Differential expression of divalent metal transporter DMT1 (Slc11a2) in the spermatogenic epithelium of the developing and adult rat testis

Kathleen P. Griffin,1 Donald T. Ward,1 Wei Liu,1 Gavin Stewart,1 Ian D. Morris,2 and Craig P. Smith1

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


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Iron is essential for male fertility, and disruptions in iron balance lead to impairment of testicular function. The divalent metal transporter DMT1 is a key modulator of transferrin- and non-transferrin-bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have characterized the pattern of DMT1 expression in the developing and adult rat testis. Northern blot analysis and RT-PCR of testis polyadenylated RNA revealed the presence of iron-responsive element (IRE) and non-IRE transcripts. Semiquantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed that DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis, DMT1 was immunolocalized to both the Sertoli and germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or the adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelium. Our data suggest an important role for DMT1 in intracellular iron handling during spermatogenesis and imply that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, as it does in other tissues, play a role in transporting iron between intracellular compartments and thus may play an important role in male fertility.

iron; spermatogenesis; immunohistochemistry


IRON IS REQUIRED BY ALL ORGANISMS and contributes to a wide range of biological functions, such as mitochondrial oxidation, DNA synthesis, oxygen transport, and nitrogen fixation (27, 29). In mammals, both iron excess and iron deficiency present as clinical conditions. As a result, body iron homeostasis is a tightly regulated process. Importantly, male fertility is affected by disruptions in iron balance. For example, hypogonadism and impaired testicular function are prevalent in patients with iron overload, exemplified by hereditary hemochromatosis (5).

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.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Animals. Sprague-Dawley rats (Charles River, Margate, UK) were bred under controlled conditions in the University of Manchester Biological Services Unit. The day of delivery of pups was designated as the first day of age. All animals were housed with controlled temperature and lighting (12:12-h light-dark cycle) and access to food and water ad libitum. All procedures were performed within the regulations of the National Research Council as detailed in the Guide for Care and Use of Laboratory Animals.

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 3–4 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 85–1,600 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% {beta}-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.


    RESULTS
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Northern blot analysis. As previously reported, Northern blot analysis of testis polyadenylated RNA, using a probe corresponding to nucleotides common to both IRE DMT1 and non-IRE DMT1, detected transcripts with molecular masses of 2.4 and 4.4 kb in kidney cortex (Fig. 1; see also Ref. 7). The 4.4-kb species corresponded to the IRE transcript, while the 2.4-kb species related to the non-IRE transcript. Although weaker, these signals were also present in adult testis.



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Fig. 1. Iron-responsive element (IRE) and non-IRE divalent metal transporter DMT1 are expressed in the adult testis (T) and are the same isoforms present in the kidney cortex (KC). Polyadenylated RNA was isolated from adult rat testis (5 µg) and dissected kidney cortex (3 µg) and was separated on a 1% agarose gel. Incubation with a 32P-labeled probe corresponding to nucleotides 85–1,600 of rat DMT1 led to detection of both IRE and non-IRE transcripts at 4.4 and 2.4 kb, respectively, in both the testis and the kidney cortex.

 
Reverse transcriptase-polymerase chain reaction. Primers specific for IRE DMT1 and non-IRE DMT1 were also used selectively to investigate the expression of these two isoforms in the immature (5-day-old) and adult testis. PCR products from the IRE and non-IRE primer pairs were detected as single bands of ~0.9 and 0.3 kb, respectively, at both ages (Fig. 2). This is consistent with the expected oligonucleotide lengths based on the primers used for each DMT1 isoform. Control reactions performed in the absence of cDNA did not give rise to PCR products. We conclude that IRE DMT1 and non-IRE DMT1 transcripts are present in immature and adult testis.



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Fig. 2. Both major DMT1 isoforms are expressed in the immature and adult rat testes. cDNA was synthesized from 3 µg total RNA from 5-day-old and adult rat testes. Using primers specific to the 2 isoforms, both IRE DMT1 (0.9-kb product) and non-IRE DMT1 (0.3-kb product) were detected in the adult rat testis (lanes 1 and 3) and also in the immature testis (lanes 5 and 7) Control reactions performed in the absence of cDNA did not give rise to PCR products (lanes 2, 4, 6, and 8).

