Localization and Action of Dragon (Repulsive Guidance Molecule b), a Novel Bone Morphogenetic Protein Coreceptor, throughout the Reproductiv
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内分泌学杂志 2005年第8期
Reproductive Endocrine Unit (Y.X., Y.S., A.M., A.S.), Neural Plasticity Research Group of the Department of Anesthesia and Critical Care (T.A.S., G.B., C.J.W.), and Program in Membrane Biology (H.Y.L.), Massachusetts General Hospital, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Schneyer.alan@mgh.harvard.edu.
Abstract
Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, L?T2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.
Introduction
MAMMALIAN REPRODUCTION is regulated by endocrine hormones such as pituitary FSH and LH as well as by locally produced growth factors, including TGF-? superfamily members activin, inhibin, and bone morphogenetic proteins (BMPs) (reviewed in Ref. 1). BMPs were originally identified by their ability to induce bone and cartilage formation (2). However, numerous studies have revealed that BMPs have a wide variety of effects on many cell types, including monocytes and epithelial, mesenchymal, and neuronal cells, and play pivotal roles in cytodifferentiation, morphogenesis, and organogenesis (3). Members of the TGF-? superfamily, including BMPs, transduce their signals through binding to type I and II serine/threonine kinase receptors. BMP signaling is mediated intracellularly by the phosphorylation of receptor-activated Smads (R-Smads) 1, 5, and 8. Activated R-Smads complex with the common partner Smad 4 and translocate to the nucleus where they initiate BMP-stimulated alterations in target gene expression. Signaling of TGF-? superfamily members including BMPs is also modulated by soluble extracellular proteins such as noggin, chordin, and gremlin. In addition, membrane-associated proteins, including betaglycan (TGF-? type III receptor), endoglin, and crypto are also critical for assisting with ligand binding to receptor or for altering receptor specificity (reviewed in Refs. 4, 5, 6).
The mRNAs encoding BMP2, 3, 3b, 4, 6, 7, and 15 have been identified in mammalian ovaries. Moreover, BMP receptors BMPRIA, IB, and II are widely expressed in the ovary, with the strongest expression in the granulosa cells and oocytes of developing follicles in normally cycling rats (7, 8). BMPs and their receptors are also expressed in uterine stroma and glandular epithelium (9). In males, BMP2, BMP4, and BMP8A and BMP8B are expressed in germ cells, and BMP4, BMP7, and BMP8A are expressed in the epididymis (reviewed in Ref. 8). Moreover, mice deficient in BMP4, BMP8A, or BMP8B show germ cell degeneration in the testis and/or epithelial cell degeneration in the epididymis (10, 11, 12). Together, these results suggest that BMPs may play important roles in regulating reproduction.
Dragon was identified through a genomic screening strategy for genes regulated by DRG11, a homeobox transcription factor that is expressed in embryonic dorsal root ganglion (DRG) (13). Independently, this gene was also cloned as RGMb, one of three mouse homologues of the chicken repulsive guidance molecule (RGM) (14). The Dragon gene encodes a 436-amino-acid glycosylphosphatidylinositol (GPI)-anchored protein, suggesting it may be associated with lipid rafts within the plasma membrane. Indeed, adhesion of DRG neurons to HEK293 cells was increased after transfection of HEK293 cells with Dragon cDNA (13). Dragon is expressed in a number of neural tissues including embryonic and adult mouse DRGs, spinal cord, and brain (13, 15, 16). Interestingly, Dragon is also involved in BMP signaling because 1) injection of Dragon mRNA into Xenopus embryos induced expression of a number of BMP-regulated genes, 2) Dragon binds directly to BMP2, BMP4, and BMP receptors, and 3) transfection of Dragon cDNA into BMP-responsive cells enhances transcription of a BMP-responsive reporter (17). These observations indicate that Dragon acts as a BMP coreceptor that regulates cellular response to BMP signals.
To understand the potential role of Dragon in BMP signaling within the reproductive tissues, we examined Dragon expression in murine reproductive tissues and cell lines. We found that Dragon is expressed and dynamically regulated in gonadal germ cells as well as in epithelial cells of the reproductive tract including epididymis and uterus. Dragon is also expressed in the pituitary. As predicted from its being anchored to the cell membrane by a GPI anchor, Dragon is indeed localized in lipid rafts where it enhances BMP2 and -4 signaling. Taken together with the overlapping expression and function of BMPs in the reproductive system, our results indicate that Dragon may play an important role in reproduction through enhancement of BMP signaling.
Materials and Methods
Animals
All animal studies were conducted in accordance with an animal use protocol approved by the Massachusetts General Hospital animal use committee in accordance with U.S. Department of Agriculture guidelines. Mice [B6C3F1 (C57BI/6 x C3H)] were maintained in the animal barrier facility and killed at different ages to collect tissues for immunohistochemistry and RNA extraction. In addition, mice at 19 d of age were injected with pregnant mare serum gonadotropin (PMSG; ip, 5 IU/mouse; Sigma Chemical Co., St. Louis, MO), and killed 48 h later to collect gonads for immunohistochemistry.
RT-PCR
Total RNA was extracted from tissues stored in RNAlater (Ambion, Austin, TX) or cells stored in Trizol (Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s protocol. Total RNA (0.5–1.0 μg) was reverse transcribed as previously described (18). Aliquots (2 μl) of first-strand cDNA mix were used in PCR (35 cycles) to amplify Dragon and ?-actin. The primers, which amplify both human and mouse Dragon cDNA, were TGT TCC AAG GAT GGA CCC ACA TC (forward) and GCA GGT CAT CTG TCA CAG CTT GG (reverse).
Immunohistochemistry and immunocytochemistry
A rabbit polyclonal antibody was raised against a peptide corresponding to the C terminus of Dragon upstream of its GPI anchor. This antibody has been shown to specifically recognize Dragon protein (13).
Immunohistochemistry on paraffin sections were performed as previously described (19). Briefly, ovaries were fixed in Bouin’s solution, and other organs were fixed in 4% paraformaldehyde. Antigen retrieval was performed on paraffin sections in 0.01 M citrate buffer (pH 6.0). Tissue sections were incubated overnight with anti-Dragon (1:4000), washed, incubated for 1 h with biotinylated goat antirabbit IgG and then 30 min with Vectastain Elite ABC (Vector Laboratories, Inc., Burlingame, CA), and developed with diaminobenzidine (DAB) for detection (ICN Biomedical, Inc., Aurora, OH). Sections were then counterstained with Harris’ hematoxylin.
