1School of Biological Sciences, University of Manchester, Manchester; and 2Hull York Medical School, University of York, Heslington, York, United Kingdom
Submitted 29 January 2004 ; accepted in final form 7 September 2004
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ABSTRACT |
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iron; spermatogenesis; immunohistochemistry
The divalent metal transporter DMT1 (Slc11a2), also known as Nramp2 (13) and DCT1 (14), is a key component of the complex physiological process regulating body iron levels. The hydrophobic protein, with 12 predicted transmembrane domains and cytoplasmic NH2 and COOH termini, has broad selectivity, mediating transport of a spectrum of divalent cations. These include iron, copper, and some toxic metals such as cadmium (14). DMT1 is expressed in many tissues, including duodenum, placenta (11), brain (22), and kidney (2, 7). In duodenum, DMT1 is expressed on the apical membrane of enterocytes, where it is primarily responsible for the non-transferrin-mediated absorption of dietary iron. Its expression is regulated by dietary iron intake in a number of tissues (3, 7, 14, 28). In other tissues, DMT1 is localized in vesicular membranes and participates in transferrin-mediated iron acquisition by transporting iron across these membranes and into the cell (1).
Alternative splicing of exons in the DMT1 gene produces four distinct DMT1 mRNA. These differ at the 3' end with respect to an iron-responsive element (IRE) present in the untranslated region (8, 20) and also at the 5' end (16). As a result of alternative splicing, the encoded proteins differ at the NH2 and COOH termini but share a common central domain.
In the testis, the acquisition of iron is of particular importance because the spermatogenic cells, which acquire iron during development, are lost as mature sperm pass into the reproductive tract (26). The testis is divided into two compartments, the interstitia and the seminiferous tubules, in which spermatogenesis takes place. Here germ cells are present at various stages of development in the form of spermatogonia, spermatocytes, and spermatids, passing further toward the lumen of the tubule as they mature (3a). The tubular epithelium, like the brain, represents a compartment functionally separate from the systemic circulation by a barrier known as the blood-testis barrier. Consequently, the spermatogenic epithelium is unable to acquire iron directly from plasma transferrin (15). However, the somatic Sertoli cells, whose tight junctions form the blood-testis barrier, synthesize their own transferrin (31) to transport iron across the cell to the spermatocytes and spermatids (4, 17, 25). Elements of transferrin-mediated iron transport have been characterized in the rat testis (10, 23, 25, 26, 32) and are particularly apparent in the mitotic and meiotic germ cells (24, 34, 37). This suggests a role for iron in cell division, differentiation, and metabolism in the testis (30).
DMT1 mRNA is present in testis (14), and DMT1 protein has been localized to the phagosomal membranes of two Sertoli cell lines (18). As a first step in determining the role of DMT1 in testicular function, we have characterized DMT1 expression in both the immature and adult rat testis. We have shown that DMT1 is present in the testis and that the expression profile in the development of the rat testis is cell specific and in the adult is highly coordinated with the spermatogenic cycle. This suggests an important role for DMT1 in spermatogenesis and implies that germ cells have a need for a precisely timed supply of iron. The stage-specific nature of expression indicates that DMT1 has a role in male fertility, and this knowledge may be useful when considering conditions of abnormal iron regulation.
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MATERIALS AND METHODS |
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Tissue preparation.
Groups of male adult rats (mean body wt of 275 g, age 60 days) and male rat pups 5, 15, 25, and 35 days of age were killed by concussion followed by cervical dislocation. After dissection, the left testes from three animals from each age group were fixed by immersion in Bouin's fluid (71% picric acid, 24% formalin, and 5% glacial acetic acid) for 34 h. These tissues were dehydrated through a gradient of ethanol and stored in absolute ethanol for 18 h before being embedded in paraffin wax. All other testes from each group were pooled and frozen at 80°C.
RNA extraction and Northern blot analysis. Total RNA was extracted from frozen testis using the acid guanidinium isothiocyanate-phenol method, and Northern blot analysis was performed according to the method of Ferguson et al. (7) using dissected kidney cortex as a positive control. The double-stranded probe was randomly primed and labeled with 32P and corresponded to nucleotides 851,600 of rat DMT1, shared by both the IRE and non-IRE transcripts (GenBank accession no. AF029757). Briefly, 5 µg of kidney cortex mRNA and 3 µg of testis mRNA were separated on 1% agarose gels containing 2.2 M formaldehyde and blotted onto Hybond-N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). Membranes were hybridized at 42°C overnight with 5x SSC, 50% formamide, 3x Denhardt's solution, 0.4% SDS, 10 µl/ml of salmon sperm DNA, and 10% wt/vol dextransulfate. Membranes were washed at 65°C with 0.1% SSC-0.1% SDS and exposed to film (Kodak Biomax; Kodak, Hemel Hempstead, UK) for 48 h.
