Specific Detection of Type III Iodothyronine Deiodinase Protein in ...

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Endocrinology 143(7):2700 –2707 Copyright © 2002 by The Endocrine Society

Specific Detection of Type III Iodothyronine Deiodinase Protein in Chicken Cerebellar Purkinje Cells C. H. J. VERHOELST, K. VANDENBORNE, T. SEVERI, O. BAKKER, B. ZANDIEH DOULABI, ¨ HN, S. VAN DER GEYTEN, AND V. M. DARRAS J. L. LEONARD, E. R. KU Laboratory of Comparative Endocrinology, K. U. Leuven, Zoological Institute (C.H.J.V., K.V., T.S., E.R.K., S.V.d.G., V.M.D.), B-3000 Leuven, Belgium; Laboratory of Endocrinology, Academic Medical Center (O.B., B.Z.D.), 1105 AZ Amsterdam, The Netherlands; and Department of Cellular and Molecular Physiology, University of Massachusetts Medical Center (J.L.B.), Worcester, Massachusetts 01655 Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T3 concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T3 levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western

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T IS WELL known that thyroid hormones play a crucial role in the regulation of different developmental processes. In birds, thyroid hormones (TH) regulate yolk sac retraction, maturation of the lungs, pipping and hatching itself (1). They are important for growth in general, thermogenesis, and several other metabolic processes (2). The thyroid gland mainly produces T4. T4 is considered to be a prohormone and in human, about 80% of it is converted peripherally into the active thyroid hormone T3 or in the inactive rT3 by means of deiodination (3). The enzymes that are responsible for these reactions are the iodothyronine deiodinases. Three types of deiodinases have been characterized, namely type I (D1), type II (D2), and type III (D3) deiodinase. These different deiodinases have been cloned from many vertebrate species like rat (4 – 6), human (7–9), mouse (10), dog (11), chicken (12, 13), tilapia (14, 15), Rana catesbeiana (16, 17), and Xenopus laevis (18). D1 and D2 catalyze outer ring deiodination, whereas inner ring deiodination is catalyzed by D1 and D3 (19). In brain, in vitro enzyme kinetics demonstrate the presence of D2 and D3 activities. Homeostasis of the available T3 concentrations is maintained in the brain due to these two enzymes. Because iodothyronine deiodinases play a crucial role in the regulation of the availability of active T3, it is important to know their cellular localization in tissues. Deiodinases are membrane-integrated proteins that could not be isolated Abbreviations: D3, Type III iodothyronine deiodinase; ICC, immunocytochemistry; TBS, Tris-buffered saline; TH, thyroid hormone.

blot analysis, a band of the expected molecular weight (⬃30 kDa) could be detected in both tissues. Using a full-length P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (ⴞ2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum. (Endocrinology 143: 2700 –2707, 2002) 32

from this membrane fraction without causing critical structural changes. This makes it difficult to show the presence of the deiodinases at the protein level. Tu et al. (20) reported the expression of D2 mRNA in tanycytes of the rat hypothalamus. In 1999 Guadan˜o-Ferraz et al. (21) also demonstrated the expression of D2 in glial cells, tanycytes of the third ventricle, and astrocytes in the rat brain by means of in situ hybridization. Hypothyroid animals were also tested, because they hypothesized that in this situation the regions that are most dependent on TH would selectively be protected against hypothyroidism by an increase in D2 mRNA expression. This was indeed the case in the relay nuclei and cortical targets of the primary somatosensory and auditory pathways. Concerning D3, Tu et al. (22) reported the expression of D3 transcripts in adult rats in a diffuse way throughout the brain, with an increased concentration in hyperthyroid rats. This was the first report showing the presence of D3 mRNA in neuronal cells, namely the pyramidal cells of the hippocampus and layers II–IV of the cerebral cortex. The presence of D3 mRNA in neurons in these regions was confirmed by cresyl-violet counterstains. Esca´mez et al. (23) studied the expression of D3 in the newborn rat brain. They found that D3 transcripts were selectively expressed in regions in the brain that were involved in sexual differentiation. Although not specifically studied in their work, the distribution of D3 transcripts suggests neuronal expression as previously pointed out by Tu et al. (22). In this study we produced polyclonal antisera against