To colocalize Dragon and FSH in the pituitary, mouse pituitaries were fixed in 4% paraformaldehyde at 4 C overnight, cryoprotected in 30% sucrose overnight, and frozen in Tissue-Tek OCT embedding compound (Electron Microscopy Sciences, Fort Washington, PA). Sections (12 μm) were incubated with a mixture of rabbit anti-Dragon serum (1:2000) and guinea pig antimouse FSH (1:1600, AFP-3080, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for 1 h, washed, and then with a mixture of fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG and tetramethyl rhodamine isothiocyanate-conjugated donkey anti-guinea pig IgG (diluted 1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
For immunocytochemistry, Ishikawa cells were grown on glass coverslips. Live cells were incubated with anti-Dragon serum (1:2000) for 1 h on ice. Cells were then fixed in 2% paraformaldehyde for 20 min. The bound antibodies were detected by incubating with FITC-conjugated donkey antirabbit IgG (diluted 1:200 in PBS) for 1 h. To demonstrate specificity, the Dragon antibody was preincubated overnight with 10 mM immunization peptide before being applied to sections or cells.
In situ hybridization
Air-dried frozen sections (14–18 μm) were fixed in 4% paraformaldehyde-PBS, digested with proteinase K, acetylated, washed, and dehydrated. Antisense and sense cRNA probes were prepared by means of in vitro transcription in the presence of [-35S]UTP, which were then hybridized in 50% deionized formamide, 10 mM Tris/HCl (pH 7.6), 600 mM NaCl, 0.25% SDS, 200 μg/ml yeast tRNA, 50 mM dithiothreitol, 1x Denhardt’s solution, and 10% dextran sulfate overnight at 55 C in a humidified chamber. After hybridization, the sections were incubated with ribonuclease A and washed in 0.1x SSC containing ?-mercaptoethanol (875 μl in 300 ml) and 0.5 mM EDTA at 65 C for 1 h. After developing, the slides were counterstained with hematoxylin and mounted for photography.
Lipid raft protein extraction
Lipid raft proteins were prepared after protocols previously described (20, 21). Briefly, Ishikawa cells were scraped and pelleted in ice-old PBS, resuspended in 2 ml ice-cold lysis buffer [10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and proteinase inhibitors], and allowed to stand on ice for 30 min. The lysate was centrifuged for 5 min at 1300 x g to remove nuclei and large cellular debris. The supernatant was mixed with an equal volume of 85% sucrose in TBS [10 mM Tris/HCl (pH 7.5), 150 mM NaCl], placed at the bottom of a 10-ml ultracentrifuge tube, and then overlaid with 5 ml of 35% sucrose and 1.4 ml of 5% sucrose. The sample was then centrifuged for 14 h at 150,000 x g in a SW41Ti rotor so that rafts could float to the tip while cytoskeletal and cytoplasmic proteins remain at the bottom. Five fractions of 1 ml and four fractions of 1.35 ml were collected from the top of the tube. The light-scattering band, an indicator of the location of lipid rafts (20), was located primarily in fraction 2. These fractions were analyzed by trichloroacetic acid precipitation of proteins from 100-μl aliquots followed by SDS-PAGE and Western blotting.
Western blotting
Western blotting analyses were performed as previously described (19). Briefly, samples from sucrose gradient fractions were subjected to SDS-PAGE under reducing conditions, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), blocked in 10% nonfat dry milk, and incubated overnight at 4 C with rabbit anti-Dragon (1:4000) or anti-caveolin-1 (1:2000; BD Biosciences, San Jose, CA) antibodies. The membranes were washed three times before being incubated for 2 h at room temperature with second antibody, which was detected with enhanced chemiluminescence (ECL) Reagent Plus (PerkinElmer Life Sciences, Boston, MA). After exposure, membranes were stripped for 30 min at 50 C and reprobed with a monoclonal ?-actin antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA).
Transfection and luciferase assay
Ishikawa and KGN cells were maintained in TT medium [1:1 mixture of DMEM and F-12, supplemented with 1% L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin sulfate, and 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD)]. To examine the effect of Dragon on BMP signaling, transfections were performed in 24-well trays using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with a total of 400 ng DNA [180 ng BRE-Luc, a BMP response element kindly provided by Dr. ten Dijke (22), 10 ng pRL-TK, and the indicated doses of Dragon cDNA and pcDNA3]. Approximately 24 h after transfection, the medium was replaced with serum-free TT medium supplemented with 0.1% BSA, with or without BMP ligands (R&D Systems, Minneapolis, MN). After treating for 16 h, the cells were lysed and assayed for luciferase activity using the dual luciferase reporter assay kit (Promega, Madison, WI).
To obtain noggin protein for neutralization of endogenous BMPs, the human noggin cDNA (IMAGE clone 4737725 from American Type Culture Collection, Rockville, MD) was subcloned into pcDNA3 (Invitrogen) and transfected into HEK-293-F suspension cultures in Freestyle serum-free medium (Invitrogen) as previously described (23). Concentrated conditioned medium was calibrated by two independent methods including 1) biological assay and 2) Western blotting. In the biological assay, increasing amounts of noggin-conditioned medium were mixed with 10 ng/ml BMP2 and used to treat HepG2 cells that were previously transfected with the BRE-Luc reporter. At the effective concentrations for half-maximal response (EC50), the amount of noggin in the culture medium was equal to that of the BMP2 concentration, assuming molar equivalent antagonism activity (24). For the Western blotting analysis, serial dilutions of noggin-conditioned medium were resolved by 12% PAGE under reducing conditions and visualized by staining with an antimouse noggin polyclonal antibody (R&D Systems). Noggin concentrations were estimated at the detection limit dose according to the manufacturer’s information. Both methods gave similar results. As a control, concentrated conditioned medium from mock-transfected HEK293 cells was tested and determined to have no BMP-inhibitory activity.
To demonstrate that Ishikawa cells transfected with Dragon cDNA actually produce Dragon protein, transfected cells were extracted in RIPA buffer [150 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 50 mM NaF, 0.5% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS], and the lysates were then analyzed by Western blotting for Dragon as described above.
Data analysis
Figure 6, A, C, and D, depicts the mean ± SE of triplicates from representative experiments. In vitro bioassay experiments in the presence or absence of transfected Dragon (Fig. 6D) represents the mean ± SE of six determinations from three independent experiments and were analyzed by two-way ANOVA. Differences between ligand doses or between presence and absence of Dragon were identified by Student-Newman-Keuls post hoc test. Differences of P < 0.05 were considered significant.