Reverse transcriptase-polymerase chain reaction. RT-PCR was performed with 3 µg of total RNA. cDNA was synthesized with oligo(dT) and Superscript II reverse transcriptase (Invitrogen, Paisley, UK). Primer sets for IRE DMT1 (GenBank accession no. AF008439) and non-IRE DMT1 (GenBank accession no. AF029757) spanning introns were designed using Primer Express (Applied Biosystems, Warrington, UK). Both isoforms used the same forward primer: 5'-CAACGGAATAGGCTGGAGGA-3'. The reverse primer sequences for IRE and non-IRE transcripts were 5'-GGCAGGAGGATCTCTGTGAG-3' and 5'-GGCACAAAAGGGCTTAGAGA-3', respectively. The amplification of IRE DMT1 cDNA was performed at 95°C for 30 s, at 55°C for 45 s, and at 72°C for 60 s for 35 cycles. The amplification of non-IRE DMT1 cDNA was performed at 95°C for 30 s, at 58°C for 30 s, and at 72°C for 30 s for 30 cycles. The PCR products were resolved on 1% agarose gels. Control reactions were performed with the primers in the absence of cDNA.
Peroxidase immunohistochemistry. Paraffin wax sections (5 µm) were cut using a Leica RM2135 rotary microtome (Leica Microsystems, Nussloch, Germany) and fixed onto Superfrost microscope slides (BDH, Poole, UK). Deparaffinized and rehydrated sections were incubated with 3% H2O2 in methanol for 20 min to quench endogenous peroxidase activity. Masked antigens were retrieved by microwaving the sections in Tris-EDTA-glycerol buffer (10 mM Tris and 0.5 mM EGTA, pH 9.0) for 2 min at 800 W and 4 min at 400 W. Sections were allowed to cool for a minimum of 2 h before being treated with 50 mM NH4Cl in PBS for 30 min. Sections were blocked with 1% BSA in PBS and incubated overnight at 4°C in a humidified chamber with a previously characterized affinity-purified polyclonal anti-DMT1 antibody raised to an epitope common to all known DMT1 isoforms (6, 7, 37). Before use, the antibody was diluted 1:500 in PBS containing 0.1% BSA and 0.3% Triton X-100. Sections from each group were also incubated with the PBS-BSA-Triton X-100 solution alone for use as negative controls.
Slides were rinsed with PBS containing 0.1% BSA before being incubated with goat anti-rabbit immunoglobulin diluted 1:200 for 1 h at room temperature. After being rinsed with the PBS solution, the immunohistochemical reaction was visualized using 3,3'diamino-benzidine-tetrahydrochloride. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted with Eukitt resin (Kindler, Freiburg, Germany). Immunostaining was examined under a Zeiss Axiophot light microscope (Carl Zeiss, Welwyn Garden City, UK), and photographs were taken using a Spot RT color digital camera (Diagnostic Instruments, Sterling Heights, MI) and analyzed with the accompanying Spot RT Advanced software, version 3.04.
Western blot analysis. Semiquantitative immunoblotting was performed according to the method described by Ferguson et al. (7). Crude membrane preparations from each group of frozen tissues were created using differential ultracentrifugation. Protein homogenates were spun at 2,500 g to remove nuclei and cell debris, and the postnuclear supernatants were centrifuged at 100,000 g to pellet the particulate proteins. These were then resolved using 8% SDS-PAGE and transferred to Biotrace nitrocellulose membranes (Paul Gelman Sciences, Northampton, UK). Adult rat kidney, proximal duodenum, and 35-day-old rat kidney were also prepared in this manner for use as positive controls. Immunoblotting was performed using the anti-DMT1 antibody diluted 1:5,000 in Tris-buffered saline-Tween 20 (TBST). The enhanced chemiluminescence system was used to visualize the signal (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Experiments were repeated in triplicate. Membranes incubated only in TBST or in antiserum preincubated with an excess of the immunizing peptide served as negative controls. In every case, staining of the gels with Coomassie blue and the membranes with Ponceau red S stain confirmed that equal amounts of proteins had been loaded and transferred to the membrane.
