Articles in PresS. Am J Physiol Cell Physiol (September 8, 2004). doi:10.1152/ajpcell.00061.2004 1
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Differential Expression of Divalent Metal Transporter DMT1
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(Slc11a2) in the Spermatogenic Epithelium of the
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Developing and Adult Rat Testis
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Kathleen P. Griffin1, Donald T. Ward1, Wei Liu1, Gavin Stewart1, Ian D. Morris2
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and Craig P. Smith1
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1
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Kingdom; 2Hull York Medical School, University of York, Heslington, York YO10 5DD
School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United
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Running title: Expression of DMT1 in testis
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Correspondence should be directed to:
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Dr. Craig P Smith
Dr Ian D. Morris
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School of Biological Sciences
Hull York Medical School
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University of Manchester
University of York
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G.38 Stopford Building
Heslington
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Oxford Road
York
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Manchester
YO10 5DD
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M13 9PT
Tel: 01904 321338
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Tel: 0161-275-5460
Fax: 01904 321696
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Fax: 0161-275-5600
Email:
[email protected]
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E-mail:
[email protected]
Copyright © 2004 by the American Physiological Society.
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Abstract
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Iron is essential for male fertility and disruptions in iron balance lead to impairment of testicular
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function. The divalent metal transporter DMT1 is a key modulator of transferrin and non-transferrin
26
bound iron homeostasis.
27
determined the pattern of DMT1 expression in the developing and adult rat testis. Northern analysis and
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RT-PCR of testis polyadenylated RNA revealed the presence of IRE and non-IRE transcripts. Semi-
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quantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct
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DMT1 protein species.
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seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis DMT1
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was immunolocalised to both the Sertoli and the germ cells.
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expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any
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plasma membranes in either the developing or adult testis, suggesting that DMT1 is not primarily
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responsible for translocating iron across this epithelia. Our data suggests an important role for DMT1 in
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intracellular iron handling during spermatogenesis and implies that germ cells have a need for a
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precisely targeted and timed supply of iron. We suggest that DMT1 may, like in other tissues, play a role
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in transporting iron between intracellular compartments and thus play an important role in male fertility.
As a first step in determining the role of DMT1 in the testis, we have
Immunohistochemistry showed DMT1 was widespread throughout each
In contrast to the immature testis,
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Key words: divalent metal transporter 1, iron, testis, spermatogenesis, immunohistochemistry
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Introduction
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Iron is required by all organisms, contributing to a wide range of biological functions such as
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mitochondrial oxidation, DNA synthesis, oxygen transport and nitrogen fixation [27, 29]. In mammals
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both iron excess and deficiency present as clinical conditions. As a result body iron homeostasis is a
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tightly regulated process.
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example, hypogonadism and impaired testicular function are prevalent in patients with iron overload,
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exemplified by hereditary hemochromatosis [5].
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Importantly, male fertility is affected by disruptions in iron balance.
For
The divalent metal transporter DMT1 (Slc11a2), also known as Nramp2 [13] and DCT1 [14] is a
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key component of the complex physiological process regulating body iron levels.
The hydrophobic
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protein, with 12 predicted transmembrane domains and cytoplasmic amino and carboxy termini, has
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broad selectivity, mediating transport of a spectrum of divalent cations. These include iron, copper and
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some toxic metals such as cadmium [14]. DMT1 is expressed in many tissues including duodenum,
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placenta [11], brain [22] and kidney [2, 7]. In duodenum, DMT1 is expressed on the apical membrane of
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enterocytes where it is primarily responsible for the non-transferrin mediated absorption of dietary iron.
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Its expression is regulated by dietary iron intake in a number of tissues [3, 7, 14, 28]. In other tissues
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DMT1 is localised in vesicular membranes and participates in transferrin mediated iron acquisition by
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transporting iron across these membranes and into the cell [1].
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Alternative splicing of exons in the DMT1 gene produces four distinct DMT1 mRNAs. These
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differ at the 3' end with respect to an iron responsive element (IRE) present in the untranslated region [8,
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20], and also at the 5' end [16]. As a result of alternative splicing, the encoded proteins differ at the N-
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and C-termini but share a common central domain.
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In the testis the acquisition of iron is of particular importance as the spermatogenic cells, which
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acquire iron during development, are lost as mature sperm pass into the reproductive tract [26]. The
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testis is divided into two compartments, the interstitium and the seminiferous tubules where
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spermatogenesis takes place. Here germ cells are present at various stages of development in the form
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of spermatogonia, spermatocytes and spermatids, passing further towards the lumen of the tubule as
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they mature [19]. The tubular epithelium, like the brain, represents a compartment functionally separate
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from the systemic circulation by a barrier, known as the blood-testis barrier.
Consequently, the
4
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spermatogenic epithelium is unable to acquire iron directly from plasma transferrin [15]. However the
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somatic Sertoli cells, whose tight junctions form the blood-testis barrier, synthesise their own transferrin
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[31] to transport iron across the cell to the spermatocytes and spermatids [4, 17, 25]. Elements of
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transferrin mediated iron transport have been characterised in the rat testis [10, 23, 25, 26, 32], being
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particularly apparent in the mitotic and meiotic germ cells [24, 34, 37]. This suggests a role for iron in
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cell division, differentiation and metabolism in the testis [30].