FIG. 6. Dragon enhances cellular response to BMPs in cell lines derived from reproductive tissues. A, Ishikawa or KGN cells were transiently transfected with BRE-Luc reporter in combination with increasing doses of Dragon cDNA and assayed for luciferase activity. Transfection with Dragon increases BRE-Luc response in the absence of exogenous ligand. Values are ratios of BRE-Luc to pRL-TK and are mean ± SE of triplicates from representative experiments. Dragon protein is detectable by Western blot in Ishikawa cells after transfection with 100 ng cDNA. The membrane was stripped and reprobed with ?-actin antibody as a control for loading (inset). B, Ishikawa cells were transfected with BRE-Luc reporter and Dragon cDNA and treated with BMP2 alone or together with noggin. Values are ratios of BRE-Luc to pRL-TK and are mean ± SE of triplicates from representative experiments. C, Control medium was used in parallel with noggin-conditioned medium to demonstrate the specificity of the inhibitory activity of noggin-conditioned medium in BMP2 signaling. Although noggin inhibited BMP2 signaling, control conditioned medium had no inhibitory effect. D, Ishikawa cells transiently transfected with BRE-Luc reporter and Dragon (0 or 10 ng) in 24-well plates were incubated with increasing doses (0–2800 pM) of BMP2. Values are fold increases of luciferase activity in treated cells relative to untreated cells and are the means ± SE of six determinations from three independent experiments in duplicate. Asterisks indicate significant differences between transfected and untransfected cells at each BMP2 dose: **, P < 0.01; *, P < 0.05.
Results
Expression of Dragon mRNA in reproductive organs
Dragon expression in mouse reproductive tract tissues was examined by RT-PCR (Fig. 1). Dragon mRNA was detected in the testis, epididymis, and seminal vesicles in males and in the ovary, uterus, and pituitary in females.
FIG. 1. Dragon mRNA expression in mouse tissues. RNA extracted from various mouse tissues was examined for Dragon mRNA expression by RT-PCR. In tissues where Dragon expression was low, ?-actin was used as a control for cDNA quality. Dragon expression was strongly detected in testis (Te), ovary (Ov), pituitary (Pit), epididymis, uterus (Ut), kidney (Kid), and brain (Br). Weaker Dragon signals were detected in seminal vesicles (SV) and adrenal (Ad).
Cellular localization of Dragon in the testis and epididymis
Immunohistochemical analyses of d 1 and 3 mouse testes showed that Dragon was localized to gonocytes both in the center and at the basement membrane of seminiferous tubules (Fig. 2A, a and b). In testes from d 9 mice, spermatogonia at the basement membrane were positive for Dragon expression (Fig. 2A, c). However, Dragon staining in spermatogonia became much weaker in testes from 21-d-old mice (Fig. 2A, d). Interestingly, a few gonocytes, which remain in the central region of the tubules from d 21 testes, were strongly stained with Dragon (Fig. 2A, d). Some interstitial cells showed weak staining in d 1 testes (Fig. 2A, a), but no staining was observed in interstitial cells in older testes. In adult (d 60) testis, Dragon was expressed in spermatocytes and round spermatids, whereas spermatogonia and Sertoli cells did not appear to express Dragon (Fig. 2A, e and f). The staining in gonocytes, spermatogonia, and spermatocytes was completely abolished when the antiserum was preincubated with the competing immunizing peptide, demonstrating the specificity of the antiserum (Fig. 2A, g and h). Dragon expression in spermatocytes and round spermatids from adult testes was confirmed by in situ hybridization (Fig. 2C).
FIG. 2. Cellular localization of Dragon in the mouse testis and epididymis during postnatal development by immunohistochemistry and in situ hybridization. For immunohistochemistry, all sections were stained with DAB (brown) and counterstained with hematoxylin (blue). Images are shown at lower (x40) or higher (x100) magnification. A, Immunolocalization of Dragon in testes at d 1 (D1, a), d 3 (D3, b), d 9 (D9, c), d 21 (D21, d), and d 60 (D60, e and f). For negative control, sections were incubated with Dragon antibody preincubated with competing immunizing peptide (g and h). Dragon is highly expressed in gonocytes and spermatogonia in testes of newborn mice and spermatocytes and round spermatids in testes of adult mice. Dragon is not expressed in spermatocytes of d 21 testes. Spermatogonia are weakly stained in d 21 testes but not stained in adult testes. B, Immunostaining of d 21 testes with (b) and without (a) PMSG (5 IU) injection 2 d earlier. Immunostaining in spermatocytes is turned on by PMSG administration. C, Localization of Dragon mRNA in mouse testes by in situ hybridization. Bright (left) and dark (right) field images are shown. D, Immunolocalization of Dragon in d 3 (D3, a–c) and d 60 (D60, d–f) epididymis. Caput (a and d), corpus (b and e), and cauda (c and f) were dissected.
Spermatocytes of d 21 testes were not stained with Dragon antibody, whereas those cells of adult testes were strongly stained (Fig. 2A, d and e). Interestingly, Dragon was highly expressed in spermatocytes of testes collected after 2 d of PMSG administration to 19-d-old mice (Fig. 2B), suggesting that Dragon expression levels are hormonally regulated.
In d 3 epididymis, Dragon protein was found on both the apical and basal sides of epithelial cells with staining in the apical side stronger compared with the basal side. Dragon staining was stronger in caudal than in caput or corpus epididymis of 3-d-old mice (Fig. 2D, a–c). In contrast, Dragon expression was primarily localized to the apical side of epithelial cells of adult epididymis, and it appeared that Dragon was more highly expressed in caput or corpus, compared with caudal epididymis (Fig. 2D, d–f). These results suggest that Dragon may be involved in regulation of spermatogenesis and epididymal epithelial function.
Cellular localization of Dragon in the ovary and uterus
Within the adult mouse ovary, Dragon protein was detected exclusively within oocytes (Fig. 3A, a, b, and d) and was more intense in oocytes from secondary follicles compared with antral follicles (Fig. 3A, a). In contrast, no Dragon staining was found in oocytes of primordial (Fig. 3A, b) or primary (Fig. 3A, c) follicles, nor in somatic cells of any follicles (Fig. 3A). In atretic follicles, oocytes showed weak Dragon staining (Fig. 3A, d). There was no staining of the ovarian surface epithelium (Fig. 3A, b–d). Oocyte staining was completely blocked by preincubating antiserum with competing immunizing peptide (Fig. 3A, e). In d 9 ovaries, Dragon staining was detected only in the oocytes of secondary follicles but not in the primordial or primary follicles (Fig. 3A, f). PMSG treatment had no effect on Dragon staining in oocytes (data not shown).
