in Sertoli-germ cell (SC-GC) cocultures. A, C) Autoradiograms of RT-PCR showing no significant changes in SODEX expression detected when freshly isolated germ cells from 20 (A)- or 60-day-old rats (C) were added to Sertoli cells at a ratio of 1:1 and cultured for short periods of time up to 24 h. Coamplification was performed using ß-actin primer pairs. Control: SC alone without germ cells at time 0. B, D) Corresponding densitometric scanning of three autoradiograms such as the one shown in A and C and normalized against ß-actin. ns, Not significantly different from control without the addition of germ cells.

Expression of SODEX in Sertoli Cells Without the Addition of Germ Cells

To rule out the possibility that Sertoli cells may produce an inhibitor(s) that may mask its own or germ cell's expression of SODEX in the cocultures, Sertoli cells were cultured alone (Fig. 6, A and B) under the same conditions as those shown in Figure 5 without the addition of germ cells. Since there were no significant changes in the expression of SODEX in Sertoli cells cultured alone (Fig. 6, A and B), these results confirm the observation that germ cells do not influence Sertoli cell SODEX expression (Fig. 5, A–D, vs. Fig. 6, A and B) and further indicate that Sertoli cells do not produce an inhibitor that may arrest their own or germ cell SODEX expression.



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  		FIG. 6. A, B) The steady-state SODEX mRNA level in Sertoli cell (SC) cultures at various time points. A) Autoradiogram of an RT-PCR showing that no significant changes in SODEX expression were detected when Sertoli cells were cultured for short periods of time for up to 24 h under the same conditions as described for Figure 5 but without the addition of germ cells. Coamplification was performed using ß-actin primer pairs. B) Densitometric scanning of three autoradiograms such as the one shown in A normalized against ß-actin. ns, Not significantly different from Sertoli cell cultures at Time 0 without the addition of germ cells.

Regulation of Sertoli Cell SODEX Expression by IL-1{alpha}, INF-{gamma}, and bFGF

It was found that the expression of SODEX mRNA by rat Sertoli cells cultured in vitro was unresponsive to either INF-{gamma} (100 U/ml) (Fig. 7, A and B) or bFGF (50 ng/ml) (Fig. 7, C and D). However, IL-1{alpha} (10 U/ml) induced a transient but significant increase in the Sertoli cell SODEX steady-state mRNA level (Fig. 8, A and B). By 2 h, the level of Sertoli cell SODEX expression was almost 4-fold that of the control but declined to the basal level by 3 h (Fig. 8, A and B) and remained at that level thereafter. The change in the expression of SODEX in Sertoli cells cultured with the addition of IL-1{alpha} was not likely to be attributable to an artifact, since Sertoli cells cultured under the same conditions without the addition of any factors exhibited no apparent changes in SODEX expression (data not shown). Figure 7, B and D, and Figure 8B show the densitometric scannings of three autoradiograms such as the ones shown in Figure 7, A and C, and Figure 8A, normalized against ß-actin, illustrating the effects of INF-{gamma}, bFGF, and IL-1{alpha}, respectively, on Sertoli cell SODEX expression.



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  		FIG. 7. A study to examine the possible effect of INF-{gamma} (A, B) and bFGF (C, D) on the steady-state SODEX mRNA level in Sertoli cells (SC). A, C) Autoradiograms of RT-PCR showing no significant changes in SODEX expression when Sertoli cells were cultured with either 100 U/ml of INF-{gamma} (A) or 50 ng/ml of bFGF (C) for various times. Coamplification was performed using ß-actin primer pairs. Control, SC alone without either INF-{gamma} or bFGF at Time 0. B, D) Corresponding densitometric scanning of three autoradiograms such as the one shown in A and C, normalized against ß-actin. ns, Not significantly different from control without the addition of either INF-{gamma} or bFGF.



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  		FIG. 8. A, B) Changes in the steady-state SODEX mRNA level in Sertoli cells (SC) with use of IL-1{alpha} (10 U/ml). A) Autoradiogram of an RT-PCR showing a transient but significant increase in SODEX expression at 2 h after the addition of IL-1{alpha} to Sertoli cells. Coamplification was performed using ß-actin primer pairs. Control: SC alone without IL-1{alpha} at Time 0. B) Densitometric scanning of three autoradiograms such as the one shown in A normalized against ß-actin. *Significantly different from control without IL-1{alpha}, p < 0.01; ns, not significantly different from control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Even though many investigators have focused heavily on understanding the effects of free radicals on various organs, the exact mechanism(s) of action of many of the antioxidant enzymes, including SOD, in many organs such as the testis remains unknown. In this study, we have described the purification of a polypeptide from SCCM, designated SODEX in view of its sequence identity with rat SODEX present in other nongonadal organs [18, 20, 37]. Rat testicular SODEX, a dimeric protein of 68 kDa as estimated by gel permeation HPLC, consists of two monomers of 35 (SODEX-{alpha}) and 33 (SODEX-ß) kDa. Analysis of the N-terminal amino acid sequences revealed that these two monomers are identical, with the exceptions that four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids are missing from SODEX-{alpha} and SODEX-ß, respectively, in comparison to the published rat SODEX sequence originally found in the epididymis [18] and other nongonadal tissues [20, 39]. In this regard, the testicular SODEX appears to be a molecular variant of the nongonadal SODEX whose monomers are formed by a mechanism analogous to that of the rat epididymal retinoic acid-binding protein (EP-RABP). EP-RABP, an androgen-dependent secretory protein, has two molecular variants of 18 and 18.5 kDa. The latter form has three extra amino acids, Thr-Glu-Gly, at its N-terminus. Both molecular forms of EP-RABP appear to be coded by the same gene with two different cleavage sites [42–44].

