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Testis |
a Department of Histology and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy
b Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, 34127 Trieste, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm maturation, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The highest level of PHGPx expression and activity is found in the testis [5, 7], where expression of this enzyme is first detected at 3 wk of age and increases thereafter [8, 9]. In particular, a stage-dependent distribution of PHGPx mRNA, as observed in the seminiferous epithelium of adult mouse [9] and rat [10] testis by in situ hybridization, revealed the presence of a very high level of expression in late round spermatids and early elongating spermatids. In rat epididymal spermatozoa, PHGPx is present in sperm heads and in tail midpiece mitochondria [11, 12]. PHGPx gene expression and enzymatic activity are hormone dependent. In hypophysectomized rats and in testosterone-deprived rats, testicular PHGPx activity and mRNA content significantly decreased and were partially restored by hCG or testosterone administration, respectively [7, 10].
Several lines of evidence strongly support the view that both PHGPx and snGPx are involved in the process of spermatogenesis. The well-established scavenger function of reducing the potentially dangerous hydroxylated phospholipids in situ has recently been enlarged by evidence of additional specific roles in sperm maturation. Ursini and colleagues [13, 14] have suggested that PHGPx is a structural protein of the mitochondrial capsule of mature spermatozoa that accounts for at least 50% of the capsule and is enzymatically inactive. In agreement with this observation, a group of infertile men with oligoasthenozoospermia had a dramatically decreased level of mitochondrial PHGPx expression in their spermatozoa and had morphologically and functionally abnormal midpiece mitochondria [15].
Biochemical and immunocytochemical evidence has indicated that PHGPx is also present in adult rat testis nuclei and binds to chromatin [11]. PHGPx also has the ability to oxidize the thiol groups of protamines, the nuclear proteins of spermatozoa, thereby suggesting that this peroxidase may play a regulatory role in chromatin condensation during the late phase of spermatogenesis [12]. In this respect, the testis-specific selenoprotein snGPx has been postulated to play a role in the stabilization of condensed chromatin by cross-linking protamine disulfides. Spermatozoa taken from the vas deferens of selenium-deficient rats show abnormal nuclei condensation [6]. However, selenium deficiency affects various selenoproteins in different ways, depending on the tissues [16, 17].
To gain further insight into the molecular functions of snGPx and PHGPx in the differentiation program of male germ cells, we investigated the expression pattern of snGPx at early stages of spermatogenesis and compared it with that of PHGPx. The results obtained in our study show that the nuclear isoform is expressed both at the mRNA and protein level during a phase of germ cell development that is temporally distinct from that of the mitochondrial isoform. These findings for the first time provide evidence that the PHGPx gene is differentially spliced in the meiotic prophase and haploid cell phases of mammalian spermatogenesis. In addition, PHGPx but not snGPx is present in the nuclear fraction of testes in which germ cell development has not gone beyond the meiotic stage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats were used in all the experiments. Animals were housed in accordance with the European Union guidelines for animal care and were killed by guillotine or CO2 asphyxia before organ removal.
Cell Preparations and Testis Fractionation
Pachytene spermatocytes and round spermatids were obtained from 36-day-old rat testis as previously described [18]. The cell suspension obtained following enzymatic digestion of testicular tissue was fractionated by velocity sedimentation at unit gravity on 0.5–3% albumin gradient (Staput method). Identity and purity of isolated cell types was assessed by both flow cytometry analysis and light microscopy. For morphologic analysis, cells were attached on polylysine-coated slides, fixed in 3:1 methanol:acetic acid, and stained with hematoxylin. The population of round spermatids at steps 1–8 was assessed at 95% purity, with spermatogonia and a few spermatids at steps 9 and 10 constituting the only source of contamination. The pachytene spermatocyte fraction was of >85% average purity, with Sertoli cells as the major contaminant. The cells were washed twice with PBS and then processed as needed.
Epididymal spermatozoa were collected from adult rats by squeezing the cauda epididymides in PBS. Cells were centrifuged at 1000 x g for 15 min and washed twice in PBS.