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DMT1 mRNA is present in testis [14] and DMT1 protein has been localised to the phagosomal
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membranes of two Sertoli cell lines [18]. As a first step in determining the role of DMT1 in testicular
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function, we have characterised DMT1 expression in both the immature and adult rat testis. We have
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shown that DMT1 is present in the testis and the expression profile in the development of the rat testis is
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cell specific and in the adult is highly co-ordinated to the spermatogenic cycle.
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important role for DMT1 in spermatogenesis and implies that germ cells have a need for a precisely
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timed supply of iron. The stage specific nature of expression indicates that DMT1 has a role in male
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fertility, and this knowledge may be useful when considering conditions of abnormal iron regulation.
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This suggests an
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Materials and Methods
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Animals
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Sprague Dawley rats (Charles River Ltd, Margate, UK) were bred under controlled conditions in
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the University of Manchester Biological Services Unit. The day of delivery of pups was designated as
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the first day of age. All animals were housed with controlled temperature and lighting (12 hours light: 12
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hours dark) and access to food and water ad libitum.
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regulations of the National Research Council as detailed in the Guide for Care and Use of Laboratory
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Animals.
All procedures were performed within the
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Tissue Preparation
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Groups of male adult rats (with a mean body weight of 275 g and an approximate age of 60 days)
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and male rat pups of 5, 15, 25 and 35 days of age were killed by concussion followed by cervical
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dislocation. Following dissection, the left testes from 3 animals from each age group were fixed by
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immersion in Bouin’s fluid (71% picric acid, 24% formalin, 5% glacial acetic acid) for 3-4 hours. These
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tissues were dehydrated through a gradient of ethanol and stored in absolute ethanol for 18 hours prior
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to embedding in paraffin wax. All other testes from each group were pooled and frozen at -80°C.
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RNA Extraction and Northern Analysis
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Total RNA was extracted from frozen testis by the acid guanidinium isothiocyanate–phenol
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method and Northern analysis performed as Ferguson et al., [7], using dissected kidney cortex as a
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positive control.
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corresponded to nucleotides 85-1600 of rat DMT1, shared by both the IRE and non-IRE transcripts
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(GenBank accession no. AF029757). Briefly, 5 Jg of kidney cortex mRNA and 3 Jg of testis mRNA were
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separated on 1% agarose gels containing 2.2 M formaldehyde, and blotted onto Hybond-N Nylon
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membranes (Amersham Pharmacia Biotech). Membranes were hybridized at 42°C overnight with 5×
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SSC-50% formamide-3× Denhardt’s solution-0.4% SDS-10 Jl/ml of salmon sperm DNA and 10% w/v
The double stranded probe was randomly primed and labelled with
32
P and
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dextransulfate.
Membranes were washed at 65°C with 0.1% SSC-0.1% SDS and exposed to film
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(Kodak Biomax, Hemel Hempstead, UK) for 48 hours.
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Reverse Transcriptase-Polymerase Chain Reaction
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Reverse transcription was performed with 3 Jg of total RNA. cDNA was synthesised with Oligo
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dT and Superscript II Reverse Transcriptase (Invitrogen Ltd., Paisley, UK). Primer sets for IRE DMT1
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(GenBank accession no. AF008439) and non-IRE DMT1 (GenBank accession no. AF029757), spanning
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introns, were designed using Primer Express
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used the same forward primer 5’-CAACGGAATAGGCTGGAGGA-3’. The reverse primer sequences for
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IRE
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GGCACAAAAGGGCTTAGAGA-3’ respectively. The amplification of IRE DMT1 cDNA was performed at
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95°C for 30 seconds, 55°C for 45 seconds and 72°C for 60 seconds for 35 cycles. The amplification of
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non-IRE DMT1 cDNA was performed at 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 30
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seconds for 30 cycles. The PCR products were resolved on 1% agarose gels. Control reactions were
123
performed with the primers in the absence of cDNA.
and
non-IRE
transcripts
(Applied Biosystems, Warrington, UK). Both isoforms
were
5’-GGCAGGAGGATCTCTGTGAG-3’
and
5’-
124 125
Peroxidase Immunohistochemistry
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Paraffin wax sections (5µm) were cut using a Leica RM2135 rotary microtome (Leica
127
Microsystems, Nussloch, Germany) and fixed onto Superfrost microscope slides (BDH, Poole, UK).
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Deparraffinized and rehydrated sections were incubated with 3% H2O2 in methanol for 20 minutes to
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quench endogenous peroxidase activity. Masked antigens were retrieved by microwaving the sections in
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TEG buffer (10mM Tris, 0.5mM EGTA, pH 9.0) for 2 minutes at 800W and 4 minutes at 400W. Sections
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were allowed to cool for a minimum of 2 hours before treatment with 50mM NH4Cl in PBS for 30 minutes.
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Sections were blocked with 1% BSA in PBS and incubated overnight at 4°C in a humidified chamber with
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a previously characterized affinity purified polyclonal anti-DMT1 antibody, raised to an epitope common
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to all known DMT1 isoforms [6, 7, 37]. Before use the antibody was diluted 1:500 in PBS containing
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0.1% BSA and 0.3% Triton X-100.