 
Semiquantitative immunoblotting. Semiquantitative Western blot analysis was used to profile DMT1 protein expression in the immature and adult rat testis. DMT1 was detected in the testis throughout the development of the immature rat (Fig. 3). The antibody detected an immunoreactive species of ~70 kDa in all of the age groups tested. This signal was strongest in the 5- and 15-day-old testis. A second, higher molecular mass species between 75 and 90 kDa was detected throughout development and was particularly apparent in the 15-day-old testis. The specificity of the DMT1 antibody was confirmed by the absence of any immunoreactive signal in the membranes incubated in TBST without the primary antibody or with antiserum preincubated with an excess of the immunizing peptide (data not shown).



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Fig. 3. DMT1 immunoreactive species are present throughout development. Membrane extracts from the testes of 5-, 15-, 25-, and 35-day-old rats were prepared and separated by performing SDS-PAGE on an 8% acrylamide gel. Incubation with a DMT1 antibody common to all known DMT1 isoforms showed that immunoreactive species were present throughout development at the ages investigated. A: 2 sets of the testis protein homogenates. In the testes of all of the age groups tested, a 70-kDa protein was detected. A second, higher molecular mass signal between 75 and 90 kDa was detected that was particularly apparent at day 15. B: the size of the signal was especially obvious after overexposure of the signal. The 70-kDa protein was also detected in the kidney of the 35-day-old rat (K).

 
Both of these immunoreactive species were detected in the testis of the adult rat (Fig. 4). The predominant DMT1 immunoreactive species had a molecular mass between 75 and 90 kDa. A band of similar size also was observed in the adult rat duodenum. The lower molecular mass species of ~70 kDa was comparable to that observed in the adult rat kidney. Several lower molecular mass bands between 30 and 60 kDa also were detected in the testis. These bands were ablated by incubation of the membranes with antiserum preincubated with an excess of immunizing peptide. Whether these bands represent functional DMT1 isoforms is currently unknown.



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Fig. 4. In the adult rat, the predominant DMT1 isoform in the testis is the same as that in the duodenum. Membrane extracts from the kidney (K), testis, and duodenum (D) were prepared and separated by performing SDS-PAGE on an 8% acrylamide gel. Incubation with the DMT1 antibody allowed us to visualize broad immunoreactive bands in each of the 3 tissue types. In the testis, the main band was detected between 75 and 90 kDa. A band corresponding to this was observed in the duodenum. A second immunoreactive species was detected in the testis at ~70 kDa. A band of similar size was observed in the kidney. Multiple lower molecular mass bands between 30 and 60 kDa also were detected in the testes.

 
To further characterize the DMT1 species expressed in the adult testis, we performed deglycosylation of testis and kidney protein homogenates with N-glycosidase F (Fig. 5). Treatment of kidney protein homogenates with N-glycosidase F reduced the molecular mass of the band to 50 kDa. In contrast, identical treatment of the testis protein reduced the predominant band from 75–90 to 60 kDa. This indicated that differences in molecular mass of the major proteins detected in testis and kidney were not due primarily to differences in the degree of glycosylation. However, these results do not rule out other forms of posttranslational modification such as phosphorylation.



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Fig. 5. Deglycosylation of DMT1 reveals that the main protein isoform in the testis is different from that in the kidney. Crude membrane extracts from the kidneys and testes of adult rats were prepared and deglycosylated with N-Glycosidase F. The deglycosylated samples (lanes 3 and 6) along with undigested protein homogenates were separated by performing SDS-PAGE on an 8% acrylamide gel. In the undigested testis samples, the main DMT1 isoform was detected between 75 and 90 kDa. The protein was detected at ~60 kDa after deglycosylation. In the undigested kidney samples, DMT1 was detected at ~70 kDa. The protein was detected at ~50 kDa after deglycosylation.