FIG. 3. Cellular localization of Dragon in the mouse ovary and uterus by immunohistochemistry and in situ hybridization. For immunohistochemistry, all sections were stained with DAB (brown) and counterstained with hematoxylin (blue). Images are shown at lower (x40) or higher (x100) magnification. A, Dragon immunostaining in ovaries at d 60 (D60, a–d) and d 9 (D9, f). The staining in oocytes of secondary follicle (arrow) is stronger than that in oocytes of antral (arrowhead) and atretic (curved arrow) follicles (a and d). Dragon is not expressed in oocytes of primordial (open arrow) and primary (triangle) follicles (b and c). For negative control, sections were incubated with Dragon antibody preincubated with competing immunizing peptide (e). B, Localization of Dragon mRNA in mouse adult ovaries by in situ hybridization. Dark (b and d) and bright (a and c) field images are shown. Signals are confined to oocytes, and signals are stronger in the secondary (arrow) than in antral (arrowhead) follicles (a and b). No signals are seen in primary follicles (triangle, c and d) and in corpus luteum (CL). C, immunohistochemistry of the mouse uterus showing protein expression of Dragon (a and b), and negative control (c), incubation with Dragon antibody preincubated with competing immunizing peptide. The solid arrow indicates luminal epithelial cells of endometrium, the arrowhead indicates glandular epithelial cells of endometrium, and the open arrow shows circular muscle.
Consistent with immunostaining, Dragon mRNA, as detected by in situ hybridization, was stronger in oocytes from secondary follicles (Fig. 3B, arrows) compared with antral (Fig. 3B, arrowheads) follicles and was undetectable in oocytes from primary follicles (Fig. 3B, triangle). Dragon mRNA was not detectable in ovarian somatic cells. These results suggest that Dragon may regulate the development of oocytes and follicles by influencing the interaction between the oocyte and granulosa cells.
In the uterus, Dragon protein was expressed in luminal and glandular epithelial cells of the endometrium (Fig. 3C, a and b). Weak staining was also found in circular muscle (Fig. 3C, a). Localization of Dragon in the luminal and glandular epithelial cells suggest Dragon may also be required for normal endometrial function.
Cellular localization of Dragon in the pituitary
In the pituitary, sporadic staining was observed in both the anterior and posterior lobes, whereas no staining was detected in the intermediate lobe (Fig. 4A). Because BMPs and their receptors are expressed in mouse pituitary gonadotropes, as well as in the L?T2 gonadotrope cell line (25, 26), and BMPs can stimulate FSH biosynthesis (27), we examined whether FSH-expressing gonadotropes also express Dragon. To this end, frozen pituitary sections were dual labeled with Dragon and FSH antibodies. FSH-expressing cells indeed overlapped extensively, albeit not completely, with Dragon-expressing cells (Fig. 4B). Interestingly, L?T2 cells also express Dragon (Fig. 5A). These results suggest that Dragon may influence BMP-mediated FSH biosynthesis in vivo and in vitro.
FIG. 4. Immunolocalization of Dragon in the pituitary and colocalization of Dragon and FSH. A, Immunostaining for Dragon (brown) in the mouse pituitary (paraffin sections). Sections were incubated with Dragon antibody (a–c) or with Dragon antibody preincubated with competing immunizing peptide (d–f). AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe. B, Colocalization of FSH? and Dragon in the mouse anterior pituitary (frozen sections). Dragon (green) and FSH? (red) were detected in the anterior pituitary. Double-labeled cells (yellow, arrows) indicate Dragon expression in the mouse pituitary gonadotrope. Note that some FSH?-positive cells are negative for Dragon staining and some Dragon-positive cells are negative for FSH staining.
FIG. 5. Expression of Dragon in cell lines derived from reproductive tissues and localization of Dragon into lipid rafts. A, RT-PCR analyses of Dragon mRNA in cell lines. ?-Actin was used as a control for cDNA quality. B, Immunochemical localization of Dragon in Ishikawa cells. The live unfixed cells were incubated with rabbit anti-Dragon serum on ice and then fixed in 2% paraformaldehyde. The bound antibodies were detected by incubating with FITC-conjugated donkey antirabbit IgG. C, Localization of Dragon in raft-enriched fractions prepared from Ishikawa cells. Cells were extracted using a buffer containing 1% Triton X-100. The lysate was mixed with 85% sucrose, sequentially layered with 35 and 5% sucrose, and centrifuged for 14 h at 150,000 x g. Nine fractions were collected and analyzed for Dragon, caveolin-1, and ?-actin by Western blot. The bands specific for Dragon are marked with asterisks.
Dragon expression in cell lines of the reproductive axis
In screening cell lines originating from reproductive organs (Fig. 5A), we found that Dragon was expressed in Hela (cervical carcinoma), MCF-7 (breast carcinoma), L?T2 (pituitary carcinoma), JEG3 (placenta carcinoma), and Ishikawa (endometrium adenocarcinoma) cells. In contrast, Dragon mRNA was undetectable in S4 spermatogonial cells (28) or KGN granulosa tumor cells (29).
Lipid raft localization of Dragon
We explored the location of Dragon on the cell surface using Ishikawa cells as a model. Live cells were incubated with Dragon antibody at 4 C and then fixed and processed for immunocytochemistry. Dragon appeared to have a punctate pattern on the cell membrane, which is typical of lipid raft proteins (Fig. 5B). To further characterize Dragon localization within membrane subdomains, cells were extracted on ice in the presence of 1% Triton X-100 and then subjected to sucrose gradient ultracentrifugation. As expected, Dragon was detectable primarily within the low-density fractions (Fig. 5C; fractions 2 and 3), along with caveolin-1, which is typically associated with lipid rafts. The high-density fractions, which include cellular and cytoskeletal proteins (fractions 6–9), contained ?-actin, some caveolin-1, and a small amount of Dragon. These results indicate that Dragon is indeed located within lipid rafts in Ishikawa cells.
Dragon enhances signaling of BMP2 and BMP4
Our results demonstrate that Dragon is expressed in gonadal germ cells and in reproductive tract epithelial cells. Moreover, we have previously shown that Dragon enhances the BMP2 response in the HepG2 liver cell and LLC-PK1 kidney cell lines (17). To examine whether Dragon has a similar role in reproductive cells, Ishikawa and KGN cells were transfected with Dragon cDNA together with BRE-Luc, a BMP-responsive luciferase reporter construct. Dragon dose-dependently increased BRE luciferase activity in both Ishikawa and KGN cells in the absence of added BMPs (Fig. 6A). Although Dragon induced similar reporter activity in both cell lines at lower doses (0.1–10 ng), it was more effective in Ishikawa cells at higher doses. Although Dragon protein was undetectable in Ishikawa cells before transfection, Dragon was detectable after transfection with 100 ng cDNA (Fig. 6A, inset), indicating that the effects of transfected Dragon are mediated by an increase in Dragon protein.
