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RESEARCH COMMUNICATION |
a Dipartimento di Chimica Biologica, I-35121 Padova, Italy
b Istituto di Anatomia Patologica, I-35121 Padova, Italy
c Deutsches Institut für Ernährungsforschung (DIFE), D-14558 Bergholz-Rehbrücke, Germany
d Department of Physiological Chemistry, Technical University of Braunschweig,D-38124 Braunschweig, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: glutathione peroxidases • selenium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The underlying mechanisms are far from clear; however, at physiological levels, selenium generally exerts its biological functions as selenocysteine residues, which have been cotranslationally incorporated into distinct selenoproteins (7). More than 20 different selenoproteins have been inferred to exist in mammals from pulse labeling experiments with 75 Se, but only a minority of them have so far been characterized as distinct gene products or enzymatic entities (8). The latter comprise four members of the glutathione peroxidase family (9–14), at least two deiodinases (15–17), thioredoxin reductase (18), and selenophosphate synthetase (19). Whenever investigated, the selenocysteine moiety of these proteins proved to be essential for catalytic function (20–22) and, correspondingly, the specific enzymatic activities in tissues or whole organisms depend on an adequate supply of selenium in a bioavailable form. Apparently, the biosynthesis of selenoproteins is not only limited by the availability of selenocysteyl-t RNA needed for the incorporation of selenocysteine (7, 23, 24), but can be regulated further at the posttranscriptional level, since specific mRNA species encoding selenoproteins were shown to be stabilized by cellular selenium (reviewed in ref 25).
In the realm of selenoproteins, two glutathione peroxidases are potentially relevant to testicular function. The `classical' cytosolic glutathione peroxidase (cGPx)2 was reported to be expressed in testis at low levels (26, 27), but has been implicated in the metabolism of H2O2 associated with steroid hormone synthesis in Leydig cells (26). Surprisingly high levels of phospholipid hydroperoxide glutathione peroxidase (PHGPx) are found in testis (14, 28), where the enzyme is at least partially directed toward the mitochondria by means of a particular leader sequence. This results from the use of an ATG start codon upstream of the ATG codon more commonly used in other tissues (29). In the rat testis, PHGPx is preferentially expressed after puberty, where it remains essentially absent after hypophysectomy and can be partially restored in hypophysectomized rats by hCG administration (28). Also, immunohistochemical studies revealed an association of PHGPx with the seminiferous epithelium (28). These intriguing observations suggested a transcriptional regulation of PHGPx either by hCG and mediated by cAMP or, indirectly, by testosterone arising from hCG-stimulated Leydig cells, an idea further strengthened by the detection of consensus sequences in the PHGPx gene reminiscent of steroid- and cAMP-responsive elements (11). However, pilot experiments with reporter gene constructs designed to validate the functional relevance of the putative regulatory elements in the known 5' flanking region of the porcine PHGPx gene are so far inconclusive. Neither testosterone nor forskolin directly activated transcription of the reporter genes nor did estradiol 17-ß inhibit transcription in hormone-responsive T47D and MCF7 cell lines (unpublished data).
In the present investigation, we rule out any short-term transcriptional regulation of PHGPx gene expression by testosterone, estrogen, or cAMP. Instead, we demonstrate that the marked increase of PHGPx activity with sexual maturation parallels the hormone-dependent increase of the spermatid layer that preferentially expresses PHGPx in the rat testis. In contrast, cGPx is expressed at low levels, which do not exhibit any unusual cell specificity pattern in the testis.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P dATP by a random primer system (Megaprime DNA labeling system, Amersham, Braunschweig, Germany). Hybridizations were performed under standard conditions (30). Results were expressed as ratio of cpm obtained for PHGPx and actin bands after an overnight exposure on Instantimager (Packard, Meriden, Conn.).