 
Immunohistochemistry of immature rat testis. Immunohistochemistry was performed to investigate the ontogenic expression of DMT1 in the rat testis. DMT1 immunostaining was observed in the testes of all of the immature rats, regardless of age. Staining was confined to the seminiferous tubules and was absent from the interstitia (Fig. 6). Despite morphological changes due to the increasing number of germ cells with age, a distinct staining pattern was observed throughout the development of the testis. In 5-day-old animals, the seminiferous tubule lumen had not formed, and the pattern of staining did not vary between tubules. Higher magnification images showed staining resided in the cytoplasm of the Sertoli cells with minor variations in intensity toward the tubule membrane. In the 15-day-old rat, germ cells were more numerous, with many more types present. Zygotene spermatocytes were observed in each of the tubules, while leptotene spermatocytes were present in a few select tubules. Despite the presence of more germ cells, the pattern of DMT1 localization matched that of the 5-day-old rat in that the Sertoli cell cytoplasm was immunopositive. In 25-day-old rats, the lumen of each tubule was evident and the number of germ cells continued to increase, with pachytene spermatocytes present in most of the tubules. Immunostaining was more punctate in some regions but remained localized to the cytoplasm of the Sertoli cells. In the 35-day-old animal, the diameter of the lumen continued to increase and round spermatids appeared. The pattern of DMT1 expression was observed to differ between tubules in that some of the tubules exhibited more intense immunostaining. This suggested the differential expression of DMT1 in the latter stages of testicular maturation before the completion of the first wave of spermatogenesis. In some tubules, layers of punctate staining were observed at the base of the epithelium, along with tracts of staining that ran from the tubule membrane and opened up toward the lumen. As in the testes of the younger rats, this staining was restricted to the cytoplasm of the Sertoli cells. No plasma membrane staining was observed in any of the groups analyzed. Controls slides incubated without the anti-DMT1 polyclonal antibody or with antiserum preincubated with an excess of the immunizing peptide were used at each age to exclude the possibility of false positives by endogenous peroxidase activity. These slides did not show any immunopositive staining for DMT1.



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Fig. 6. DMT1 is expressed in the Sertoli cell cytoplasm of the immature rat testis. These images show immunohistochemical staining of DMT1 in 5-µm sections of immature rat testis at low and high magnification. Scale bars, 100 µm. Insets in A, C, E, and G depict control staining. A and B: immunostaining at day 5. Immunostaining is present in all of the tubules (*) but not in the interstitia (arrows). B: the cytoplasm of the tubular (Sertoli) cells, which fill the lumen completely, is stained. The germ cells themselves are unstained. C and D: immunostaining at age 15 days. Both photomicrographs show obvious development of the epithelium, with many more cell types present. Zygotene (Z) spermatocytes are present in some tubules, while in others the most advanced germ cell is the leptotene (L) spermatocyte. The cytoplasm of the tubular cells is uniformly stained. E and F: immunostaining at age 25 days. The lumen (*) of the seminiferous tubule is developed, and pachytene (P) spermatocytes are present in many of the tubules. Immunostaining is present in all tubules but is more punctate (arrows). The cytoplasm and nuclei of the zygotene (Z) and pachytene spermatocytes are unstained. G and H: immunostaining at age 35 days. Immunostaining is not as intense or as uniform here as it was at the earlier ages, and some tubules are more heavily stained (1) than others (2). The cytoplasm and nuclei of the germ cells, including the round (R) spermatids that appear at this age, are not immunostained. The apical surface of the epithelium shows layers of strong punctate staining toward the base of the epithelium (arrows). Tracts of staining (<) are visible between the basal and apical stained areas.

 
Immunohistochemistry of adult rat testis. In the adult rat, with a fully developed spermatogenic cycle, the pattern of immunostaining (Fig. 7) was in marked contrast to that observed in 5-, 15-, and 25-day-old rats. First, both Sertoli cells and germs cells of the adult rat testis stained positively for DMT1. Second, while the germ cells exhibited mostly cytoplasmic staining, DMT1 could be localized not to all but to only a select number of nuclei. Third, the Sertoli cell nuclei also were clearly stained throughout all of the stages, in contrast to the developing animal, in which staining was confined to the cytoplasm.



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Fig. 7. DMT1 is differentially expressed in the seminiferous tubules of the adult rat testis. This low-magnification image shows immunohistochemical staining of DMT1 in a 5-µm section of adult rat testis. Inset depicts control staining. Staining varies in intensity in each of the tubules and is associated with different stages of the spermatogenic cycle of the seminiferous epithelium. There is no staining in the interstitial cells. Scale bar, 100 µm.