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chicken D3 using D3 synthetic peptides and used these antisera to visualize the cellular localization of this enzyme in the brain. A Western blot analysis was performed to confirm the presence of D3 at the protein level. Furthermore, we studied D3 mRNA expression by means of RT-PCR and Northern blot analysis with a specific 32P-labeled cRNA probe.

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Before the embedding in paraffin, they were treated as follows: 50% ethanol for 2 h, 70% ethanol for 2 h, 95% ethanol for 2 h, 100% ethanol for 2 h, 100% ethanol for 16 h, ethanol/xylol (1:1) for 4 h, 100% xylol for 4 h, 100% xylol for 16 h, xylol/paraffin (1:1) for 4 h, 100% paraffin for 4 h, and 100% paraffin for 16 h.

Immunocytochemistry

The Quick Prep Micro mRNA isolation kit was obtained from Amersham Pharmacia Biotech (Aylesbury, UK). SuperTaq DNA polymerase was purchased from HT Biotechnology Ltd. (Cambridge, UK). AMV reverse transcriptase and pCI-Neo were obtained from Promega Corp. (Madison, WI). Synthetic oligonucleotides and primers were purchased from Life Technologies, Inc. (Merelbeke, Belgium). Hybond membranes and [␣-32P]deoxy-CTP were purchased from NEN Life Science Products (Boston, MA). pCRII was obtained from Invitrogen (San Diego, CA). All other reagents were of the highest purity commercially available. All experimental procedures were approved by the K. U. Leuven ethical experimental animal committee.

The paraffin slices were hydrated using 100% xylol (twice, 15 min each time), 100% ethanol (3 min), 96% ethanol (3 min), 80% ethanol (3 min), and 70% ethanol (3 min) and washed in distilled water for 10 sec. Subsequently, they were microwaved for 6 min at 600 watts in Trisbuffered saline (TBS). After cooling, blocking buffer (TBS-Triton X-100 and 5% low fat milk) was applied for 1 h to block the nonspecific binding sites. Thereafter, the tissue slices were incubated with a 1:200 dilution of the primary antiserum in TBS-Triton X-100 for 1 h at room temperature and overnight at 4 C. Both preimmune and exhausted primary antiserum were tested as negative controls. The slices were washed twice for 10 min each time with TBS-Triton X-100. Next, they were incubated with goat antirabbit alkaline phosphatase-linked IgG (1:250) for 1 h at room temperature. Again, they were washed twice with TBS for 10 min each time and twice with AP detection buffer for 10 min each time. Then the substrate was applied (45– 60 min). The slices were washed in methanol and enclosed with chromoglycerine.

Antiserum production

D3 activity assay

Two different peptides were chosen according to the hydropathy profile of the entire chicken D3 amino acid sequence (12), and possible cross-reactivity with the other deiodinases was avoided by choosing amino acid sequences with a low homology degree: peptide D3a [NH2(243)YKTRLQSPGAVVIQV(259)-COOH] and peptide D3b [NH2(41)TAGEGPPPDDPPV(53)-COOH]. Peptide D3a was coupled to keyhole limpet hemocyanin. Peptide D3b was linked to BSA. New Zealand White rabbits where injected every 3 wk with the conjugates, and blood samples were taken within 7–10 d after each injection. The serum was collected, snap-frozen in a mixture of dry ice and ethanol, and stored at ⫺80 C until further use.

An in vitro D3 activity test was performed on liver microsomes according to the method previously described by Van der Geyten et al. (25).