Sections from each group were also incubated with the
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PBS/BSA/Triton X-100 solution alone for use as negative controls.
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Slides were rinsed with PBS containing 0.1% BSA before incubation with goat anti-rabbit
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immunoglobulin, diluted 1:200, for an hour at room temperature. After rinsing with the PBS solution, the
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immunohistochemical reaction was visualised using 3,3’diamino-benzidine-tetrahydrochloride (DAB).
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Sections were counterstained with Mayer’s haematoxylin, dehydrated and mounted with Eukitt resin
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(Kindler GmBH & Co., Freiburg, Germany). Immunostaining was examined using the Zeiss Axiophot
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light microscope (Carl Zeiss Ltd, Welwyn Garden City, Hertfordshire, UK) and photographs were taken
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using a Spot RT Colour digital camera (Diagnostic Instruments Inc, Michigan, USA) and analysed with
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the accompanying Spot RT Advanced software, Version 3.04.
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Western Analysis Semi-quantitative immunoblotting was performed according to Ferguson et al. [7].
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Crude
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membrane preparations from each group of frozen tissues were prepared by differential
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ultracentrifugation. Protein homogenates were spun at 2,500xg to remove nuclei and cell debris and the
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post-nuclear supernatants were centrifuged at 100,000xg to pellet the particulate proteins. These were
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then resolved by 8% SDS-PAGE and transferred to Biotrace nitrocellulose membranes (Paul Gelman
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Sciences, Northampton, UK). Adult rat kidney, proximal duodenum and 35 day old rat kidney were also
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prepared in this manner for use as positive controls. Immunoblotting was performed using the anti-
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DMT1
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chemoluminescence (ELC) system was used to visualise the signal (Amersham Pharmacia Biotech,
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Little Chalfont, UK) according to the manufacturer’s instructions. Experiments were repeated in triplicate.
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Membranes incubated in only TBST or with antiserum pre-incubated with an excess of the immunizing
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peptide served as negative controls. In every case, staining of the gels with Coomassie Blue and the
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membranes with Red Ponceau S confirmed that equal amounts of proteins had been loaded and
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transferred to the membrane.
antibody,
diluted
1:5000
in
Tris-buffered
saline-Tween
20
(TBST).
The
enhanced
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Protein Deglycosylation
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Particulate proteins were deglycosylated using N-glycosidase F (PNGase F; New England
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Biolabs) according to the manufacturer’s instructions. Briefly, particulate fractions were denatured at
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65oC in buffer containing 0.5% SDS and 1% Q-mercaptoethanol, then incubated at 37oC for 1 hour in
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buffer supplemented with NP40 (1% final) and PNGase F (25U / µg particulate protein). Following
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deglycosylation, samples were solubilized using 5X Laemmli buffer and immunoblotted as before. The
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electrophoretic mobility of deglycosylated DMT1 was compared to that obtained from a sample
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processed identically but in the absence of PNGase F and also to a sample of particulate proteins stored
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on ice throughout.
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Results
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Northern Analysis
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As previously reported, Northern analysis of testis polyadenylated RNA, using a probe
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corresponding to nucleotides common to both IRE DMT1 and non-IRE DMT1, detects transcripts with
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molecular masses of 2.4 kb and 4.4 kb in kidney cortex (Figure 1, [7]). The 4.4 kb species corresponds
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to the IRE transcript while the 2.4 kb species relates to the non-IRE transcript. Although weaker, these
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signals were also present in adult testis
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Figure 1
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Reverse Transcriptase-Polymerase Chain Reaction
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Primers specific for IRE DMT1 and non-IRE DMT1 were also used selectively to investigate the
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expression of these two isoforms in the immature (5 day) and adult testis. PCR products from the IRE
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and non-IRE primer pairs were detected as single bands of approximately 0.9kb and 0.3kb respectively
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at both ages (Figure 2). This is consistent with the expected oligonucleotide lengths based on the
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primers used for each DMT1 isoform. Control reactions, performed in the absence of cDNA, did not give
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rise to PCR products. We conclude that IRE DMT1 and non-IRE DMT1 transcripts are present in
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immature and adult testis.
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Figure 2
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Semi-Quantitative Immunoblotting
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Semi-quantitative Western analysis was used to profile DMT1 protein expression in the immature
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and adult rat testis. DMT1 was detected in the testis throughout development of the immature rat (Figure
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3). The antibody detected an immunoreactive species of approximately 70 kDa in all of the age groups
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tested. This signal was strongest in the 5 and 15 day testis. A second higher molecular weight species
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was detected throughout development of between 75-90 kDa, which was particularly apparent in the 15
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day testis. The specificity of the DMT1 antibody was confirmed by the absence of any immunoreactive
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signal in the membranes that were incubated TBST without the primary antibody, or with antiserum pre-
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incubated with an excess of the immunizing peptide (data not shown).
200 201
Figure 3
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Both of these immunoreactive species were detected in the testis of the adult rat (Figure 4). The
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predominant DMT1 immunoreactive species had a molecular weight of between 75-90 kDa. A band of
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similar size was also observed in the adult rat duodenum. The lower molecular weight species of
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approximately 70 kDa was comparable with that observed in the adult rat kidney.