Testis mitochondria and nuclei were isolated from 20-day-old rats as previously described [11, 19].
Northern Blot Analysis
Total RNA was extracted from testes of rats of different ages (10, 20, 25, 30, 45, and 60 days) or from isolated cell populations using the guanidinium thiocyanate-caesium chloride ultracentrifugation method [20]. Northern blotting was performed as previously described [21]. The blots were hybridized overnight at 42°C with cDNA probes labeled with 32P-dCTP by random priming (Gibco BRL, Grand Island, NY). The DNA fragment corresponding to nucleotides 31–268 and coding for the alternative exon Ea1 of snGPx (accession no. AF274028) [6] was prepared by reverse transcription polymerase chain reaction (RT-PCR) and cloned in pUC18 (Amersham Pharmacia, Piscataway, NJ). The whole PHGPx cDNA [5] was generously supplied by Dr. D. Driscoll (Cleveland, OH). After probing, the membranes were washed with 2x saline-sodium citrate, 0.1% SDS at room temperature for 30 min, followed by two 15-min washes at 65°C. For the standardization of different lanes, blots were rehybridized with an rRNA cDNA probe.
RT-PCR Analysis
Total RNA (5 µg) of spermatocytes and spermatids was reverse transcribed with the SuperScript II RT kit (Gibco BRL) according to the manufacturer's instructions. PCR was performed in a 50-µl reaction volume as follows: 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 62°C for 45 sec, and extension at 72°C for 45 sec. Within the range of linear amplification, this cycle number allowed a linear cDNA dose response. The cDNA of snGPx and PHGPx was separately amplified by the following sets of primers: snGPx sense primer 5'-ATGGGCCGCGCGGCCG-3' and antisense primer 5'-CCAGGAACTCGTGGCTGTTGC-3' yielding a 278-base pair (bp) fragment; PHGPx sense primer 5'-GCCTCGCGCGTCCATTGG-3' and antisense primer 5'-GGCTGAGAACTCTTCGTGCATGG-3' yielding a 192-bp fragment. As an internal control for the amount of cDNA used, S16 amplification was performed as previously described [21]. A fraction of the PCR products was analyzed by restriction endonuclease digestion.
Protein Extraction and Immunoblotting
After isolation, epididymal spermatozoa were first incubated in 50 mM Tris-HCl, pH 8.00, containing 100 mM dithiothreitol (DTT) for 30 min at 4°C. Cells were washed by centrifugation at 1000 x g for 20 min, lysed, and sonicated in ice 12 times for 15 sec each time. This procedure was repeated twice (Ultrasonics, Leicester, U.K.). The lysis buffer contained 150 mM NaCl, 1% Triton X-100, 12 mM sodium deoxycholate, 2% hexadecyltrimethyl-ammonium bromide, 40 mM DTT, 2 mM PMSF, and a protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO).
After fractionation, meiotic and postmeiotic cells were lysed in the same lysis buffer as described above, except for the absence of DTT, and sonicated 12 times for 15 sec each time. Testicular tissue from 60-day-old rats was homogenized (Ultra-turrax T25; Janke & Kunkel, Staufen, Germany) in lysis buffer without DTT and centrifuged at 1000 x g for 10 min. The supernatant was sonicated 12 times for 15 sec each time and centrifuged at 20 000 x g for 40 min at 4°C.
Protein quantification was performed by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL) with albumin as the standard. Cell proteins were separated on 15% SDS-polyacrylamide gels [22] and then transferred onto a nitrocellulose membrane (Hybond-C extra; Amersham Pharmacia). Blotted membranes were incubated with 1:4000 rabbit polyclonal anti-PHGPx antibody for 1 h at room temperature in 5% nonfat dry milk, 0.1% Tween 20. The affinity chromatography-purified anti-PHGPx antibody was prepared as previously described [23]. After several washes, the filters were incubated with biotin goat anti-rabbit IgG diluted 1:6000 (Zymed, South San Francisco, CA) and avidin-biotinylated horseradish peroxidase (Vectastain; Vector, Burlingame, CA). To assess the purity of the nuclear fraction, membranes were washed overnight in 5% nonfat dry milk, 0.1% Tween 20 and incubated with mouse monoclonal anti-cytochrome c antibody (BD Bioscience, Heidelberg, Germany) at 1:500 dilution followed by a secondary goat anti-mouse IgG diluted 1:1000. Bands were visualized by an enhanced chemiluminescence system (Amersham Pharmacia) according to the manufacturer's recommendations.