 
At Stage II/III of the cycle (Fig. 8, A and B), the pachytene spermatocytes had abundant DMT1, while the cytoplasm of the elongate spermatids exhibited light immunopositive staining. At Stage V of the cycle (Fig. 8, C and D), the tubule was very darkly stained, which was attributable mainly to the tails of the elongate spermatids. The nuclei of the round spermatids exhibited faint staining, while the pachytene spermatocytes remained unstained, highlighting the stage-specific nature of DMT1 expression. In tubules at Stage VIII (Fig. 8, E and F) elongate spermatids were stained intensely. The residual bodies that accompany these mature germ cells also showed strong DMT1 staining. Pachytene spermatocytes were once again stained, while the pattern of immunostaining in the spermatids matched that in the Stage V tubule. At Stage X (Fig. 8, G and H), DMT1 was not detected in the elongating spermatids but was abundant in the pachytene spermatocytes. At Stage XII/XIII of the cycle (Figs. 8, I and J), the location and intensity of DMT1 expression was similar to that at Stage X but exhibited the occasional staining of zygotene spermatocytes. Importantly, we did not detect plasma membrane staining of Sertoli cells at any stage, suggesting that in the adult rat, DMT1 is not the primary means of iron transport across the blood-testis barrier. The control slides of adult testis did not exhibit any immunopositive staining for DMT1.



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Fig. 8. DMT1 expression is highly coordinated with the spermatogenic cycle in the adult rat. These images show immunohistochemical staining of DMT1 in 5-µm sections of adult rat testis at medium and high magnification. All scale bars, 100 µm. A: a tubule at Stage II/III of the cycle of the seminiferous epithelium. B: same tubule shown in A at higher magnification. The predominant staining is associated with the nuclei of pachytene (P) spermatocytes. There is also some faint staining associated with the cytoplasm of the elongating (E) spermatids. The Sertoli cell nuclei also stain positively for DMT1 (arrows). C and D: a Stage V tubule showing intense staining at the apical surface of the epithelium associated with the tails of the elongate (E) spermatids. The nuclei of round (R) spermatids are stained faintly. The nuclei of the Sertoli cells are again stained (arrows), while the pachytene (P) spermatocytes are unstained. E and F: a Stage VIII tubule showing intense immunostaining, which can be attributed to both the tails of the elongate (E) spermatids and their accompanying residual bodies (<). There is also positive staining of the round (R) spermatids, the pachytene (P) spermatocytes, and the Sertoli cell nuclei (arrows). G and H: a Stage X tubule showing no immunostaining of the characteristic elongating spermatids (Sp), while the pachytene (P) spermatocytes and Sertoli cell nuclei (arrows) remain heavily stained. I and J: a tubule at Stage XII/XIII of the cycle of the seminiferous epithelium. The elongated (E) spermatids and their associated cytoplasm are devoid of any staining for DMT1. However, the nuclei of the pachytene (P) spermatocytes and occasional zygotene spermatocytes (<) are strongly stained. Again, Sertoli cell nuclei are positively stained (arrows).

 

    DISCUSSION
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 MATERIALS AND METHODS
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 DISCUSSION
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It is well established that iron plays an important role in spermatogenesis and in the normal function of the testis (5). In other tissues, the mechanism of iron sequestration is becoming clearer. For example, in the duodenum, the broad spectrum divalent metal transporter DMT1 mediates iron movement across the apical enterocyte membrane as part of the process of dietary iron absorption. Although DMT1 was previously shown to be present in the testis, its cellular localization and role in testicular function remains unreported. As a first step in determining its role in this tissue, we determined the pattern of DMT1 expression in the developing and mature rat testis using semiquantitative immunoblotting and immunohistochemistry.

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 75–90 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 75–90 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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Fig. 9. Summary graph showing the stage-specific expression of DMT1 in the germ cells of the adult rat testis. DMT1 was located in the spermatocytes of the seminiferous tubules throughout the spermatogenic cycle but was absent at Stage V (hatched bars). The round spermatids expressed DMT1 between Stages V and VIII, while the elongate spermatids expressed DMT1 from Stage I though Stage VIII. The spermatogonia were devoid of any immunostaining throughout the cycle.