Protein deglycosylation.
Particulate proteins were deglycosylated using N-glycosidase F (PNGase F; New England Biolabs) according to the manufacturer's instructions. Briefly, particulate fractions were denatured at 65°C in buffer containing 0.5% SDS and 1% -mercaptoethanol, then incubated at 37°C for 1 h in buffer supplemented with NP-40 (1% final) and PNGase F (25 U/µg of particulate protein). After deglycosylation, samples were solubilized using 5x Laemmli buffer and immunoblotted as before. The electrophoretic mobility of deglycosylated DMT1 was compared with that obtained from a sample processed identically but in the absence of PNGase F and also to a sample of particulate proteins stored on ice throughout.
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RESULTS |
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DISCUSSION |
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Immature testis. Using a combination of Northern blot analysis and RT-PCR, we detected both IRE and non-IRE DMT1 transcripts in the immature testis. The presence of the IRE transcript indicates that like other tissues, including kidney and duodenum, the testis can modulate iron translocation via DMT1 in response to cellular demands.
Semiquantitative immunoblotting of immature rat testis revealed that two distinct DMT1 protein species were differentially expressed. The lower molecular mass isoform of 70 kDa showed strong expression at days 5 and 15, but thereafter expression decreased. At 15 days, a 75- to 90-kDa protein was also particularly visible. This isoform has a molecular mass similar to that of the major DMT1 protein found in the adult testis (see below) and is analogous to the predominant isoform found in the duodenum (3). In contrast, the lower molecular mass protein expressed at days 5 and 15 is of the same molecular mass as the kidney isoform (7).
The pattern of DMT1 expression in the immature testis was found to be similar in all ages up to 35 days. Immunohistochemistry localized the DMT1 protein to the cytoplasm of the Sertoli cells. Despite the age-related changes in seminiferous tubule morphology that include an increase in tubule diameter and the number of germ cell types, no age-related changes in the cellular pattern of DMT1 expression were detected. In addition, the overall pattern of DMT1 immunoreactivity was unaffected by the changes in the levels of expression of the two isoforms as revealed by Western blot analysis. This may indicate changes in the required function of DMT1 by Sertoli cells over time.
In the 35-day-old rat testis, immunostaining was restricted to the cytoplasm of the Sertoli cells. As the number of germ cells increased, the overall staining of the tubules decreased. Also, at this age, the pattern of DMT1 expression was first observed to vary between some of the tubules in that some tubules exhibited intense regions of staining in the form of tracts running from the tubular membrane to the lumen. As the first wave of spermatogenesis approached completion, immunostaining began to appear more like that observed in the adult rat, indicating the onset of stage-specific DMT1 expression as seen in the adult testis. This pattern of expression was similar to that of transferrin mRNA, although differential tubular expression of transferrin was noted much earlier, at 14 days (21).
The Sertoli cell is responsible for the biochemical and structural support of the germ cells embedded within the epithelium, providing the factors needed for spermatogenesis (3a). In the immature rat, both before and after the establishment of the Sertoli cell tight junctions, DMT1 was present in the cytoplasm of the Sertoli cells but absent from the plasma membranes. Therefore, the results of the present study suggest that in the Sertoli cells of immature rat testis, DMT1 is not the primary route of iron transport across the blood-testis barrier. DMT1 has been localized to late endosomes in a number of transfected cell lines, including Chinese hamster ovary, RAW 264.7 (12), human embryonic kidney-293T (33), and Hep-2 cells (35), where it also has been observed in lysosomes. Without higher resolution images, which might be obtained using immunogold electron microscopy, it was not possible to resolve the intracellular location of DMT1 and therefore speculate on the regions of punctate staining observed. However, it is possible that DMT1 serves to transport iron between the intracellular compartments of the testis as it does in other cell types.
Adult testis. In the fully developed rat testis, Northern blot analysis and RT-PCR showed that both IRE and non-IRE DMT1 transcripts were present. This finding corroborates data reported by Gunshin et al. (13), who isolated a 4.4-kb cDNA encoding a 561-amino acid protein. On the basis of Northern blot analysis, Gunshin reported the expression of the 4.4-kb transcript in adult rat testis. This transcript encodes an iron-responsive DMT1 isoform expressed in the duodenum.