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molecular weight bands were also detected in the testis between 30-60 kDa. These bands were ablated
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by incubation of the membranes with antiserum pre-incubated with an excess of immunizing peptide.
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Whether these bands represent functional DMT1 isoforms is currently unknown.
Several lower
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210 211
Figure 4
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To further characterize the DMT1 species expressed in the adult testis, we performed
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deglycosylation of testis and kidney protein homogenates with N-glycosidase F (Figure 5). Treatment of
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kidney protein homogenates with N-glycosidase F reduced the molecular weight of the band to 50 kDa.
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In contrast, identical treatment of the testis protein reduced the predominant band from 75-90 kDa to 60
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kDa. This indicated that differences in molecular weight of the major proteins detected in testis and
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kidney were not primarily due to differences in the degree of glycosylation. However these results do not
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rule out other forms of post-translational modification such as phosphorylation.
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Figure 5
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Immunohistochemistry of Immature Rat Testis
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Immunohistochemistry was used to investigate the ontogenic expression of DMT1 in the rat
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testis. DMT1 immunostaining was observed in the testes of all of the immature rats, irrespective of the
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age studied. Staining was confined to the seminiferous tubules and was absent from the interstitium
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(Figure 6). Despite morphological changes due to the increasing number of germ cells with age, a
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distinct staining pattern was observed throughout the development of the testis. In 5 day old animals the
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seminiferous tubule lumen had not formed and the pattern of staining did not vary between tubules.
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Higher magnification images showed staining resided in the cytoplasm of the Sertoli cells with minor
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variations in intensity towards the tubule membrane.
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numerous with many more types present.
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tubules while leptotene spermatocytes were present in a few select tubules. Despite the presence of
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more germ cells, the pattern of DMT1 localisation matched that of the 5 day old rat in that the Sertoli cell
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cytoplasm was immunopositive. In 25 day rats, the lumen of each tubule was evident and the number of
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germ cells continued to increase with pachytene spermatocytes present in most of the tubules.
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Immunostaining was more punctate in some regions, but remained localised to the cytoplasm of the
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Sertoli cells. In the 35 day old animal, the diameter of the lumen continued to increase and round
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spermatids appeared. The pattern of DMT1 expression was observed to differ between tubules in that
In the 15 day old rat, germ cells were more
Zygotene spermatocytes were observed in each of the
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some of the tubules exhibited more intense immunostaining. This suggested the differential expression
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of DMT1 in the latter stages of testicular maturation, prior to the completion of the first wave of
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spermatogenesis.
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epithelium along with tracts of staining that ran from the tubule membrane and opened up towards the
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lumen. As in the testis of the younger rats, this staining was restricted to the cytoplasm of the Sertoli
245
cells. No plasma membrane staining was observed in any of the groups that were analyzed. Controls
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slides, incubated without the anti-DMT1 polyclonal antibody or with antiserum pre-incubated with an
247
excess of the immunizing peptide, were used at each age to exclude the possibility of false positives by
248
endogenous peroxidase activity. These slides did not show any immunopositive staining for DMT1.
In some tubules layers of punctate staining were observed at the base of the
249 250
Immunohistochemistry of Adult Rat Testis
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In the adult rat, with a fully developed spermatogenic cycle, the pattern of immunostaining (Figure
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7) was in marked contrast to the pattern of immunostaining observed in 5, 15 and 25 day old rats. In the
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first instance, both Sertoli cells and germs cells of the adult rat testis stained positively for DMT1.
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Secondly, whilst the germ cells exhibited mostly cytoplasmic staining, DMT1 could be localised to not all,
255
but a select number of nuclei. Finally, the Sertoli cell nuclei were also clearly stained throughout all of
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the stages, in contrast to the developing animal where staining was confined to the cytoplasm.
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At Stage II/III of the cycle (Figures 8A and 8B), the pachytene spermatocytes had abundant
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DMT1 while the cytoplasm of the elongate spermatids exhibited light immunopositive staining. At Stage
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V of the cycle (Figures 8C and 8D) the tubule was very darkly stained which was mainly attributable to
260
the tails of the elongate spermatids. The nuclei of the round spermatids exhibited faint staining while the
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pachytene spermatocytes remained unstained, highlighting the stage specific nature of DMT1
262
expression. In tubules at Stage VIII (Figures 8E and 8F) elongate spermatids were stained intensely.
263
The residual bodies that accompany these mature germ cells also showed strong DMT1 staining.
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Pachytene spermatocytes were once again stained while the pattern of immunostaining in the
265
spermatids matched that in the Stage V tubule. At Stage X (Figures 8G and 8H), DMT1 was not
266
detected in the elongating spermatids but was abundant in the pachytene spermatocytes. At Stage
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XII/XIII of the cycle (Figures 8I and 8J) the location and intensity of DMT1 expression is similar to that at
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Stage X but exhibits the occasional staining of zygotene spermatocytes. Importantly, we did not detect
269
plasma membrane staining of Sertoli cells at any stage, suggesting that in the adult rat DMT1 is not the
270
primary means of iron transport across the blood-testis barrier. The control slides of adult testis did not
271
exhibit any immunopositive staining for DMT1.