Materials and Methods Materials

Antibody specificity We tested the specificity of the polyclonal antisera by means of an immunospotting test. To examine possible cross-reactivity, a specific D1 peptide was chosen [NH2-(235)YHPQEIRAVLEKLK(250)-COOH]. One microliter of each peptide (D1, D3a, or D3b) was spotted onto a Hybond C membrane, followed by a blocking step for 1 h in 5% dry milk in PBS at 37 C. Thereafter, the membrane was incubated with the primary antiserum to be examined, and binding was detected by means of an alkaline phosphatase-conjugated secondary antibody. We also tested the specificity of the polyclonal antisera D3a and D3b using a competitive ELISA in which the D3a/b-specific peptide was coated to the plate (2.5 ␮g/ml). The D1 or D3a/b peptide (0 – 8,000 ng/ml) was brought into competition with the coated peptide to bind to the D3a/b-specific polyclonal antiserum (1/1,000 for anti-D3a and 1/10,000 for anti-D3b).

Western blot analysis Western blot analysis was performed according to a modification of the protocol described by Laemmli (26). Extracts of chicken cerebellum and liver were made in 20% sucrose in the presence of protease inhibitor and Triton X-100. Protein concentrations were determined according to the Bradford method (27). The extracts were diluted 1:1 in sample buffer [25% Tris-Cl/sodium dodecyl sulfate (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate, 3.1% dithiothreitol, and 0.1% bromophenol blue] and denatured by heating for 5 min at 90 C. Ten microliters of each sample (7 mg protein/ml) were applied to a 12.5% acrylamide-bisacrylamide gel (30%/0.8%), followed by electrophoresis at 120 V. Thereafter the samples were blotted electrophoretically onto a Hybond C membrane (3 h at 150 mA). The blots were incubated with blocking buffer (5% low fat milk in TBS) for 1 h. Subsequently, the primary antiserum was diluted 1:200 in blocking buffer for cerebellum extracts and in TBS-Tween for liver extracts and added to the blots. The blots were incubated overnight at room temperature. Four wash steps in TBS-Tween 20 of 15 min each were included next. The blots were then incubated with goat antirabbit alkaline phosphatase-linked IgG (1:1000) for 1 h, and the wash steps were repeated. The substrate (nitro blue tetrazolium/5-bromo-4-chloro3-indolyl-phosphate) was added, and the coloring was stopped by rinsing the membranes with distilled water.

Northern blot analysis

Fertilized chicken eggs from a rapidly growing broiler strain (Hybro) were purchased from Euribrid (Aarschot, Belgium) and incubated in a laboratory incubator as described previously (24). The chickens were decapitated on the day after hatching. Brain tissue for immunocytochemistry (ICC) was immediately fixed in Bouin-Hollande. Liver and brain tissue for Western and Northern blot analyses were isolated, frozen in liquid nitrogen, and stored at ⫺80 C until further analysis. In addition, liver tissue was collected from 17-d-old embryos and from newly hatched chicks for comparison of D3 activity levels with Western blot staining intensities.

mRNA was isolated using the Quick Prep Micro mRNA isolation kit. mRNA (0.5 ␮g) was separated on a 1% (wt/vol) formaldehyde-agarose gel and blotted onto a Hybond-N⫹ membrane. First, the blot was hybridized for 30 min at 62 C with Northernmax prehybridization buffer (Ambion, Sanbio, Uden, The Netherlands). Subsequently, the blot was hybridized overnight at 62 C with the specific 32P-labeled D3 cRNA probe. The blot was washed twice for 15 min each time with 1⫻ standard saline citrate (0.15 m NaCl and 0.01 m sodium citrate in 1 liter, pH 7)-0.1% sodium dodecyl sulfate and twice at 62 C with 0.01% standard saline citrate-0.1% sodium dodecyl sulfate. Finally, the blot was exposed on phosphor imager (Fujix BAS1000, Fuji, Tokyo, Japan) for 3 d for signal detection.