Treatment of Ishikawa cells with noggin-containing conditioned medium (100 ng/ml) resulted in partial inhibition of Dragon-dependent BRE-Luc activity and completely inhibited signaling by BMP2 (10 ng/ml) (Fig. 6B). Conditioned medium from mock-transfected HEK293 cells had no inhibitory activity (Fig. 6C), indicating that the BMP-inhibiting activity in the noggin-conditioned medium was caused by noggin itself. These results also suggest that the effect of Dragon on BMP reporter activity is dependent on endogenous ligand. We next examined the effect of Dragon on BMP signaling in the presence of added BMP ligands. Ishikawa cells were transfected with Dragon cDNA and treated with increasing doses of BMP ligands. Dragon significantly increased BMP2 signaling at 11–700 pM BMP2 doses (P < 0.05), but had no effect at 2800 pM (Fig. 6D). Thus, at lower BMP2 doses (i.e. 11 or 44 pM), Dragon transfection resulted in detectable reporter activity that is not seen in the absence of Dragon (Fig. 6D). Similar results were observed with BMP4 and in KGN cells (data not shown). These results indicate that Dragon acts to increase sensitivity of BMP-responsive cells to low concentrations of endogenous or exogenous BMP ligands.
Discussion
Although originally discovered as a GPI-anchored cell surface protein involved in neuronal differentiation and cell-cell contact in the developing nervous system (13), it is now evident that Dragon can also act as a coreceptor for BMP2 and BMP4 (17). In addition to previously reported sites of expression in neural tissues (13, 17), our studies demonstrate that Dragon is also expressed in many specific cell types throughout the reproductive system. In addition, we found that Dragon was expressed in numerous cell lines derived from reproductive tissues and that Dragon expression enhanced responsiveness of Ishikawa and KGN cells to BMP2 and BMP4. Thus, our results suggest that Dragon may have important roles in mediating BMP signaling in reproduction.
To define specific sites where Dragon-mediated BMP signaling might be important in reproduction, we explored cell-specific expression in the male and female reproductive tracts. In males, BMP8a and BMP8b are expressed in maturing spermatocytes, and BMP8b knockout males are infertile because of developmental arrest and degeneration of spermatocytes (11, 12), suggesting that these BMPs are critical for normal spermatocyte development. BMP receptors ALK3 and BMPR-II are localized in postnatal spermatogonia, and BMP4 is produced by Sertoli cells very early in postnatal development, consistent with an ongoing requirement for BMP signaling in the testis (30). In addition, BMP2 primarily stimulates spermatogonial proliferation, whereas BMP7 acts mainly on Sertoli cells in the testis from 7-d-old mice (31). Our results extend these findings to a novel BMP coreceptor that enhances BMP signaling, because in 3-d-old male mice, Dragon was highly expressed in gonocytes before and after they migrated from the tubule lumen to their basal position, with this immunoreactivity being maintained as spermatogonia in d 9 animals. By d 21, staining in spermatogonia was substantially diminished, but gonocytes remaining within the tubule lumen were still positive. In adults, Dragon staining appeared in maturing spermatocytes but not in other testicular cell types. The shift in expression from gonocytes and spermatogonia in juvenile animals to spermatocytes in mature males suggests that the role of BMPs may change as the testes mature to produce active sperm. Taken together, these results suggest a critical role for BMPs in regulating testis development and spermatogenesis and suggest that Dragon may be an important mediator of these processes.
BMP4, BMP7, and BMP8A are expressed in the epididymis, and knockout of each gene by itself resulted in degeneration of the epididymal epithelium (10, 11, 12). These results indicate a role for BMPs in the control of epididymal function. Interestingly, Dragon was strongly expressed on the apical surface of polarized epididymal epithelium in immature and mature males consistent with a role for Dragon in enhancing this essential BMP signaling.
In females, both BMPs and their receptors have been identified in numerous ovarian cell types, including oocytes and granulosa cells (8). In vitro studies have demonstrated that BMP2, 4, 6, 7, and 15 regulate granulosa cell functions, and BMP4 and 7 promote the primordial-to-primary follicle transition during follicle maturation (reviewed in Ref. 8). The significance of BMP signaling in ovarian function is also underscored by the altered ovulation rates in Inverdale sheep with a natural point mutation in the BMP15 gene (32) and in Booroola sheep with a point mutation in the BMPRIB gene (33). Our results demonstrate that in the ovary, Dragon is expressed exclusively in oocytes and most prominently in oocytes within secondary follicles. This is a time of oocyte growth and cytoplasmic maturation (34), suggesting that BMP signaling in general, and Dragon enhancement of this signaling in particular, may be important for growth and maturation of oocytes.
BMP signaling components are expressed in a variety of cells within the rat uterus (9). BMP2 mRNA is restricted to periluminal stroma, and BMP7 is expressed in periluminal stroma and glandular epithelial cells, whereas BMP4 and BMP6 are expressed in blood vessels in the uterus. BMPRIA, BMPRIB, and BMPRII are expressed in a number of cell types in the uterus including luminal and glandular epithelial cells. We observed Dragon expression in luminal and glandular epithelial cells of the mouse endometrium, suggesting that Dragon may enhance BMP signals involved in regulating uterine maturation in preparation for implantation.
BMP2, 4, 6, 7, and 15 are expressed in mouse pituitary, and BMP6, 7, and 15 have been shown to stimulate FSH synthesis and secretion (25, 27, 35). BMP6 and BMP7 can also stimulate FSH mRNA biosynthesis in L?T2 mouse pituitary cells in culture (27). We observed Dragon expression in L?T2 cells as well as in numerous cells within the mouse pituitary, some of which also stained for FSH. These results suggest that BMPs may act in an autocrine manner to modulate FSH biosynthesis and that Dragon may enhance this process.