Immunochemical Electron Microscopy
Isolated spermatocytes and spermatids were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 h at 4°C. Cells were washed several times in cacodylate buffer, dehydrated, and embedded in LR White M acrylic resin (Sigma). Ultrathin sections were treated with a blocking buffer (1% BSA, 20% normal goat serum [NGS]) in 0.1 M Tris-HCl, pH 8.4, for 1 h at room temperature and then incubated overnight at 4°C in Tris-buffered saline (TBS) containing 1% BSA, 1% NGS, 4% fetal calf serum, 0.1% Tween 20 and anti-PHGPx rabbit polyclonal antibody (final dilution 1:10). After several washes in TBS to remove the excess antibody, the sections were incubated for 2 h at room temperature with colloidal (20 nm) gold-conjugated secondary goat anti-rabbit IgG (British BioCell, Cardiff, U.K.) diluted 1:100 in the same TBS medium. Sections were finally counterstained with uranyl acetate and lead citrate and examined with a Philips EM 208 electron microscope. Anti-PHGPx antibody was omitted in the control sections.
Enzymatic Assay
Germ cells were lysed with a hand-driven glass-teflon Potter in 50 mM Tris-HCl (pH 7.4) containing 1 mM PMSF, 0.1 mM deferoxamine mesylate (desferal), and 0.1 mM resveratrol. The PHGPx activity was measured spectrophotometrically (340 nm, using homemade phosphatidylcholine hydroperoxide as a substrate: 1 mU = 1 µmol NADPH/min) according to the method of Maiorino et al. [24].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the stage-specific mRNA expression pattern of PHGPx and its alternative spliced variant snGPx during germ cell differentiation, we performed a Northern blot analysis of total RNA isolated from fractions of highly enriched pachytene spermatocyte germ cells and from fractions of highly enriched round spermatids of steps 1–8. When the membrane was hybridized with a cDNA probe corresponding to the whole PHGPx, a transcript of 1 kilobase (kb) was detected in both cell types but predominantly in spermatids (Fig. 1). In contrast, when a probe recognizing the alternative snGPx exon was used, a striking difference in the level of expression was observed (Fig. 1). Little, if any, signal was seen in spermatocytes, whereas an intense and broad mRNA band was visible in the haploid cells, suggesting the presence of two transcripts of 1.2 and 1 kb. Because the cDNA probe for PHGPx recognized both isoforms, we used a more sensitive and specific RT-PCR strategy to confirm the differential presence of the two variants in meiotic and postmeiotic cells. One set of primers was selected from the first exon (E1) of PHGPx and one from the first exon (Ea) of snGPx to separately detect transcripts of these two isoenzymes. The results obtained (not shown) were in close agreement with the Northern blot data.
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To evaluate correlations between the developmental appearance of the two isoform transcripts and a definite stage of spermatogenesis, testicular RNAs from rats of different ages were analyzed by Northern hybridization with probes specific for PHGPx or snGPx. In agreement with previous findings [10], PHGPx mRNA appeared in the testis at 20 days of age, then gradually increased, and was retained in the adult (Fig. 2A). In contrast, snGPx transcript was first detected at 30 days of age and was highly accumulated in the adult (Fig. 2B). These data indicate that snGPx mRNA is a unique postmeiotic transcript and that PHGPx mRNA is, by contrast, also transcribed from the diploid genome.