Fixation and embedding of tissues

RT-PCR

Tissues meant for ICC were fixed in Bouin-Hollande for approximately 24 h. Thereafter, they were washed thoroughly in distilled water.

mRNA was isolated using the Quick Prep Micro mRNA isolation system of Amersham Pharmacia Biotech. Oligo(deoxythymidine)-

Experiments

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primed cDNA was obtained using avian myeloblastosis virus reverse transcriptase. PCR was performed using the sense primer 5⬘-CTG CGT GTC CGA CTC CAA-3⬘ (nucleotides 158 –175), the antisense primer 5⬘-AGG CAG CGC TGG AAG CGT-3⬘ (nucleotides 601– 618), and SuperTaq DNA polymerase. The products were isolated and ligated into pCRII-TOPO vector. One-shot chemocompetent Escherichia coli were transformed with this vector. Plasmid DNA was isolated and sequenced by means of the dideoxy method (28) using universal primers T7 (5⬘AAT ACG ACT CAC TAT AGG GCG A-3⬘) and SP6 (5⬘-TTT AGG TGA CAC TAT AGA ATA C-3⬘).

Results Antibody specificity

As expected, no cross-reactivity could be revealed between the D1 peptide and the D3a and D3b antisera by means of the immunospotting test. The competitive ELISA clearly indicated that the D3a/b antiserum only bound to the free D3a/b peptide that was brought into competition. The D1 peptide was not recognized and did not compete with the coated D3a/b peptide for binding to the antiserum. This resulted in a maximum staining of the wells regardless of the amount of


free D1 peptide that was added (Fig. 1, A and B). Finally, no cross-reactivity could be found between the D3a peptide and the D3b antiserum or vice versa (Fig. 1, C and D). ICC

It is clear that the use of anti-D3a and anti-D3b resulted in a clear staining of cells in the cerebellum, as shown in the figures, whereas coloring remained very diffuse in other brain parts (Fig. 2, A and F). Alkaline phosphatase staining with the D3a antiserum revealed an even band of colored cells in the chicken cerebellar lobes (Fig. 2, B and C). The molecular cell layer, including the Purkinje cells that border this specific layer, is entirely stained using this antiserum. Staining with the D3b antiserum also led to a positive signal in the chick cerebellum. Interestingly though, no even band was seen, but the Purkinje cells were very specifically stained. The cell bodies and the dendrites of these Purkinje cells were clearly visible (Fig. 2, G–I). The controls we used were all negative. Figure

FIG. 1. A, ELISA performed with 2.5 ␮g/ml D3a peptide coated to the plate and 0 – 8,000 ng/ml D1 or D3a peptide brought into competition for binding to the D3a antiserum (1:1,000). B, ELISA performed with 2.5 ␮g/ml D3b peptide coated to the plate and 0 – 8,000 ng/ml D1 or D3b peptide brought into competition for binding to the D3b antiserum (1:10,000). C, ELISA performed with 2.5 ␮g/ml D3a peptide coated to the plate and 0 – 8,000 ng/ml D3a or D3b peptide brought into competition for binding to the D3a antiserum (1:1,000). D, ELISA performed with 2.5 ␮g/ml D3b peptide coated to the plate and 0 – 8,000 ng/ml D3a or D3b peptide brought into competition for binding to the D3b antiserum (1:10,000).