272 273
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Discussion
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It is well established that iron plays an important role in spermatogenesis and the normal function
275
of the testis [5]. In other tissues the mechanism of iron sequestration is becoming clearer. For example,
276
in the duodenum, the broad spectrum divalent metal transporter DMT1 mediates iron movement across
277
the apical enterocyte membrane as part of the process of dietary iron absorption. Although DMT1 has
278
previously been shown to be present in the testis, its cellular localisation and role in testicular function
279
remains unreported. As a first step in determining its role in this tissue, we determined the pattern of
280
DMT1 expression in the developing and mature rat testis using semi-quantitative immunoblotting and
281
immunohistochemistry.
282 283
Immature Testis
284
Using a combination of Northern analysis and RT-PCR we detected both IRE and non-IRE DMT1
285
transcripts in the immature testis. The presence of the IRE transcript indicates that like other tissues,
286
including kidney and duodenum, the testis can modulate iron translocation via DMT1 in response to
287
cellular demands.
288
Semi-quantitative immunoblotting of immature rat testis revealed that two distinct DMT1 protein
289
species were differentially expressed. The lower molecular weight isoform of approximately 70 kDa
290
showed strong expression at days 5 and 15, thereafter expression decreased. At day 15 a 75-90 kDa
291
protein was also particularly visible. This isoform is of a similar molecular weight to the major DMT1
292
protein found in the adult testis (see below) and is also analogous to the predominant isoform found in
293
the duodenum [3]. In contrast, the lower molecular weight protein expressed at days 5 and 15 is of the
294
same molecular weight as the kidney isoform [7].
295
The pattern of DMT1 expression in the immature testis was found to be similar in all ages up to
296
day 35. Immunohistochemistry localised the DMT1 protein to the cytoplasm of the Sertoli cells. Despite
297
the age related changes in seminiferous tubule morphology, that include an increase in tubule diameter
298
and the number of germ cell types, no age related changes in the cellular pattern of DMT1 expression
299
were detected.
Additionally, the overall pattern of DMT1 immunoreactivity was unaffected by the
16
300
changes in the levels of expression of the two isoforms, as revealed by Western analysis. This may
301
indicate changes in the required function of DMT1 by Sertoli cells over time.
302
In the 35 day old rat testis, immunostaining was restricted to the cytoplasm of the Sertoli cells.
303
As the number of germ cells increased, the overall staining of the tubules decreased. At this age also,
304
the pattern of DMT1 expression was first observed to vary between some of the tubules, in that some
305
tubules exhibited intense regions of staining in the form of tracts running from the tubular membrane to
306
the lumen. As the first wave of spermatogenesis approached completion, immunostaining began to
307
appear more like that observed in the adult rat, indicating the onset of stage specific DMT1 expression
308
as seen in the adult testis. This pattern of expression is similar to that of transferrin mRNA, although
309
differential tubular expression of transferrin was noted much earlier at 14 days [21].
310
The Sertoli cell is responsible for the biochemical and structural support of the germ cells
311
embedded within the epithelium, providing the necessary factors needed for spermatogenesis [19]. In
312
the immature rat, both prior to and after the establishment of the Sertoli cell tight junctions, DMT1 was
313
present in the cytoplasm of the Sertoli cells but was absent from the plasma membranes. Therefore the
314
results of this study suggest that in the Sertoli cells of immature rat testis, DMT1 is not the primary route
315
of iron transport across the blood-testis barrier. DMT1 has been localized to late endosomes in a
316
number of transfected cell lines, including CHO, RAW 264.7 [12], HEK293T [33] and Hep-2 cells [35],
317
where it has also been observed in lysosomes. Without higher resolution images, possibly utilizing
318
immunogold electron microscopy, it is not possible to resolve the intracellular location of DMT1 and
319
therefore speculate on the regions of punctate staining observed. However, it is possible that DMT1
320
serves to transport iron between the intracellular compartments of the testis, as it does in other cell
321
types.
322 323
Adult Testis
324
In the fully developed rat testis, Northern analysis and RT-PCR showed that both IRE and non-
325
IRE DMT1 transcripts were present. This finding corroborates with data reported by Gunshin et al. [13]
326
who isolated a 4.4 kb cDNA encoding a 561 amino acid protein. Using Northern analysis Gunshin
17
327
reported expression of the 4.4 kb transcript in adult rat testis. This transcript encodes an iron-responsive
328
DMT1 isoform expressed in duodenum.
329
Western analysis showed that the predominant isoform of DMT1 in the adult testis has a
330
molecular weight of 75-90 kDa, although proteins of lower molecular weight, like those observed in the
331
immature testis, were also present. Deglycosylation of protein isolated from the adult testis reduced the
332
molecular weight from 75-90 kDa to 60 kDa. This is consistent with the predicted molecular weights of
333
the IRE and non-IRE DMT1 isoforms, of 61 kDa and 62 kDa respectively. The predominant isoform in
334
the adult testis had a different molecular weight to that expressed in the immature testis and kidney. The
335
predicted difference in molecular weights between the IRE and non-IRE forms of DMT1 is approximately
336
1 kDa, whereas the difference between the molecular weights of the deglycosylated proteins was 10
337
kDa. Therefore, it is unlikely that expression of predominantly IRE or non-IRE proteins can explain these
338
variations. It is more likely that the renal isoform, and possibly that expressed in the immature testis, are
339
truncated splice variants or undergo posttranslational modification other than glycosylation.