 
From the basement membrane to the lumen of the seminiferous tubules, germ cells are classified into three types: spermatogonia, spermatocytes, and spermatids, which are either round or elongate. Throughout the 14 stages of spermatogenesis, DMT1 was not observed in any of the spermatogonia. DMT1 was detected in all of the spermatocytes but was absent in these cells at Stage V of the cycle. DMT1 immunostaining was observed in the round spermatids between Stages V and VIII of the cycle and in the elongate spermatids between Stages I and VIII of the cycle. The most intense immunostaining was detected in the elongate spermatids, particularly between Stages V and VIII. This suggests that these cells have a particularly high requirement for iron, which may be of importance in the maturation and long-term survival of the sperm as it passes out of the seminiferous tubule. These data are consistent with those of Morales et al. (25), who reported that levels of radiolabeled transferrin were highest in the elongate spermatids and residual bodies of the testis.

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We thank The Royal Society, the Wellcome Trust, and the Medical Research Council for funding this work. K. P. Griffin is a Medical Research Council Co-operative Award in Science and Engineering student. D. T. Ward is the recipient of National Kidney Research Fund Training Fellowship TF6/2002. C. P. Smith is the recipient of a Royal Society university fellowship.


    FOOTNOTES
 

I. D. Morris, Hull Medical School, Univ. of York, Heslington, York YO10 5DD, United Kingdom (E-mail: ian.morris{at}hyms.ac.uk)


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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
1. Andrews NC. Disorders of iron metabolism. N Engl J Med 341: 1986–1995, 1999.[Free Full Text]

2. Canonne-Hergaux F and Gros P. Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice. Kidney Int 62: 147–156, 2002.[CrossRef][ISI][Medline]

3. Canonne-Hergaux F, Gruenheid S, Ponka P, and Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 93: 4406–4417, 1999.[Abstract/Free Full Text]

3. De Krester DM and Kerr JB. The cytology of the testis. In: The Physiology of Reproduction (2nd ed.), edited by Knobil E and Neill JD. New York: Raven, 1994, vol. 1, p. 1177–1290.

4. Dym M and Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 3: 308–326, 1970.[Medline]

5. Edwards CQ, Cartwright GE, Skolnick MH, and Amos DB. Homozygosity for hemochromatosis: clinical manifestations. Ann Intern Med 93: 519–525, 1980.[ISI][Medline]

6. Ferguson CJ, Wareing M, Delannoy M, Fenton R, McLarnon SJ, Ashton N, Cox AG, McMahon RF, Garrick LM, Green R, Smith CP, and Riccardi D. Iron handling and gene expression of the divalent metal transporter, DMT1, in the kidney of the anemic Belgrade (b) rat. Kidney Int 64: 1755–1764, 2003.[CrossRef][ISI][Medline]

7. Ferguson CJ, Wareing M, Ward DT, Green R, Smith CP, and Riccardi D. Cellular localization of divalent metal transporter DMT-1 in rat kidney. Am J Physiol Renal Physiol 280: F803–F814, 2001.[Abstract/Free Full Text]

8. Fleming MD, Romano MA, Garrick LM, Garrick MD, and Andrews NC. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 95: 1148–1153, 1998.[Abstract/Free Full Text]

9. Garrick MD, Dolan KG, Horbinski C, Ghio AJ, Higgins D, Porubcin M, Moore EG, Hainsworth LN, Umbreit JN, Conrad ME, Feng L, Lis A, Roth JA, Singleton S, and Garrick LM. DMT1: a mammalian transporter for multiple metals. Biometals 16: 41–54, 2003.[CrossRef][ISI][Medline]

10. Gelly JL, Richoux JP, and Grignon G. Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. Cell Tissue Res 276: 347–351, 1994.[CrossRef][ISI][Medline]

11. Georgieff MK, Wobken JK, Welle J, Burdo JR, and Connor JR. Identification and localization of divalent metal transporter-1 (DMT-1) in term human placenta. Placenta 21: 799–804, 2000.[CrossRef][ISI][Medline]

12. Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, and Gros P. The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med 189: 831–841, 1999.[Abstract/Free Full Text]

13. Gruenheid S, Cellier M, Vidal S, and Gros P. Identification and characterization of a second mouse Nramp gene. Genomics 25: 514–525, 1995.[CrossRef][ISI][Medline]

14. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Cloning and characterisation of a mammalian proton-coupled metal-ion transporter. Nature 388: 482–488, 1997.[CrossRef][ISI][Medline]

15. Holash JA, Harik SI, Perry G, and Stewart PA. Barrier properties of testis microvessels. Proc Natl Acad Sci USA 90: 11069–11073, 1993.[Abstract]

16. Hubert N and Hentze MW. Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function. Proc Natl Acad Sci USA 99: 12345–12350, 2002.[Abstract/Free Full Text]