In cell culture studies using cell lines from the reproductive tract, we found that Dragon expression enhanced the response to endogenous BMP ligand as well as low doses of exogenous BMP2 and BMP4, results that agree with our earlier observations in nonreproductive cell lines (17). Although the precise mechanism for this signaling enhancement has not yet been fully elucidated, our immunocytochemical analysis indicates that Dragon is located on the plasma membrane in discrete patches, consistent with its belonging to the class of GPI-anchored proteins that are known to localize in lipid rafts (36). Moreover, our earlier studies indicate that Dragon can interact directly with BMPRII, ActRII, and the Alk3 and Alk6 type I receptors (17). Taken together, these results suggest a model for enhanced BMP signaling in which Dragon acts as a BMP coreceptor in collecting type II and type I receptors into lipid rafts where they are optimized to respond to low doses of BMP ligands. Because Dragon can bind BMP2 and BMP4 directly, it is also possible that Dragon acts to stabilize the ligand-receptor complex in lipid rafts, thereby facilitating endocytosis and signaling. Of course, these two possibilities are not mutually exclusive. Based on the localized expression of Dragon in developing and maturing germ cells, as well as specific epithelial cells within the reproductive tract that are known to be BMP responsive, our results support the concept that BMPs play an important role in regulating reproduction in mammals and that this role may be regulated by Dragon.
Acknowledgments
Dr. Ernestina Schipani and Janet Saxton provided expertise and access to critical facilities for the immunocytochemical and in situ hybridization analyses. Dr. Carla Boitani provided helpful suggestions for germ cell classifications in the testis from newborn mice. Cell lines were generously provided by Dr. Martin Dym (S4 spermatogonial cells), Dr. Pamela Mellon (L?T2 cells), and Dr. Hajime Nawata (KGN cells). Dr. Peter ten Dijke provided the BRE-Luc reporter.
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Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Schneyer.alan@mgh.harvard.edu.
Abstract
Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, L?T2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.
Introduction
MAMMALIAN REPRODUCTION is regulated by endocrine hormones such as pituitary FSH and LH as well as by locally produced growth factors, including TGF-? superfamily members activin, inhibin, and bone morphogenetic proteins (BMPs) (reviewed in Ref. 1). BMPs were originally identified by their ability to induce bone and cartilage formation (2). However, numerous studies have revealed that BMPs have a wide variety of effects on many cell types, including monocytes and epithelial, mesenchymal, and neuronal cells, and play pivotal roles in cytodifferentiation, morphogenesis, and organogenesis (3). Members of the TGF-? superfamily, including BMPs, transduce their signals through binding to type I and II serine/threonine kinase receptors. BMP signaling is mediated intracellularly by the phosphorylation of receptor-activated Smads (R-Smads) 1, 5, and 8. Activated R-Smads complex with the common partner Smad 4 and translocate to the nucleus where they initiate BMP-stimulated alterations in target gene expression. Signaling of TGF-? superfamily members including BMPs is also modulated by soluble extracellular proteins such as noggin, chordin, and gremlin. In addition, membrane-associated proteins, including betaglycan (TGF-? type III receptor), endoglin, and crypto are also critical for assisting with ligand binding to receptor or for altering receptor specificity (reviewed in Refs. 4, 5, 6).
The mRNAs encoding BMP2, 3, 3b, 4, 6, 7, and 15 have been identified in mammalian ovaries. Moreover, BMP receptors BMPRIA, IB, and II are widely expressed in the ovary, with the strongest expression in the granulosa cells and oocytes of developing follicles in normally cycling rats (7, 8). BMPs and their receptors are also expressed in uterine stroma and glandular epithelium (9). In males, BMP2, BMP4, and BMP8A and BMP8B are expressed in germ cells, and BMP4, BMP7, and BMP8A are expressed in the epididymis (reviewed in Ref. 8). Moreover, mice deficient in BMP4, BMP8A, or BMP8B show germ cell degeneration in the testis and/or epithelial cell degeneration in the epididymis (10, 11, 12). Together, these results suggest that BMPs may play important roles in regulating reproduction.
Dragon was identified through a genomic screening strategy for genes regulated by DRG11, a homeobox transcription factor that is expressed in embryonic dorsal root ganglion (DRG) (13). Independently, this gene was also cloned as RGMb, one of three mouse homologues of the chicken repulsive guidance molecule (RGM) (14). The Dragon gene encodes a 436-amino-acid glycosylphosphatidylinositol (GPI)-anchored protein, suggesting it may be associated with lipid rafts within the plasma membrane. Indeed, adhesion of DRG neurons to HEK293 cells was increased after transfection of HEK293 cells with Dragon cDNA (13). Dragon is expressed in a number of neural tissues including embryonic and adult mouse DRGs, spinal cord, and brain (13, 15, 16). Interestingly, Dragon is also involved in BMP signaling because 1) injection of Dragon mRNA into Xenopus embryos induced expression of a number of BMP-regulated genes, 2) Dragon binds directly to BMP2, BMP4, and BMP receptors, and 3) transfection of Dragon cDNA into BMP-responsive cells enhances transcription of a BMP-responsive reporter (17). These observations indicate that Dragon acts as a BMP coreceptor that regulates cellular response to BMP signals.
To understand the potential role of Dragon in BMP signaling within the reproductive tissues, we examined Dragon expression in murine reproductive tissues and cell lines. We found that Dragon is expressed and dynamically regulated in gonadal germ cells as well as in epithelial cells of the reproductive tract including epididymis and uterus. Dragon is also expressed in the pituitary. As predicted from its being anchored to the cell membrane by a GPI anchor, Dragon is indeed localized in lipid rafts where it enhances BMP2 and -4 signaling. Taken together with the overlapping expression and function of BMPs in the reproductive system, our results indicate that Dragon may play an important role in reproduction through enhancement of BMP signaling.
Materials and Methods
Animals
All animal studies were conducted in accordance with an animal use protocol approved by the Massachusetts General Hospital animal use committee in accordance with U.S. Department of Agriculture guidelines. Mice [B6C3F1 (C57BI/6 x C3H)] were maintained in the animal barrier facility and killed at different ages to collect tissues for immunohistochemistry and RNA extraction. In addition, mice at 19 d of age were injected with pregnant mare serum gonadotropin (PMSG; ip, 5 IU/mouse; Sigma Chemical Co., St. Louis, MO), and killed 48 h later to collect gonads for immunohistochemistry.
RT-PCR
Total RNA was extracted from tissues stored in RNAlater (Ambion, Austin, TX) or cells stored in Trizol (Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s protocol. Total RNA (0.5–1.0 μg) was reverse transcribed as previously described (18). Aliquots (2 μl) of first-strand cDNA mix were used in PCR (35 cycles) to amplify Dragon and ?-actin. The primers, which amplify both human and mouse Dragon cDNA, were TGT TCC AAG GAT GGA CCC ACA TC (forward) and GCA GGT CAT CTG TCA CAG CTT GG (reverse).