Animal treatment
Male Wistar rats were maintained on a standard diet containing 0.38 mg/kg selenium and allowed food and water ad libitum. Ninety-day-old rats received ethane dimethane sulfonate (EDS; prepared according to ref 31 and dissolved in DMSO/H2O, 1:3) in a single intraperitoneal injection (75 mg/kg body weight) and were killed at the times indicated. Supplementation of EDS-treated rats with testosterone was performed with a long-acting preparation of testosterone esters (Sustanon, Organon Laboratories, Oss, The Netherlands) injected subcutaneously every 3 days at a dosage of 25 mg/animal. Animal treatments were approved by the `Comitato di Bioetica' of the Medical Faculty of Padova and met the highest standard for animal care by humans.
Ex vivo enzyme determinations
Testis homogenate was prepared as follows. After decapsulation, testes were weighed and diluted 1/4 w/vol. with 50 mM Tris-HCl, pH 7.5, 0.3 M KCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 5 mM ß-mercaptoethanol, and homogenized. After freeze-thawing, the homogenate was centrifuged at 2000 x g for 10 min on a tabletop centrifuge to eliminate unbroken material. The supernatant was assayed for protein content (32) and used for enzyme determinations. For separation of cGPx and PHGPx, 5 ml of the supernatant containing 25 mg/ml protein was applied onto a gel permeation chromatography column (Superdex 75 prep. grade, Pharmacia, Uppsala, Sweden). Fractions of 2.5 ml were collected and assayed for glutathione peroxidase and phenyl esterase activity. Commercially available red blood cell cGPx (Sigma), and purified pig heart PHGPx (33) were used as molecular weight markers in two independent runs.
Glutathione peroxidase activity was measured spectrophotometrically at 340 nm as described (33). Glutathione concentration was 3 mM, final Triton X-100 concentration was adjusted to 0.1%, and the reaction was started with 16 µM PCOOH or 150 µM H2O2. To calculate activity, an 
of 6.22 mM-1 cm-1 was used. The nonspecific NADPH oxidation was subtracted only with the substrate H2O2. PCOOH was prepared and titrated as described (33).
Nonspecific phenyl esterase activity was measured spectrophotometrically at 420 nm, following the rate of hydrolysis of 1 mM p-nitrophenyl acetate in 0.1 M Tris-HCl buffer, pH 7.5 (34). As the indicator enzyme for Leydig cells, only phenyl esterase activity eluting in the exclusion volume from Superdex 75 was measured. Aliquots (5–10 µl) of the single fractions from column chromatography were analyzed. Activity rate measurements never exceeded 0.25 OD/min. For calculation of activity, an of 18.8 mM-1 cm-1 was used. The nonspecific hydrolysis of p-nitrophenyl acetate observed in the absence of enzyme was subtracted. One unit is the amount of enzyme catalyzing the transformation of 1 µmol min-1 at room temperature.
In situ hybridization studies for expression of cGPx and PHGPx
Testes were fixed with paraformaldehyde and serial slices 4 µm in thickness were prepared. For RNA in situ hybridization, a mouse PHGPx-cDNA (180 bp) and a mouse cGPx-cDNA (290 bp) probe cloned into pcDNA3 were used. `Antisense' and `sense' RNA probes of PHGPx were generated with BglII and BamHI linearized vectors using the T7 and SP6 promoters, respectively, and labeled with 35S-UTP to a specific activity of 
109 dpm/µg probe. The PHGPx probe corresponded to position 545–738 (Acc. No. D87896). In the case of cGPx, the vector was linearized with XbaI and BamHI. The probe comprised position 289–543 and position 760–95, excluding the intron sequence (ref 35; Acc. No. X03920). The slices were prehybridized at 54°C in a solution containing 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 10 mM Tris, 10 mM sodium phosphate pH 6.8, 20 mM dithiothreitol, 0.2x Denhardt's solution, 0.1% Triton X-100, 0.1 mg/ml Escherichia coli RNA, and `cold' 0.1 mM S-UTP. For hybridization, 90,000 dpm/µl 35S-UTP labeled RNA probe was added to the hybridization mix and the hybridization was continued at 56°C for 16 h in a humid chamber. The slices were washed in hybridization salt solution with dithiothreitol. After RNase digestion, the slices were washed for 30 min with 2x SSC, 0.1% sodium dodecyl sulfate, 30 min with 0.1% SSC at 37°C, and dehydrated by increasing concentrations of ethanol. The slices were coated with Ilford K5 photoemulsion for autoradiography. After 1–4 wk of exposure at 4°C, the slides were developed in Kodak D19b and slices were Giemsa