FIG. 2. A, Alkaline phosphatase staining of paraffin slices of the chick brain with D3a antiserum. B, Alkaline phosphatase staining of paraffin slices of the chick cerebellum (C) with D3a antiserum (magnification, ⫻12.4). C, Alkaline phosphatase staining of paraffin slices of the chick cerebellum with D3a antiserum (MC, molecular cell layer; PC, Purkinje cells; magnification, ⫻84). D, Alkaline phosphatase staining of paraffin slices of the chick cerebellum negative control (magnification, ⫻19). E, Cerebral cortex region stained with D3a antiserum (magnification, ⫻336). F, Alkaline phosphatase staining of paraffin slices of the chick brain with D3b antiserum. G, Alkaline phosphatase staining of paraffin slices of the chick cerebellum with D3b antiserum (magnification, ⫻42). H, Alkaline phosphatase staining of paraffin slices of the chick cerebellum with D3b antiserum (magnification, ⫻84). I, Alkaline phosphatase staining of paraffin slices of the chick cerebellum with D3b antiserum (CB, cell body; D, dendrites; magnification, ⫻168). J, Cerebral cortex region stained with D3b antiserum (magnification, ⫻336).

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2D represents the preimmune treated slice. Figure 2, E and J, represent a magnification of the cerebral cortex stained with anti-D3a and anti-D3b, respectively. As mentioned above only a very diffuse and unevenly spread staining could be detected in this part of the brain, and no specifically stained cell bodies could be identified. Western blot analysis

Because the iodothyronine deiodinases are membraneintegrated proteins, it was essential to solubilize these enzymes. Previous experiments on chicken tissue extracts using an anti-D1 antiserum showed that the addition of Triton X-100 resulted in a good solubilization of the deiodinase. The signal increased gradually as more Triton X-100 was used. By analogy with these earlier experiments, extracts of the chicken cerebellum were made in the presence of different concentrations of Triton X-100 (0%, 0.4%, 0.8%, 1.2%, and 1.6% in extraction buffer) to use in the Western blot analysis of D3. Different heating temperatures and times were tested to denature the enzymes, but heating them for 5 min at 90 C gave the best results. Ten microliters per sample were loaded on the gel. Seven microliters of a prestained molecular mass marker were loaded in the first lane. Satisfying results were obtained after blotting the gel for 3 h at 150 mA. Using this method we were able to reveal three protein bands in cerebellum extracts when we incubated the blot with the D3a antiserum: a medium-sized fragment of 30 kDa, a larger fragment of about 61 kDa, and a smaller signal of 14 kDa (Fig. 3). The other half of the blot, containing the same samples, was incubated with preimmune serum instead of primary antiserum D3a and was thus used as a negative control. No bands were revealed. When Western blots were incubated with the D3b antiserum, the background signal ratio was very high. Increasing the blocking times and the concentration of blocking medium led to a decrease in the background, but only very weak specific signals were obtained. The estimated molecular mass of the band thus obtained appeared to be the same as that of the predominant band with the D3a antiserum, namely, 30 kDa, but no bands of higher or lower molecular mass were revealed. Western blot analysis of liver extracts confirmed the re-

FIG. 3. Western blot analysis of cerebellum extract using anti-D3a and preimmune serum. Lane 1, Molecular mass marker; lane 2, extract without Triton X-100; lane 3, extract with 0.4% Triton X-100; lane 4, extract with 0.8% Triton X-100; lane 5, extract with 1.2% Triton X-100; lane 6, 1.6% Triton X-100; lane 7, extract without Triton X-100; lane 8, extract with 0.4% Triton X-100; lane 9, extract with 0.8% Triton X-100; lane 10, extract with 1.2% Triton X-100; lane 11, extract with 1.6% Triton X-100.


sults obtained by an in vitro D3 activity test of these tissues. A significant decrease in both the D3 activity level and the D3 protein staining intensity could be detected in newly hatched animals compared with embryonic d 17 chicks (Fig. 4). Northern blot analysis

Northern blot analysis was performed on 0.5 ␮g chicken cerebellar and hepatic mRNA. After 3 d of exposure to a phosphor imager, a single mRNA species was detected in both liver and cerebellum samples. Both fragments were approximately 2.4 kb long (Fig. 5). RT-PCR