Immunohistochemistry and immunocytochemistry
A rabbit polyclonal antibody was raised against a peptide corresponding to the C terminus of Dragon upstream of its GPI anchor. This antibody has been shown to specifically recognize Dragon protein (13).
Immunohistochemistry on paraffin sections were performed as previously described (19). Briefly, ovaries were fixed in Bouin’s solution, and other organs were fixed in 4% paraformaldehyde. Antigen retrieval was performed on paraffin sections in 0.01 M citrate buffer (pH 6.0). Tissue sections were incubated overnight with anti-Dragon (1:4000), washed, incubated for 1 h with biotinylated goat antirabbit IgG and then 30 min with Vectastain Elite ABC (Vector Laboratories, Inc., Burlingame, CA), and developed with diaminobenzidine (DAB) for detection (ICN Biomedical, Inc., Aurora, OH). Sections were then counterstained with Harris’ hematoxylin.
To colocalize Dragon and FSH in the pituitary, mouse pituitaries were fixed in 4% paraformaldehyde at 4 C overnight, cryoprotected in 30% sucrose overnight, and frozen in Tissue-Tek OCT embedding compound (Electron Microscopy Sciences, Fort Washington, PA). Sections (12 μm) were incubated with a mixture of rabbit anti-Dragon serum (1:2000) and guinea pig antimouse FSH (1:1600, AFP-3080, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for 1 h, washed, and then with a mixture of fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG and tetramethyl rhodamine isothiocyanate-conjugated donkey anti-guinea pig IgG (diluted 1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
For immunocytochemistry, Ishikawa cells were grown on glass coverslips. Live cells were incubated with anti-Dragon serum (1:2000) for 1 h on ice. Cells were then fixed in 2% paraformaldehyde for 20 min. The bound antibodies were detected by incubating with FITC-conjugated donkey antirabbit IgG (diluted 1:200 in PBS) for 1 h. To demonstrate specificity, the Dragon antibody was preincubated overnight with 10 mM immunization peptide before being applied to sections or cells.
In situ hybridization
Air-dried frozen sections (14–18 μm) were fixed in 4% paraformaldehyde-PBS, digested with proteinase K, acetylated, washed, and dehydrated. Antisense and sense cRNA probes were prepared by means of in vitro transcription in the presence of [-35S]UTP, which were then hybridized in 50% deionized formamide, 10 mM Tris/HCl (pH 7.6), 600 mM NaCl, 0.25% SDS, 200 μg/ml yeast tRNA, 50 mM dithiothreitol, 1x Denhardt’s solution, and 10% dextran sulfate overnight at 55 C in a humidified chamber. After hybridization, the sections were incubated with ribonuclease A and washed in 0.1x SSC containing ?-mercaptoethanol (875 μl in 300 ml) and 0.5 mM EDTA at 65 C for 1 h. After developing, the slides were counterstained with hematoxylin and mounted for photography.
Lipid raft protein extraction
Lipid raft proteins were prepared after protocols previously described (20, 21). Briefly, Ishikawa cells were scraped and pelleted in ice-old PBS, resuspended in 2 ml ice-cold lysis buffer [10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and proteinase inhibitors], and allowed to stand on ice for 30 min. The lysate was centrifuged for 5 min at 1300 x g to remove nuclei and large cellular debris. The supernatant was mixed with an equal volume of 85% sucrose in TBS [10 mM Tris/HCl (pH 7.5), 150 mM NaCl], placed at the bottom of a 10-ml ultracentrifuge tube, and then overlaid with 5 ml of 35% sucrose and 1.4 ml of 5% sucrose. The sample was then centrifuged for 14 h at 150,000 x g in a SW41Ti rotor so that rafts could float to the tip while cytoskeletal and cytoplasmic proteins remain at the bottom. Five fractions of 1 ml and four fractions of 1.35 ml were collected from the top of the tube. The light-scattering band, an indicator of the location of lipid rafts (20), was located primarily in fraction 2. These fractions were analyzed by trichloroacetic acid precipitation of proteins from 100-μl aliquots followed by SDS-PAGE and Western blotting.
Western blotting
Western blotting analyses were performed as previously described (19). Briefly, samples from sucrose gradient fractions were subjected to SDS-PAGE under reducing conditions, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), blocked in 10% nonfat dry milk, and incubated overnight at 4 C with rabbit anti-Dragon (1:4000) or anti-caveolin-1 (1:2000; BD Biosciences, San Jose, CA) antibodies. The membranes were washed three times before being incubated for 2 h at room temperature with second antibody, which was detected with enhanced chemiluminescence (ECL) Reagent Plus (PerkinElmer Life Sciences, Boston, MA). After exposure, membranes were stripped for 30 min at 50 C and reprobed with a monoclonal ?-actin antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA).
Transfection and luciferase assay
Ishikawa and KGN cells were maintained in TT medium [1:1 mixture of DMEM and F-12, supplemented with 1% L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin sulfate, and 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD)]. To examine the effect of Dragon on BMP signaling, transfections were performed in 24-well trays using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with a total of 400 ng DNA [180 ng BRE-Luc, a BMP response element kindly provided by Dr. ten Dijke (22), 10 ng pRL-TK, and the indicated doses of Dragon cDNA and pcDNA3]. Approximately 24 h after transfection, the medium was replaced with serum-free TT medium supplemented with 0.1% BSA, with or without BMP ligands (R&D Systems, Minneapolis, MN). After treating for 16 h, the cells were lysed and assayed for luciferase activity using the dual luciferase reporter assay kit (Promega, Madison, WI).
To obtain noggin protein for neutralization of endogenous BMPs, the human noggin cDNA (IMAGE clone 4737725 from American Type Culture Collection, Rockville, MD) was subcloned into pcDNA3 (Invitrogen) and transfected into HEK-293-F suspension cultures in Freestyle serum-free medium (Invitrogen) as previously described (23). Concentrated conditioned medium was calibrated by two independent methods including 1) biological assay and 2) Western blotting. In the biological assay, increasing amounts of noggin-conditioned medium were mixed with 10 ng/ml BMP2 and used to treat HepG2 cells that were previously transfected with the BRE-Luc reporter. At the effective concentrations for half-maximal response (EC50), the amount of noggin in the culture medium was equal to that of the BMP2 concentration, assuming molar equivalent antagonism activity (24). For the Western blotting analysis, serial dilutions of noggin-conditioned medium were resolved by 12% PAGE under reducing conditions and visualized by staining with an antimouse noggin polyclonal antibody (R&D Systems). Noggin concentrations were estimated at the detection limit dose according to the manufacturer’s information. Both methods gave similar results. As a control, concentrated conditioned medium from mock-transfected HEK293 cells was tested and determined to have no BMP-inhibitory activity.