stained. The sections were analyzed with bright- or dark-field illumination using a Olympus B 201 microscope. Photographs were made using Kodak Ectachrome 160T Professional film. Adjacent slices were stained with hematoxylin/eosin (HE) or processed for macrophage visualization by a monoclonal antibody against murine macrophages (ED2, Serotec, Indianapolis, Ind.). To this end, sections were deparaffinized and incubated in phosphate-buffered saline with 0.3% H2O2 for 10 min to destroy endogenous peroxidase activity. Tissue sections were further incubated with 0.1% trypsin (Sigma) for 15 min for antigen retrieval. After treatment with 1% normal horse serum for 10 min, the slices were incubated with the primary antibody at a dilution of 1:400 for 30 min. Treatment with biotinylated anti-mouse immunoglobulin followed, and sections were incubated with peroxidase-conjugated streptavidin for another 30 min. After a final wash, the sections were immersed in a solution containing 0.06 mM 3-3' diamino-benzidine and 2 mM H2O2 in 0.05% Tris-HCl, pH 7.6, for 5 min. Finally, the sections were counterstained with Mayer's hematoxylin. As negative control, normal mouse immunoglobulin G was used instead of the primary antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1A, B,
the PHGPx mRNA level was found to be lower in early puberal rats than in middle puberal ones, corroborating the previous observation that PHGPx in testis indeed depends on sexual maturation. A roughly fivefold increase in PHGPx mRNA can be estimated between the untreated testes of rats 25 and 60 days old, which compares reasonably with the sixfold increase of PHGPx activity 30 to 60 days after birth reported previously (28). The conventional normalization of the mRNA under investigation by actin mRNA ( Fig. 1C) led to an underestimation of the increase of PHGPx mRNA, since the actin mRNA in whole testis also increased with sexual maturation.
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Incubations with the hormones or forskolin, however, did not significantly affect PHGPx mRNA levels. Thus, a direct transcriptional control of the PHGPx gene by testosterone, hCG, or forskolin, i.e., cAMP, was not detectable by this approach.
The specific activity of PHGPx, not of cGPx, correlates with testicular growth
Left without any evidence corroborating the idea that testosterone, hCG, or cAMP might directly activate PHGPx gene transcription, we decided to reinvestigate the development of PHGPx and cGPx activities of the testis during the prepuberal phase of intact rats. Figure 2
confirms that the specific activity of PHGPx increases linearly with testis growth during sexual development. In this experiment, GPx activity was measured in unfractionated supernatants with the PHGPx-specific substrate PCOOH and with H2O2, which can be reduced by both PHGPx and cGPx. Measurements of both types of activity, when related to testis weight, yielded straight regression lines that differed significantly in slopes and intercepts (P<0.05).
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Apparently the main activity may be attributed to PHGPx, since the activities measured with H2O2 ranged only slightly above those obtained with the PHGPx-specific substrate. The convergence of the lines further suggests that the relative contribution of cGPx to the total GPx activity shrinks with increased age. To check the accuracy of these assumptions, testis homogenates of early puberal and adult rats were subjected to gel permeation chromatography, taking the difference in molecular mass of the tetrameric cGPx (80 kDa) and the monomeric PHGPx (20 kDa) as an additional criterion of discrimination. As evident from Fig. 3,
a high and a low molecular mass GPx activity is detected with H2O2. With PCOOH, only the low molecular mass GPx activity (i.e., PHGPx) is seen. Comparing the activities of cGPx and PHGPx in early puberal ( Fig. 3A) and adult rat testis ( Fig. 3B), it is evident that PHGPx is increased in the sexually mature testis whereas cGPx remains essentially the same as in early puberal rats. The activity units underestimate the amount of PHGPx roughly by a factor of 10, which implies that PHGPx is already more abundant than cGPx in the testis of young rats and exceedingly high in adults. Thus, the original observation that PHGPx is the main selenoperoxidase in rat testis and correlates with sexual maturation (28) is confirmed, and a relevant change in cGPx is ruled out.