Using the gene-specific D3 primers, a fragment of approximately 500 bp was expected for D3. By performing an RTPCR on 200 ng chicken cerebellum mRNA we were indeed able to amplify a single DNA fragment of approximately 500 bp. This fragment was isolated and transformed into bacteria. Subsequent subcloning and sequencing showed a DNA sequence that was 99% homologous to the D3 cDNA sequence published previously (12) (see Fig. 6). Discussion

Our findings clearly demonstrate the presence of D3 in Purkinje cells of chick cerebellum. Two antisera against different D3 peptides stain these cells, although with different intensity. The D3b antiserum stains individual Purkinje cells very clearly. The staining with D3a antiserum does the same thing, but apparently at a higher intensity, so that the individual cells cannot be distinguished. The D3a antiserum seems to have developed a greater affinity to the D3 compared with the D3b antiserum, because the latter only stains Purkinje cells, which probably contain the highest concentration of D3 protein. Because the different negative controls showed no staining, it can be concluded that D3 staining is indeed specific. The microwave treatment used in the current ICC protocol is believed to lead to a better exposure of the antigens to their antisera and has already been applied successfully to stain specific cells around the hepatic central blood veins of the rat with a polyclonal antiserum against rat D1 (29). In the present study a microwave treatment enhanced the results obtained in the cerebellum, but no supplementary cells were stained. The expression of D3 in the chicken brain and more specifically in the cerebellum was previously reported by Van den Eynde et al. (30) by means of Northern blot analysis. Because no other cells in the brain could be stained by means of ICC, it can be presumed that D3 is more abundantly present in cerebellar Purkinje cells than elsewhere in chicken brain. Until now little was known about the expression of D3 in different regions of the chicken brain and, more specifically, about the cellular localization of the deiodinase protein in neuronal cells. It has been shown that Purkinje cells are very dependent on thyroid hormone, especially during the early phases of morphogenesis. Bouvet et al. (31) demonstrated this by studying the dendritic arborization of Purkinje cells in thyroid-deficient chickens. In chickens injected



FIG. 4. A, In vitro D3 hepatic activity in liver microsomes of 17-d-old embryos (E17) and newborn chicks (C0) in femtomoles per milligrams per minute. Values are the mean ⫾ SEM. B, Western blot analysis of liver extracts (E17 and C0) using anti-D3a and preimmune serum. C, Relative mean concentration of D3 protein in liver extracts represented on Western blot in B.

FIG. 5. Northern blot analysis of hepatic and cerebellar mRNA hybridized with a 32P-labeled D3 cRNA probe. Lane 1, Liver; lane 2, cerebellum; lane 3, cerebellum.

with tetramethylthiourea, the development of Purkinje cells was the most affected process of cerebellar cortex maturation. Strait et al. (32), on the other hand, localized the TH ␤1 receptor (TR␤1) in the Purkinje cells of rat cerebellum using antisera to unique peptide regions of ␤1. Their study clearly showed that Purkinje cells are a direct target for T3, because the cerebellum contains significant concentrations of the TR␤1 protein despite a low TR␤1 mRNA content. The signal was localized to the nucleus of these cells and was greater than that seen in the liver. A positive, but weaker, signal was found in the granule cells (32, 33). In the beginning, deiodinases (in contrast to TRs) were hypothesized to be only expressed in nonneuronal cells, such as astrocytes. Astrocytes are capable of contributing to the

transfer of nutrients between blood and neurons. For example, they absorb glucose out of blood and metabolize it to lactate, which is then released for neuronal use. A similar mechanism was postulated for T3 production by the deiodinases (34). However, our results indicate for the first time the presence of the D3 protein in neuronal cells of the brain, namely, cerebellar Purkinje cells, which is in agreement with the results of Tu et al. (22), who showed that D3 mRNA is present in neurons. Because Purkinje cells in chick cerebellum were previously stated to be TH dependent and moreover to be a direct target for T3, it can be presumed that the TH homeostasis in the brain is not only preserved by the D3 enzyme present in nonneuronal glial cells by means of a metabolic interaction between these cells and the neurons, but also by the neurons themselves. Concerning the Western blot analysis, many attempts have been previously described to solubilize the iodothyronine deiodinases out of the membrane fraction. Hummel and Walfish (35) were able to solubilize the deiodinases out of rat liver microsomes by adding the detergent Triton X-100 (0.1%) to the microsomal membranes. Leonard and Rosenberg (36) also solubilized deiodinases out of rat kidney microsomes by using 0.2% deoxycholate. Deoxycholate revers-