340
The data from the immature and adult testis together can be summarized as follows: Throughout
341
development to maturation the testis differentially expresses two predominant DMT1 isoforms; a 70 kDa
342
protein that based on molecular weight is the same as the major DMT1 isoform expressed in the kidney,
343
and a 75-90 kDa protein that based on molecular weight is the same as the predominant DMT1 isoform
344
expressed in the duodenum.
345
Immunohistochemistry showed that DMT1 has a differential pattern of expression in the mature
346
rat testis, in contrast to the immature animal. In the adult staining was both cytoplasmic and nuclear, and
347
both Sertoli cells and germ cells were stained. DMT1 expression was cell specific; therefore the pattern
348
observed was dependent on the stage of the spermogenic cycle.
349
specific expression of DMT1 in the germ cells of the adult testis.
350
Figure 9 summarises the stage
18
351 352
Figure 9
353 354
From the basement membrane to the lumen of the seminiferous tubules, germ cells are classified
355
into 3 types; spermatogonia, spermatocytes and spermatids, which are either round or elongate.
356
Throughout the 14 stages of spermatogenesis, DMT1 was not observed in any of the spermatogonia.
357
DMT1 was detected in all of the spermatocytes but was absent in these cells at Stage V of the cycle.
358
DMT1 immunostaining was observed in the round spermatids between Stages V and VIII of the cycle
359
and in the elongate spermatids between Stages I and VIII of the cycle.
360
immunostaining was detected in the elongate spermatids, particularly between Stages V and VIII. This
361
suggests that these cells have a particularly high requirement for iron, which may be of importance in the
362
maturation and long-term survival of the sperm as it passes out of the seminiferous tubule. These data
363
are consistent with that of Morales et al. [25] who reported that levels of radiolabelled transferrin were
364
highest in the elongate spermatids and residual bodies of the testis.
The most intense
19
365
DMT1 was clearly immunolocalised to a number of nuclei in the germ cells of the adult testis.
366
The nuclear stain was not present in all of the spermatogenic cells of the testis, indicating that DMT1 is
367
expressed specifically in these structures and its presence is not an artefact of the methodology. DMT1
368
has been discovered in the nuclei of other cell types and while its function in the nucleus remains
369
unknown, this may indicate a new role for DMT1 in metal ion transport [9].
370
As the testis is functionally separate from the systemic circulation and cannot sequester iron
371
directly from plasma transferrin, the Sertoli cells synthesize their own transferrin [31]. A theoretical
372
model has been proposed to explain how iron crosses the blood testis barrier for use by the germ cells in
373
the seminiferous tubules [17, 25]. Firstly the basal membranes of the tubules are exposed to iron laden
374
plasma transferrin, which binds to transferrin receptors on the Sertoli cells.
375
internalised within the Sertoli cells into endosomes.
376
detachment of iron from the transferrin. Iron is then transported out of the endosomes into the Sertoli
377
cell cytoplasm. Here the iron binds with testicular transferrin and these complexes are secreted to the
378
intracellular spaces between the germ cells. Transferrin mediated iron transport has been localised to all
379
structures within the seminiferous tubule [23, 26, 34], but not all investigations have addressed stage
380
specificity.
These complexes are
These contain proton pumps, which aid the
381
We currently do not know the precise intracellular location of DMT1 in testicular cells, but the fact
382
that the protein is expressed in Sertoli cells and in a stage specific manner by germ cells supports a role
383
for DMT1 in iron handling. By analogy to its role in other cells and because we have found no evidence
384
for expression of DMT1 on plasma membranes, DMT1 expressed within Sertoli and germ cells could
385
mediate proton-coupled export of iron out of the endocytic compartment and into the cell.
386
In this investigation we have localised DMT1 to the Sertoli cells, spermatocytes and spermatids of
387
the adult rat testis. There is different special and temporal expression of DMT1 in the adult rat, in
388
contrast to the immature rat, which exhibits widespread expression of DMT1 throughout development.
389
Expression of DMT1 in the adult rat testis is cell specific and highly co-ordinated to the spermatogenic
390
cycle, being particularly apparent in the elongate spermatids between Stages V and VIII of the cycle.
391
This suggests that the germ cells within the seminiferous tubules of the testis have a need for a precisely
392
timed supply of iron. The stage specific nature of DMT1 expression implies an important role for DMT1
20
393
in spermatogenesis and male fertility. This may be useful when considering conditions of abnormal iron
394
regulation. The broad specificity of DMT1 may also implicate these findings in the transport of other
395
divalent and toxic metals and could help explain their testicular toxicity.