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When normally developing rats were treated with hCG (500 IU once a day for 1–2 wk), the rise in PHGPx activity reported for hypophysectomized rats (28) was not observed. In fact, the specific PHGPx activities of hCG-treated rats were consistently lower than that of age- or weight-matched controls (data not shown). This apparent discrepancy in the effects of hCG on testicular PHGPx parallels the differential impact of hCG on the integrity of the seminiferous epithelium. Whereas hCG partially restores the seminiferous epithelium in hypophysectomized rats by substituting for the pituitary stimulation of Leydig cells, it reportedly damages the seminiferous epithelium in normal rats (36, 37).
Testicular PHGPx expression in vivo is mediated by Leydig cell-derived testosterone
The observations reported so far—i.e., lack of evidence for a short-term transcriptional activation of the PHGPx gene, positive correlation of PHGPx activity and mRNA with testicular maturation, and the model-dependent differential effects of hCG on testicular PHGPx—prompted us to reconsider our working hypothesis. Instead of looking for a direct hormonal regulation of PHGPx gene transcription or translation, we turned to the alternative possibility of hormone dependency of a particular cell type that preferentially expresses PHGPx. To evaluate this hypothesis, we treated mature rats with EDS, thereby interrupting the pituitary-gonadal axis. EDS is known to selectively eradicate Leydig cells, which results in a time-delayed atrophy of the seminiferous epithelium and, finally, in sterility (38). In this experimental design, a fast decline of PHGPx activity and/or expression paralleling the loss of Leydig cells would support a direct control of the enzyme by Leydig cell-derived hormones, whereas a delayed response paralleling the atrophy of the seminiferous epithelium would favor the assumption of a prevailing expression in a hormone-dependent cell type.
As shown in Fig. 4A,
unspecific phenyl esterase, a marker enzyme of Leydig cells, rapidly declines after EDS treatment, reaching negligible levels by day 3 and recovering only marginally by day 28. PHGPx activity also declines upon EDS treatment ( Fig. 4A), but with considerable delay. Similarly, PHGPx mRNA does not decrease significantly by 4 wk after EDS treatment ( Fig. 4C). The considerable discrepancy in the time course of Leydig cell eradication and PHGPx activity/mRNA decrease demonstrates that 1) Leydig cells themselves do not significantly contribute to overall PHGPx content in testis, 2) testicular PHGPx is obviously not under a direct and short-term control of Leydig cells-derived hormones, but 3) nevertheless cannot be sustained over time without Leydig cell-derived factors. In view of the data reported above and the known relevance of testosterone to functional integrity of the seminiferous epithelium, we investigated whether the lost Leydig cell function in EDS-treated animals could be substituted by testosterone treatment; indeed, Fig. 4B, C shows that, despite a complete and sustained loss of phenyl esterase (or Leydig cells, respectively), the PHGPx activity and mRNA remained completely unchanged for the 4 wk period of observation in the testosterone-substituted animal.
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Not unexpectedly, cGPx activities were not altered accordingly by EDS treatment. In fact, they increased shortly after EDS treatment (at the time of ongoing Leydig cell destruction) and returned to control values during the ensuing weeks (
Fig. 4A). Leydig cells can therefore also be ruled out as a testicular compartment harboring substantial amounts of cGPx.
The round spermatid layer is the site of preferential PHGPx expression
The sites of predominant expression of cGPx and PHGPx were finally clarified by mRNA in situ hybridization. The cGPx mRNA distribution appeared rather diffuse, with a slight predominance in the intertubular interstitium ( Fig. 5A–D).