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FIG. 6. Sequence alignment of D3 cerebellar fragment (upper line) and D3 chicken sequence from hepatic cDNA (12) (lower line). The underlined parts represent the primers.

ibly inhibits the deiodinases, but removal of the detergent restores their activity. Because previous research using a deiodinase antiserum pointed out that the use of nonextracted liver microsomal membranes in the Western analysis did not give satisfying results (Verhoelst, C. H. J., and V. M. Darris, unpublished results), we decided to make cerebellum extracts by adding different concentrations of Triton X-100. Administration of increasing Triton X-100 concentrations did not enhance the signals obtained for the cerebellum, but in all samples we were able to detect three differently sized bands for D3 by means of Western blot analysis. Until now, no reports have been published about the molecular size of chicken D3. Schoenmakers et al. (37) demonstrated that chicken hepatic D1 corresponds to a 25.7-kDa protein by means of affinity labeling with N-bromoacetyl-[125I]T3. In rats too, a 27-kDa protein has been reported for D1 (38). Our findings with chicken D3 show a fragment of 30 kDa, which is in the same range as these previous results for D1. The two other molecular mass fragments in the Western blot analysis are of an unknown origin, and further research is required to determine their origin. However, the hypothesis that they might be truncated proteins can most likely be excluded, because they were detected using the D3a antiserum, which is a C-terminal antiserum, and not the D3b antiserum (which is an Nterminal antiserum). Another possible explanation for the appearance of two additional protein fragments might be an insufficient or only partial solubilization of the deiodinases out of the membrane fractions. Finally, in the immunohistological staining the contributions of these two other fragments, if present, would be minor, given the high intensity of the 30-kDa fragment in the Western blot analysis compared with the other fragments. Moreover, the identity of the 30-kDa fragment as D3 protein could also be confirmed by comparison of protein staining intensity and D3 activity

levels in liver of 17-d-old embryos compared with newly hatched chicks. These results are clearly in accordance with the previously described decreasing hepatic D3 activity from embryonic day 17 toward hatching during the ontogeny of the chicken (12, 24). Because RT-PCR revealed a single fragment that shows a homology of 99% with the chicken D3 cDNA sequence cloned from liver (12), this again is an indication of the presence and expression of the D3 enzyme in the cerebellum. Northern blot analysis also supports this by demonstrating a single mRNA species of the same length as the hepatic control mRNA, namely, 2.4 kb. In conclusion, this study showed for the first time the presence of type III iodothyronine deiodinase protein in cerebellar Purkinje cells of the chicken using D3 polyclonal antisera. By means of Western blot analysis a 30-kDa fragment corresponding to D3 could be detected. Acknowledgments We thank Prof. Dr. F. Vandesande (K. U. Leuven, Leuven, Belgium) for synthesis of the peptides. We also thank L. Noterdaeme, W. Van Ham, and F. Voets for their valuable technical assistance. Received August 21, 2001. Accepted March 4, 2002. Address all correspondence and requests for reprints to: Dr. Carla H. J. Verhoelst, Laboratory of Comparative Endocrinology, Zoological Institute, K. U. Leuven, Naamsestraat 61, B-3000 Leuven, Belgium. Email: [email protected]. This work was supported by the K. U. Leuven Research Council (OT/97/11), Fonds voor Wetenschappelijk Onderzoek Vlaanderen Grant G.0295.99, and European Union Grant (QLG3-CT2000-00930).

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