396
21
396
Acknowledgments
397
The authors thank The Royal Society, Wellcome Trust and MRC for funding this work. Kathleen
398
P. Griffin is a MRC CASE student. Donald T. Ward is a recipient of a National Kidney Research Fund
399
Training Fellowship (TF6/2002). Craig P. Smith is recipient of a Royal Society University Fellowship
400 401
22
401
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402
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2. Canonne-Hergaux F, Gros P. Expression of the iron transporter DMT1 in kidney from normal
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4. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 3: 308-326, 1970. 5. Edwards CQ, Cartwright GE, Skolnick MH, Amos DB. Homozygosity for hemochromatosis: Clinical manifestations. Ann Intern Med 93: 519-525, 1980. 6. Ferguson CJ, Wareing M, Delannoy M, Fenton R, McLarnon SJ, Ashton N, Cox AG,
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12. Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P. The iron
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14. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S,
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Gollan JL Hediger MA. Cloning and characterisation of a mammalian proton-coupled metal-ion
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15. Holash JA, Harik SI, Perry G, Stewart PA. Barrier properties of testis microvessels. Proc Natl Acad Sci USA 90: 11069-11073, 1993. 16. Hubert N, Hentze MW.
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(DMT)-1: Implications for regulation and cellular function. Proc Natl Acad Sci USA 99: 12345-
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17. Huggenvik J, Sylvester SR, Griswold MD. Control of transferrin mRNA synthesis in Sertoli cells. Ann N Y Acad Sci 438: 1-7, 1984. 18. Jabado N, Canonne-Hergaux F, Gruenheid S, Picard V, Gros P.
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21. Maguire SM, Millar MR, Sharpe RM, Gaughan J, Saunders PT. Investigation of the potential
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role of the germ cell complement in control of the expression of transferrin mRNA in the
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prepubertal and adult rat testis. J Mol Endocrinol 19: 67-77, 1997.
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22. Moos T, Trinder D, Morgan EH. Cellular distribution of ferric iron, ferritin and divalent metal
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transporter 1 (DMT1) in substantia nigra and basal ganglia of normal and beta2-microglobulin
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deficient mouse brain. Cell Mol Biol 46: 549-561, 2000.
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23. Morales C, Clermont Y. Receptor-mediated endocytosis of transferrin by Sertoli cells of the rat. Biol Reprod 35: 393-405, 1986. 24. Morales C, Hugly S, Griswold MD. Stage-dependent levels of specific mRNA transcripts in Sertoli cells. Biol Reprod 36: 1035-1046, 1987. 25. Morales C, Sylvester SR, Griswold MD. Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo. Biol Reprod 37: 995-1005, 1987. 26. Petrie RG Jr, Morales CR. Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis. Cell Tissue Res 267: 45-55, 1992. 27. Ponka P, Lok CN. The transferrin receptor: Role in health and disease. Int J Biochem Cell Biol 31: 1111-1137, 1999.
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28. Rolfs A, Bonkovsky HL, Kohlroser JG, McNeal K, Sharma A, Berger UV, Hediger, MA.
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Intestinal expression of genes involved in iron absorption in humans. Am J Physiol Gastrointest
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Liver Physiol 282: G598-G607, 2002.
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29. Rouault TA. Systemic iron metabolism: A review and implications for brain iron metabolism. Pediatr Neurol 25: 130-137, 2001.
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30. Segretain D, Egloff M, Gerard N, Pineau C, Jegou B. Receptor-mediated and absorptive
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endocytosis by male germ cells of different mammalian species. Cell Tissue Res 268: 471-478,
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31. Skinner MK, Griswold MD. Sertoli cells synthesize and secrete transferrin-like protein. J Biol Chem 255: 9523-9525, 1980. 32. Stallard BJ, Collard MW, Griswold MD. A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis. Mol Cell Biol 11: 1448-453, 1991. 33. Su MA, Trenor CC, Fleming JC, Fleming MD, Andrews NC. The G185R mutation disrupts function of the iron transporter Nramp2. Blood 92: 2157-2163, 1998.
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34. Sylvester SR, Griswold MD. Localization of transferrin and transferrin receptors in rat testes. Biol Reprod 31: 195-203, 1984.
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35. Tabuchi M, Yoshimori T, Yamaguchi K, Yoshida T, Kishi F. Human NRAMP2/DMT1, which
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mediates iron transport across endosomal membranes, is localized to late endosomes and
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lysosomes in HEp-2 cells. J Biol Chem 275: 22220-22228, 2000.
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36. Wareing M, Ferguson CJ, Delannoy M, Cox AG, McMahon RF, Green R, Riccardi D, Smith
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CP. Altered dietary iron intake is a strong modulator of renal DMT1 expression. Am J Physiol
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Renal Physiol 285: F1050-F1059, 2003.
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37. Wright WW, Paryinen M, Musto NA, Gunsalus GL, Phillips DM, Mather JP, Bardin CW.
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Identification of stage-specific proteins synthesized by rat seminiferous tubules. Biol Reprod 29:
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257-270, 1983.