Immunostaining of adjacent serial slices with a monoclonal antibody against rat macrophages revealed that interstitial cGPx mRNA was largely associated with macrophages and not with Leydig cells ( Fig. 5E, F). Accordingly, a similar distribution of cGPx mRNA was observed in testes of rats treated with EDS 28 days earlier and thus largely devoid of Leydig cells ( Fig. 5E). When hybridized to a probe of comparable size and specific radioactivity, PHGPx mRNA proved to be much more abundant ( Fig. 6).
In the intertubular interstitium, no particular enrichment of PHGPx mRNA in Leydig cells ( Fig. 6E, F; Fig. 7E, F)
or mast cells ( Fig. 7E, F) was detectable. At best, some association with macrophages was sporadically observed. Instead, a heavily stained inner layer of tubular cells was seen in the majority of tubules ( Fig. 6A–D). Comparison with hematoxylin/eosin-stained slices, in which the cellular morphology is better conserved than in those exposed to the more drastic in situ hybridization conditions, revealed that the characteristic label has to be attributed to late round spermatids, whereas the layer containing Sertoli cells, spermatogonia, and primary spermatocytes (but also the elongate spermatids and mature spermatozoa) exhibits a comparable and low background label. This peculiar pattern remains virtually unchanged by EDS treatment ( Fig. 7). The small quantitative difference in PHGPx mRNA content found by transcript quantitation in whole testis of normal and EDS-treated rats (see Fig. 4C) could not be verified histologically due to the various stages of the spermatogenic cycle presented in the tubules of a given slice. In fact, the only obvious changes observed histologically in the EDS-exposed testes were decrease of elongate spermatids and mature spermatozoa, lack of Leydig cells, and enrichment with mast cells ( Fig. 5E, 7E), which is in agreement with earlier findings (38).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The irrelevance of cGPx to specific testicular function may also be inferred from recent gene disruption experiments. cGPx-negative mice developed normally and were obviously fertile even in the homozygous state (39). Only their susceptibility to oxidative stress, as demonstrated by paraquat exposure, was dramatically increased (40). These observations classify cGPx as an emergency device to balance unspecific oxidative stress. This role of cGPx may be considered important for the prevention of hydroperoxide-mediated mutagenic events also in the germ line, as it probably is in somatic cells.
In contrast, PHGPx appears to be intimately involved in the process of spermatogenesis. Admittedly, an essential role of PHGPx in spermatogenesis awaits final proof, since specific PHGPx inhibitors are not available, genetic deficiencies have not yet been discovered, and PHGPx knock-out animals have not been constructed. But the peculiar expression pattern of PHGPx in the seminiferous epithelium as well as its dependency on testosterone is intriguing. Preferential PHGPx gene transcription is observed in the late round spermatids, which ceases abruptly upon transition to elongated spermatids. It therefore is tempting to speculate about a specific role of PHGPx in the prefinal stage of spermatogenesis comprising the change in shape from round spermatids to the elongated forms. We have no idea how PHGPx may trigger this process of differentiation. We can, however, safely state that this differentiation process itself depends on testosterone and that PHGPx expression exhibits persuasive parallelism. Both phenomena occur only after puberty; both are prevented by hypophysectomy, are decreased by pharmacological Leydig cell destruction, and are fully restored by administration of testosterone alone in Leydig cell-deprived testis. It may also be inferred from the data presented here that the sudden transcription of the PHGPx gene at a particular phase of spermatogenesis does not likely result from a direct transcriptional control by testosterone, but rather is due to a testosterone-dependent factor selectively expressed in the spermatids, that still awaits identification.