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Western Blot Analysis of PHGPx and snGPx in Differentiating Germ Cells
Because of the proposed functions of snGPx and PHGPx in the late stages of spermatogenesis [6, 14], we investigated the expression pattern of the two enzymes at the protein level during germ cell development. To identify the differentiation stage at which each isoenzyme is synthesized, total proteins extracted from meiotic pachytene spermatocytes and haploid round spermatids were analyzed by immunoblotting. Epididymal spermatozoa and adult testis were used as positive controls. The anti-PHGPx polyclonal antibody used in this experiment specifically recognized a series of testis-specific selenoproteins with molecular masses ranging from 34 kDa to 20 kDa. A major immunoreactive band of 20 kDa, comigrating with rPHGPx (not shown), was detected in both meiotic and haploid germ cells and in control extracts (Fig. 3A). The adult testis displayed two additional bands of 34 kDa and 24 kDa, respectively. To assess whether these bands were also present in pachytene spermatocytes and round spermatids, we overloaded the gel with 20 µg protein/lane (Fig. 3B). The 34-kDa band became apparent, although less so than 20-kDa band, only in round spermatids. These results are consistent with the pattern of mRNA expression and demonstrate that snGPx transcription and translation begin in a phase of spermatogenesis distinct from and later than that for PHGPx.
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Morphological and Functional Characterization of PHGPx in Differentiating Germ Cells
A morphological analysis of PHGPx intracellular distribution was performed by enzyme immunocytolocalization at the electron microscope level in isolated spermatocytes and round spermatids. The polyclonal antibody used did not specifically recognize either PHGPx or snGPx. In keeping with biochemical evidence, specific immunostaining was evident in round spermatids, within the nucleus (Fig. 4C), on the border between the nucleus and cytoplasm (Fig. 4D), in the acrosomal vesicle (Fig. 4F), and within the mitochondria (Fig. 4E). In addition, spermatocytes were also specifically immunostained, with signals localized both in the mitochondria and, unexpectedly, in the nucleus (Fig. 4B).
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The morphological observation that PHGPx and/or snGPx were localized in the nuclei of pachytene spermatocytes prompted us to investigate the subcellular distribution of the two proteins in this phase of testis differentiation. Nuclear and mitochondrial fractions from 20-day-old rat testis were assayed for the presence of PHGPx by immunoblotting (Fig. 5). A band corresponding to the 20-kDa PHGPx was clearly present in both the nuclei and mitochondria fractions, whereas no signal was observed at 34 kDa (not shown). Nuclear fraction purity was determined on the same membrane by incubation with an anti-cytochrome c antibody, which reacted as expected only with the mitochondrial fraction (Fig. 5). This result is in close agreement with the morhological observation, demonstrating conclusively that in addition to the mitochondria PHGPx is also located in the nuclei isolated from a testis lacking haploid spermatid stages.
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To determine whether the immunoreactive selenoproteins present in spermatocytes and spermatids were functional peroxidases, we evaluated the PHGPx activity of isolated germ cells at those developmental stages (Table 1). Both cell types contained catalytically active PHGPx, with the round spermatids having the highest level of specific activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our observation that the highest levels of PHGPx expression and activity were found in round spermatids is in agreement with other reports published previously [7, 9, 10]. By using a specific probe, we were able to demonstrate that round spermatids are also the site of snGPx expression. Therefore, the high levels of transcripts obtained in haploid germ cells using a probe chosen in the common region of the two isoforms should be reinterpreted as a reflection of the combination of the two transcripts.
We also demonstrated that snGPx is switched on in round spermatids at both the mRNA level and the protein level, which indicates that snGPx transcripts are efficiently translated at the same time they are synthesized. More importantly, our data provide evidence of an additional site of expression of the novel selenoprotein during spermatogenesis. Pfeifer et al. [6] purified and identified the 34-kDa peroxidase in the nuclei of late spermatids (steps 13–19). We demonstrated here that expression of the protein begins as early as steps 1–8 of spermiogenesis, providing new information on the timing of the appearance of this selenoprotein during spermatid differentiation. Although the presence of a 34-kDa protein in spermatids of steps 9–13 remains to be investigated, the intense band corresponding to the nuclear form detected in adult testis likely reflects protein accumulation during all stages of elongation. The detection of snGPx in round spermatids coincides with the activation of several genes encoding structural proteins required for spermatozoon assembly (transition proteins, protamines). Hence, the temporal expression of snGPx, its localization in the nucleus, and its catalytic activity all strongly suggest that snGPx might be one of the factors involved in the striking morphological and molecular transformations that occur during spermiogenesis, including arrest of transcription, histone replacement by protamine, and chromatin condensation. The observation that sperm nuclear DNA compaction was severely impaired in selenium-depleted rats lends further support to this hypothesis [6].