Western blot analysis showed that the predominant isoform of DMT1 in the adult testis has a molecular mass of 7590 kDa, although proteins of lower molecular mass, such as those observed in the immature testis, were also present. Deglycosylation of protein isolated from the adult testis reduced the molecular mass from 7590 to 60 kDa. This is consistent with the predicted molecular masses of 61 and 62 kDa of the IRE and non-IRE DMT1 isoforms, respectively. The predominant isoform in the adult testis had a molecular mass different from that expressed in the immature testis and kidney. The predicted difference in molecular mass between the IRE and non-IRE forms of DMT1 is 1 kDa, whereas the difference between the molecular mass of the deglycosylated proteins was 10 kDa. Therefore, it is unlikely that expression of predominantly IRE or non-IRE proteins can explain these variations. It is more likely that the renal isoform and possibly that expressed in the immature testis are truncated splice variants or undergo posttranslational modification other than glycosylation.
The data from the immature and adult testes together can be summarized as follows. Throughout development to maturation, the testis differentially expresses two predominant DMT1 isoforms: a 70-kDa protein that, based on molecular mass, is the same as the major DMT1 isoform expressed in the kidney, and a 75- to 90-kDa protein that, based on molecular mass, is the same as the predominant DMT1 isoform expressed in the duodenum.
Immunohistochemistry showed that, in contrast to the immature animal, DMT1 in the mature rat testis has a differential pattern of expression. In the adult, staining was both cytoplasmic and nuclear, and both Sertoli cells and germ cells were stained. DMT1 expression was cell specific; therefore, the pattern observed was dependent on the stage of the spermogenic cycle. Figure 9 summarizes the stage-specific expression of DMT1 in the germ cells of the adult testis.
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DMT1 was clearly immunolocalized to a number of nuclei in the germ cells of the adult testis. The nuclear stain was not present in all of the spermatogenic cells of the testis, indicating that DMT1 is expressed specifically in these structures and that its presence is not an artifact of the methodology. DMT1 has been discovered in the nuclei of other cell types, and while its function in the nucleus remains unknown, this may indicate a new role for DMT1 in metal ion transport (9).
Because the testis is functionally separate from the systemic circulation and cannot sequester iron directly from plasma transferrin, the Sertoli cells synthesize their own transferrin (31). A theoretical model has been proposed to explain how iron crosses the blood-testis barrier for use by the germ cells in the seminiferous tubules (17, 25). First, the basal membranes of the tubules are exposed to iron-laden plasma transferrin, which binds to transferrin receptors on the Sertoli cells. These complexes are internalized within the Sertoli cells into endosomes. These contain proton pumps, which aid the detachment of iron from the transferrin. Iron is then transported out of the endosomes into the Sertoli cell cytoplasm. Here the iron binds with testicular transferrin, and these complexes are secreted into the intracellular spaces between the germ cells. Transferrin-mediated iron transport has been localized to all structures within the seminiferous tubule (23, 26, 34), but not all investigations have addressed stage specificity.
We currently do not know the precise intracellular location of DMT1 in testicular cells, but the fact that the protein is expressed in Sertoli cells and in a stage-specific manner by germ cells supports a role for DMT1 in iron handling. By analogy to its role in other cells and because we have found no evidence for expression of DMT1 on plasma membranes, DMT1 expressed within Sertoli and germ cells could mediate proton-coupled export of iron out of the endocytic compartment and into the cell.
In this investigation, we have localized DMT1 to the Sertoli cells, spermatocytes, and spermatids of the adult rat testis. There is different special and temporal expression of DMT1 in the adult rat, in contrast to the immature rat, which exhibits widespread expression of DMT1 throughout development. Expression of DMT1 in the adult rat testis is cell specific, highly coordinated with the spermatogenic cycle, and particularly apparent in the elongate spermatids between Stages V and VIII of the cycle. This suggests that the germ cells within the seminiferous tubules of the testis have a need for a precisely timed supply of iron. The stage-specific nature of DMT1 expression implies an important role for DMT1 in spermatogenesis and male fertility. This may be useful when considering conditions of abnormal iron regulation. The broad specificity of DMT1 may also implicate these findings in the transport of other divalent and toxic metals and could help to explain their testicular toxicity.
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, Univ. of Manchester, G.38 Stopford Bldg., Oxford Road, Manchester M13 9PT, United Kingdom (E-mail: cpsmith{at}man.ac.uk)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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