Unlike intracellular Cu/Zn SOD and Mn SOD, which are expressed by virtually all cell types examined to date, SODEX is secreted and/or expressed by only a few tissues, which include the epididymis [18], heart [45], lung [46], and ovary [47]. Using specific primers coding for SODEX, we were able to demonstrate the expression of SODEX in the heart, brain, lung, kidney, epididymis, testis, and Sertoli and germ cells, with low expression in the liver and ovary and no expression in the uterus, spleen, or thymus. Numerous studies have already shown that germ cells synthesize and/or express cytosolic Cu/Zn SOD and Mn SOD [48, 49]; however, there is no mention of Sertoli cells and/or germ cells synthesizing and/or expressing an extracellular form of SOD. Furthermore, previous studies do not seem to report any significant SOD activity in Sertoli cells. For instance, Bauche et al. [48] reported very low activity of Cu/Zn SOD and Mn SOD in Leydig, peritubular myoid, and Sertoli cells, whereas Jow et al. [50] demonstrated a uniform distribution of Cu/Zn SOD in prepubertal rats but a stage-specific pattern of expression in older animals by in situ hybridization. The highest level of Cu/Zn SOD mRNA was detected in seminiferous tubules of stages VI–VIII just prior to spermiation [50], suggestive of its involvement in spermiation. In the present study, we have demonstrated unequivocally that both Sertoli and germ cells express SODEX. The expression of SODEX in the germ cell preparations used in this study has been validated by Southern blots using an oligonucleotide internal to the 447-bp SODEX PCR product (data not shown) in two different batches of germ cells. Moreover, the somatic cell contamination was minimal as judged by DNA flow cytometry and RT-PCR of a Sertoli cell product such as testin, as previously described [27]. The fact that germ cells are known to express Cu/Zn SOD and Mn SOD [48] illustrates that developing germ cells may participate in a wide range of physiological activities, which may include the regulation of their own development in the epithelium. The synthesis and expression of SODEX by Sertoli cells (the major phagocytic component in the seminiferous epithelium [51, 52]) and germ cells may serve, in fact, to protect them and the seminiferous epithelium from self-inflicted damage caused by superoxide radicals during spermatogenesis. Numerous investigators have proposed that phagocytes activated during inflammation are likely to be the major producer of substrates for SODEX [3, 9]. Therefore, it is very likely that SODEX is important in protecting the Sertoli and/or germ cell surface and extracellular proteins from superoxide radical-mediated damage.

Germ cells isolated from either 20- or 60-day-old rats, when cocultured with Sertoli cells for a period of up to 24 h prior to the establishment of Sertoli-germ cell specialized junctions, failed to elicit a change in SODEX expression in the cocultures. We hypothesize that the increase in the testicular SODEX steady-state mRNA level during maturation may not be a result of up-regulation by germ cells. It is possible that the increase in SODEX in the developing testis may be due to an increase in Sertoli-germ cell interactions as a result of the onset of spermatogenesis. It has been reported that short-term interactions of Sertoli and germ cells in vitro preceding the formation of specialized junctions are associated with significant changes in the expression of several proteases and protease inhibitors [29]. The increase in testicular SODEX expression during maturation may also be ascribed to Sertoli, germ, Leydig, or peritubular myoid cells or a combination of these cell types whose SODEX expression may be enhanced as a result of differentiation. Moreover, some other yet-to-be-identified factor(s) may be responsible for the enhanced stimulation of testicular SODEX expression during maturation. It is not known from this study whether other testicular cells such as Leydig or peritubular myoid cells produce and/or express SODEX.

The studies presented in this paper indicate that INF-{gamma} and bFGF, both of which are germ cell-derived cytokines, were unable to influence the mRNA expression of Sertoli cell SODEX in vitro, since the addition of either one of these factors failed to increase/decrease SODEX expression in Sertoli cells. However, the addition of IL-1{alpha} (10 U/ml), a known Sertoli cell product possibly also released by germ cells [53], in vitro induced a transient but significant increase in SODEX expression. This transient but significant increase in the expression of SODEX in Sertoli cells incubated with IL-1{alpha} is not likely due to an artifact, since Sertoli cells cultured under the same conditions failed to show a change in SODEX expression. Therefore, it is possible that cytokines or growth factors released by germ cells are capable of influencing the production and/or expression of some Sertoli cell products, since culture of Sertoli cells in the presence of 50 ng/ml of bFGF [54] for up to 24 h resulted in a significant increase in cathepsin L expression (unpublished results), which is a known Sertoli cell secretory product [55]. Nonetheless, until free radical damage and its effect(s) on spermatogenesis are deciphered in greater detail, the exact mechanism(s) of action of SODEX in the testis will remain unknown.


   FOOTNOTES
 
1 This work was supported in part by grants from the Rockefeller Foundation (PS9528, PS9601, PS9721), Conrad Program (CIG96-05), Noopolis Foundation, NIH (HD-13541), and Hong Kong Research Grant Council (HKU7235/97M). This work was derived from a dissertation to be submitted by D.M. to the University of Hong Kong for the partial fulfillment for the requirements of Doctor of Philosophy. Back

2 Correspondence: C. Yan Cheng, Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021. FAX: (212) 327-7678; yan{at}popcbr.rockefeller.edu Back

Accepted: March 17, 1998.

Received: September 30, 1997.


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