We previously reported the presence of PHGPx in the nuclear fraction of adult rat testis [11] and in the heads of elongated spermatids and epididymal spermatozoa [23]. The present study produces morphological and biochemical evidence that part of PHGPx is localized in the nucleus of pachytene spermatocytes, which only express the mitochondrial variant. In accordance with this finding, Arai et al. [25] reported that although PHGPx is mostly imported into mitochondria thanks to its specific targeting signal, the 20-kDa PHGPx protein is also located in the nucleus of cells overexpressing the mitochondrial type of PHGPx. This unexpected result, along with our experimental observation, argues in favor of additional nuclear selenoproteins besides snGPx. It remains to be determined whether the 20-kDa PHGPx is also present in haploid cell nuclei in addition to snGPx. Although the functional significance of the nuclear PHGPx is still elusive, this isoenzyme might contribute to the enzymatic defenses against oxidative damage to the nucleus [26]. Alternatively, it might be involved in different phases of the complex process of chromatin structure remodeling that takes place during production of spermatozoa. The latter possibility is supported by our previous finding of thiol-oxidizing activity of pure PHGPx on protamine [27]. A possible difference between nuclear and mitochondrial isoforms as regards their ability to oxidize germ cell-specific nuclear or mitochondrial protein thiols should be investigated. In this regard, Ursini et al. [13] showed that PHGPx is oxidatively cross-linked in epididymal spermatozoa, functioning as a major structural protein of the sperm mitochondrial capsule but also active as a peroxidase in a mixed population of differentiating spermatogenic cells. In keeping with the latter finding, we observed abundant catalytic activity of PHGPx and/or snGPx in purified populations of pachytene spermatocytes and round spermatids, with the haploid cells showing higher activity. The functional relevance of PHGPx in sperm maturation, whether as an antioxidant or as a protein of the mitochondrial capsule, is further demonstrated by the dramatic reduction in the level of expression of PHGPx, accompanied by an abnormal morphology of midpiece mitochondria, observed in spermatozoa of infertile men with oligoasthenozoospermia [15]. This finding suggests that these defects might be one of the causes of infertility in such patients.
Our data provide evidence that the single-copy PHGPx gene is differentially spliced in meiotic and postmeiotic cells during male germ cell maturation, thereby representing an important example of developmental modulation in gene expression by alternative splicing. Several genes, including those for cAMP response element binding protein (CREB) [28, 29], cAMP response element modulator (CREM) [30], Sox 17 [31], and soluble adenyl cyclase [32], encode functionally different mRNA isoforms by alternative splicing. During spermatogenesis, splicing variant forms with different developmental patterns of expression and functions are produced. For example, the CREM transcript is expressed in the repressor form in premeiotic germ cells, but an activator isoform is produced exclusively at the pachytene spermatocyte stage. CREM protein, however, is detected only after meiosis [33].
In the present study, we demonstrated that snGPx expression is restricted to a specific type of germ cells, the haploid spermatid, and that PHGPx is instead present from the pachytene spermatocyte stage onward. These observations reveal a strict developmental regulation of gene expression by alternative splicing as part of the physiological mechanisms responsible for differentiation of male germ cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Carla Boitani, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via Scarpa 14, 00161 Rome, Italy. FAX: 39 06 4462854; carla.boitani{at}uniroma1.it
3 These authors contributed equally to this study
Received: 19 April 2002.
First decision: 13 May 2002.
Accepted: 22 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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