The regulatory role of PHGPx we infer here is not as remote as it may appear at first glance. PHGPx is indeed an unusual representative of the GPx family of proteins, and is not likely to be an enzyme responsible simply for the defense against oxidative stress. Although it is homologous to and shares the catalytic center with its congeners (21), it has been questioned whether PHGPx is adequately classified as a glutathione peroxidase, because all residues implicated in the specific binding of glutathione by cGPx are missing and the rate of the reaction with GSH is comparatively low (14). It therefore remains uncertain whether PHGPx uses the major cellular reductant GSH efficiently enough to constitute a defense system against oxidative damage. The tissue distribution of PHGPx, being low in the lung, liver, and kidney but high in testis (41, 42), does not parallel any obvious demand for antioxidants. Similarly, the only nonvertebrate homologue of PHGPx discovered so far is almost exclusively associated with the female gender of Schistosoma mansoni and with the vitelline glands there, which are indispensable for reproduction (43, 44). A role of PHGPx distinct from defense against peroxide damage is further suggested by the lack of correlation of protein levels determined immunologically and pertinent glutathione peroxidase activities (45). Considerable protein levels with low or absent enzymatic activity are detectable, for example, in liver and spermatozoa (28), respectively, and this PHGPx protein appears to be closely associated with other proteins yet to be identified (F. Ursini, unpublished observation). A putative regulatory role of PHGPx has also been discussed due to its variable subcellular distribution (14) and its unique ability to reduce hydroperoxides of complex biomembrane lipids generated by 15-lipoxygenase (46), an enzyme implicated in reticulocyte differentiation processes (47). Clear evidence for the involvement of PHGPx in cellular responses has been reported in two cases: 1) PHGPx levels manipulated by time-controlled selenium deprivation and resupplementation correlated inversely with endotoxin-triggered leukotriene biosynthesis in vivo (48); and 2) overexpression of PHGPx in a human endothelial cell line abrogated the interleukin 1-dependent NF
B activation (49). Whether such regulatory events are achieved by modulating the cellular peroxide tone is still debatable. It may as well be envisaged that the selenium moiety of PHGPx is oxidized by particular hydroperoxides and then reacts with susceptible thiols of proteins subject to redox regulation.
The evident potential of PHGPx to regulate responses in various cellular systems strengthens the idea that its peculiar expression pattern in testis is somehow related to spermatid differentiation. If so, its selenoprotein nature might explain the male infertility observed in selenium deficiency. However, beyond the two selenoperoxidases discussed so far, other links of selenium biochemistry with testicular function have to be considered. Certainly, a decline in thioredoxin reductase recently shown to contain a selenocysteine residue (18) and to respond to selenium deprivation in tissue culture (23) would inevitably lead to an impairment of the rapidly dividing seminiferous epithelium due to disturbance of the thioredoxin-dependent deoxynucleotide metabolism (50). Thioredoxin reductase activity, however, is less easily affected by selenium deprivation (23) than the glutathione peroxidases under consideration (25). Still, selenoprotein P, a protein containing 10–12 selenocysteine residues of unknown function, is expressed in Leydig cells (51); pulse-labeling experiments with 75Se showed an additional selenoprotein of 34 kDa to be expressed in rat testis after onset of puberty (52), as is PHGPx (28). In view of this emerging complexity, we anticipate other selenoproteins to complement PHGPx in the testicular maturation process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: EDS, ethane dimethane sulfonate; cGPx, `classical' cytosolic glutathione peroxidase (EC 1.11.1.9); PHGPx, phospholipid hydroperoxide glutathione peroxidase (EC 1.11.1.12); HE, hematoxylin/eosin; PCOOH, phosphatidylcholine hydroperoxide.
Received for publication November 24, 1997. Accepted for publication April 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation is inhibited by overexpression of PHGPx in a human endothelial cell line. Biochem. J. In press
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), or H2O2 (
) as substrates directly from homogenates. Results represent the mean of two measurements (one rat each). The two regression lines are significantly different (P<0.05). The age of the rats was, from left to right: 32, 40, 47, 57, 54, 64, and 121 days.
), and PHGPx (
) activity is reported. C) Results obtained by Northern blotting with the same experimental model performed identically three times. The blotting was probed with radiolabeled PHGPx and actin cDNAs and then quantified as described in Fig. 1. *P < 0.01 vs. day 0 and day 28 after EDS plus testosterone administration. Note that PHGPx activity (A, 
) decline much later than phenyl esterase activity (A, 
= macrophage, LC = Leydig cell. C, D) Sense probe (control) otherwise as in panels A, B. E) Intertubular tissue in testis of rats treated with EDS 28 days earlier. Macrophages (deep brown, 