17. Huggenvik J, Sylvester SR, and Griswold MD. Control of transferrin mRNA synthesis in Sertoli cells. Ann NY Acad Sci 438: 1–7, 1984.[ISI][Medline]

18. Jabado N, Canonne-Hergaux F, Gruenheid S, Picard V, and Gros P. Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells. Blood 100: 2617–2622, 2002.[Abstract/Free Full Text]

20. Lee PL, Gelbart T, West C, Halloran C, and Beutler E. The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis 24: 199–215, 1998.[CrossRef][ISI][Medline]

21. Maguire SM, Millar MR, Sharpe RM, Gaughan J, and Saunders PT. Investigation of the potential role of the germ cell complement in control of the expression of transferrin mRNA in the prepubertal and adult rat testis. J Mol Endocrinol 19: 67–77, 1997.[Abstract]

22. Moos T, Trinder D, and Morgan EH. Cellular distribution of ferric iron, ferritin and divalent metal transporter 1 (DMT1) in substantia nigra and basal ganglia of normal and {beta}2-microglobulin deficient mouse brain. Cell Mol Biol (Noisy-le-grand) 46: 549–561, 2000.

23. Morales C and Clermont Y. Receptor-mediated endocytosis of transferrin by Sertoli cells of the rat. Biol Reprod 35: 393–405, 1986.[Abstract]

24. Morales C, Hugly S, and Griswold MD. Stage-dependent levels of specific mRNA transcripts in Sertoli cells. Biol Reprod 36: 1035–1046, 1987.[Abstract]

25. Morales C, Sylvester SR, and Griswold MD. Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo. Biol Reprod 37: 995–1005, 1987.[Abstract]

26. Petrie RG Jr and Morales CR. Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis. Cell Tissue Res 267: 45–55, 1992.[ISI][Medline]

27. Ponka P and Lok CN. The transferrin receptor: role in health and disease. Int J Biochem Cell Biol 31: 1111–1137, 1999.[CrossRef][ISI][Medline]

28. Rolfs A, Bonkovsky HL, Kohlroser JG, McNeal K, Sharma A, Berger UV, and Hediger MA. Intestinal expression of genes involved in iron absorption in humans. Am J Physiol Gastrointest Liver Physiol 282: G598–G607, 2002.[Abstract/Free Full Text]

29. Rouault TA. Systemic iron metabolism: a review and implications for brain iron metabolism. Pediatr Neurol 25: 130–137, 2001.[CrossRef][ISI][Medline]

30. Segretain D, Egloff M, Gerard N, Pineau C, and Jegou B. Receptor-mediated and absorptive endocytosis by male germ cells of different mammalian species. Cell Tissue Res 268: 471–478, 1992.[CrossRef][ISI][Medline]

31. Skinner MK and Griswold MD. Sertoli cells synthesize and secrete transferrin-like protein. J Biol Chem 255: 9523–9525, 1980.[Abstract/Free Full Text]

32. Stallard BJ, Collard MW, and Griswold MD. A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis. Mol Cell Biol 11: 1448–453, 1991.[ISI][Medline]

33. Su MA, Trenor CC III, Fleming JC, Fleming MD, and Andrews NC. The G185R mutation disrupts function of the iron transporter Nramp2. Blood 92: 2157–2163, 1998.[Abstract/Free Full Text]

34. Sylvester SR and Griswold MD. Localization of transferrin and transferrin receptors in rat testes. Biol Reprod 31: 195–203, 1984.[Abstract]

35. Tabuchi M, Yoshimori T, Yamaguchi K, Yoshida T, and Kishi F. Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells. J Biol Chem 275: 22220–22228, 2000.[Abstract/Free Full Text]

36. Wareing M, Ferguson CJ, Delannoy M, Cox AG, McMahon RFT, Green R, Riccardi D, and Smith CP. Altered dietary iron intake is a strong modulator of renal DMT1 expression. Am J Physiol Renal Physiol 285: F1050–F1059, 2003.[Abstract/Free Full Text]

37. Wright WW, Paryinen M, Musto NA, Gunsalus GL, Phillips DM, Mather JP, and Bardin CW. Identification of stage-specific proteins synthesized by rat seminiferous tubules. Biol Reprod 29: 257–270, 1983.[Abstract]





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