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Figure Legends
495
Figure 1. IRE and non-IRE DMT1 are Expressed in the Adult Testis and are the Same
496
Isoforms Present in the Kidney Cortex. Polyadenylated RNA was isolated from adult rat testis (5µg)
497
and dissected kidney cortex (3µg) and was separated on a 1% agarose gel. Incubation with a
498
labelled probe corresponding to nucleotides 85-1600 of rat DMT1 led to detection of both IRE and non-
499
IRE transcripts, at 4.4kb and 2.4kb respectively, in both the testis (T) and kidney cortex (KC). Sizes (in
500
kb pairs) of molecular weight markers are shown on the left
32
P
501
Figure 2. Both Major DMT1 Isoforms are Expressed in the Immature and Adult Rat Testis.
502
cDNA was synthesized from 3µg total RNA from 5 day old and adult rat testis. Using primers specific to
503
the two isoforms, both IRE (0.9kb product) and non-IRE DMT1 (0.3kb product) were detected in the adult
504
rat testis (lanes 1 and 3) and also in the immature testis (lanes 5 and 7) Control reactions, performed in
505
the absence of cDNA did not give rise to PCR products (lanes 2, 4, 6 and 8). Sizes (in kb pairs) of
506
molecular weight markers are shown on the left
507
Figure 3. DMT1 Immunoreactive Species are Present throughout Development. Membrane
508
extracts from the testes of 5, 15, 25 and 35 day old rats were prepared and separated by SDS-PAGE on
509
an 8% acrylamide gel. Incubation with a DMT1 antibody, common to all known DMT1 isoforms, shows
510
immunoreactive species are present throughout the ages investigated. Two sets of the testis protein
511
homogenates are displayed on Panel A. In the testes of all of the age groups tested a 70 kDa protein
512
was detected. A second, higher molecular weight signal of between 75-90 kDa was detected, being
513
particularly apparent at day 15. The size of this is especially obvious following overexposure of the
514
signal, shown in Panel B. The 70 kDa protein was also detected in the kidney of the 35 day old rat (K).
515
Sizes (in kDa) of molecular weight markers are shown on the left.
516
Figure 4. In the Adult Rat the Predominant DMT1 Isoform in the Testis is the same as that
517
in the Duodenum. Membrane extracts from the kidney (K), testis (T) and duodenum (D) were prepared
518
and separated by SDS-PAGE on an 8% acrylamide gel. Incubation with the DMT1 antibody recognised
519
broad immunoreactive bands in each of the three tissue types. In the testis, the main band was detected
520
between 75-90 kDa.
A band corresponding to this was observed in the duodenum.
A second
27
521
immunoreactive species was detected in the testis at approximately 70 kDa. A band of similar size was
522
also observed in the kidney. Multiple lower molecular weight bands were also detected in the testes
523
between 30-60 kDa. Sizes (in kDa) of molecular weight markers are shown on the left.
524
Figure 5.
Deglycosylation of DMT1 Reveals the Main Protein Isoform in the Testis is
525
Different to that in the Kidney. Crude membrane extracts from the kidney (K) and testis (T) of adult
526
rats were prepared and deglycosylated with N-Glycosidase F. The deglycosylated samples (lanes 3 and
527
6); along with undigested protein homogenates were separated by SDS-PAGE on an 8% acrylamide gel.
528
In the undigested testis samples, the main DMT1 isoform was detected between 75-90 kDa. The protein
529
was detected at approximately 60 kDa following deglycosylation. In the undigested kidney samples,
530
DMT1 was detected at approximately 70 kDa. The protein was detected at approximately 50 kDa
531
following deglycosylation. Sizes (in kDa) of molecular weight markers are shown on the right.
532
Figure 6. DMT1 is Expressed in the Sertoli Cell Cytoplasm of the Immature Rat Testis.
533
These images show immunohistochemical staining of DMT1 in 5µm sections of immature rat testis at low
534
and high magnification. The scale bars represent 100 µm. The panel insets depict control staining.
535
Figures 6A and 6B show immunostaining at age 5 days. Immunostaining is present in all of the tubules
536
(*) but not in the interstitium (?). Figure 6B demonstrates that the cytoplasm of the tubular cells, which fill
537
the lumen completely, is stained. This is the cytoplasm of the Sertoli cells. The germ cells themselves
538
are unstained. Immunostaining at age 15 days is depicted in Figures 6C and 6D. Both micrographs
539
show obvious development of the epithelium with many more cell types present.
540
spermatocytes are present in some tubules whilst in others the most advanced germ cell is the leptotene
541
(L) spermatocyte. The cytoplasm of the tubular cells is uniformly stained. Figures 6E and 6F show
542
immunostaining at age 25 days. The lumen (*) of the seminiferous tubule is developed and pachytene
543
(P) spermatocytes are present in many of the tubules. Immunostaining is present in all tubules but is
544
more punctate (?). The cytoplasm and nuclei of the zygotene (Z) and pachytene spermatocytes are
545
unstained. Figures 6G and 6H show immunostaining at age 35 days. Immunostaining is not as intense
546
or as uniform as in the earlier ages and some tubules are more heavily (1) stained than others (2). The
547
cytoplasm and nuclei of the germ cells, including the round spermatids (R) that appear at this time, are
548
not immunostained. The apical surface of the epithelium is strongly stained with layers of punctate
Zygotene (Z)
28
549
staining towards the base of the epithelium (?). Tracts of staining (