To demonstrate that Ishikawa cells transfected with Dragon cDNA actually produce Dragon protein, transfected cells were extracted in RIPA buffer [150 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 50 mM NaF, 0.5% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS], and the lysates were then analyzed by Western blotting for Dragon as described above.
Data analysis
Figure 6, A, C, and D, depicts the mean ± SE of triplicates from representative experiments. In vitro bioassay experiments in the presence or absence of transfected Dragon (Fig. 6D) represents the mean ± SE of six determinations from three independent experiments and were analyzed by two-way ANOVA. Differences between ligand doses or between presence and absence of Dragon were identified by Student-Newman-Keuls post hoc test. Differences of P < 0.05 were considered significant.
FIG. 6. Dragon enhances cellular response to BMPs in cell lines derived from reproductive tissues. A, Ishikawa or KGN cells were transiently transfected with BRE-Luc reporter in combination with increasing doses of Dragon cDNA and assayed for luciferase activity. Transfection with Dragon increases BRE-Luc response in the absence of exogenous ligand. Values are ratios of BRE-Luc to pRL-TK and are mean ± SE of triplicates from representative experiments. Dragon protein is detectable by Western blot in Ishikawa cells after transfection with 100 ng cDNA. The membrane was stripped and reprobed with ?-actin antibody as a control for loading (inset). B, Ishikawa cells were transfected with BRE-Luc reporter and Dragon cDNA and treated with BMP2 alone or together with noggin. Values are ratios of BRE-Luc to pRL-TK and are mean ± SE of triplicates from representative experiments. C, Control medium was used in parallel with noggin-conditioned medium to demonstrate the specificity of the inhibitory activity of noggin-conditioned medium in BMP2 signaling. Although noggin inhibited BMP2 signaling, control conditioned medium had no inhibitory effect. D, Ishikawa cells transiently transfected with BRE-Luc reporter and Dragon (0 or 10 ng) in 24-well plates were incubated with increasing doses (0–2800 pM) of BMP2. Values are fold increases of luciferase activity in treated cells relative to untreated cells and are the means ± SE of six determinations from three independent experiments in duplicate. Asterisks indicate significant differences between transfected and untransfected cells at each BMP2 dose: **, P < 0.01; *, P < 0.05.
Results
Expression of Dragon mRNA in reproductive organs
Dragon expression in mouse reproductive tract tissues was examined by RT-PCR (Fig. 1). Dragon mRNA was detected in the testis, epididymis, and seminal vesicles in males and in the ovary, uterus, and pituitary in females.
FIG. 1. Dragon mRNA expression in mouse tissues. RNA extracted from various mouse tissues was examined for Dragon mRNA expression by RT-PCR. In tissues where Dragon expression was low, ?-actin was used as a control for cDNA quality. Dragon expression was strongly detected in testis (Te), ovary (Ov), pituitary (Pit), epididymis, uterus (Ut), kidney (Kid), and brain (Br). Weaker Dragon signals were detected in seminal vesicles (SV) and adrenal (Ad).
Cellular localization of Dragon in the testis and epididymis
Immunohistochemical analyses of d 1 and 3 mouse testes showed that Dragon was localized to gonocytes both in the center and at the basement membrane of seminiferous tubules (Fig. 2A, a and b). In testes from d 9 mice, spermatogonia at the basement membrane were positive for Dragon expression (Fig. 2A, c). However, Dragon staining in spermatogonia became much weaker in testes from 21-d-old mice (Fig. 2A, d). Interestingly, a few gonocytes, which remain in the central region of the tubules from d 21 testes, were strongly stained with Dragon (Fig. 2A, d). Some interstitial cells showed weak staining in d 1 testes (Fig. 2A, a), but no staining was observed in interstitial cells in older testes. In adult (d 60) testis, Dragon was expressed in spermatocytes and round spermatids, whereas spermatogonia and Sertoli cells did not appear to express Dragon (Fig. 2A, e and f). The staining in gonocytes, spermatogonia, and spermatocytes was completely abolished when the antiserum was preincubated with the competing immunizing peptide, demonstrating the specificity of the antiserum (Fig. 2A, g and h). Dragon expression in spermatocytes and round spermatids from adult testes was confirmed by in situ hybridization (Fig. 2C).
FIG. 2. Cellular localization of Dragon in the mouse testis and epididymis during postnatal development by immunohistochemistry and in situ hybridization. For immunohistochemistry, all sections were stained with DAB (brown) and counterstained with hematoxylin (blue). Images are shown at lower (x40) or higher (x100) magnification. A, Immunolocalization of Dragon in testes at d 1 (D1, a), d 3 (D3, b), d 9 (D9, c), d 21 (D21, d), and d 60 (D60, e and f). For negative control, sections were incubated with Dragon antibody preincubated with competing immunizing peptide (g and h). Dragon is highly expressed in gonocytes and spermatogonia in testes of newborn mice and spermatocytes and round spermatids in testes of adult mice. Dragon is not expressed in spermatocytes of d 21 testes. Spermatogonia are weakly stained in d 21 testes but not stained in adult testes. B, Immunostaining of d 21 testes with (b) and without (a) PMSG (5 IU) injection 2 d earlier. Immunostaining in spermatocytes is turned on by PMSG administration. C, Localization of Dragon mRNA in mouse testes by in situ hybridization. Bright (left) and dark (right) field images are shown. D, Immunolocalization of Dragon in d 3 (D3, a–c) and d 60 (D60, d–f) epididymis. Caput (a and d), corpus (b and e), and cauda (c and f) were dissected.
Spermatocytes of d 21 testes were not stained with Dragon antibody, whereas those cells of adult testes were strongly stained (Fig. 2A, d and e). Interestingly, Dragon was highly expressed in spermatocytes of testes collected after 2 d of PMSG administration to 19-d-old mice (Fig. 2B), suggesting that Dragon expression levels are hormonally regulated.
In d 3 epididymis, Dragon protein was found on both the apical and basal sides of epithelial cells with staining in the apical side stronger compared with the basal side. Dragon staining was stronger in caudal than in caput or corpus epididymis of 3-d-old mice (Fig. 2D, a–c). In contrast, Dragon expression was primarily localized to the apical side of epithelial cells of adult epididymis, and it appeared that Dragon was more highly expressed in caput or corpus, compared with caudal epididymis (Fig. 2D, d–f). These results suggest that Dragon may be involved in regulation of spermatogenesis and epididymal epithelial function.
Cellular localization of Dragon in the ovary and uterus
Within the adult mouse ovary, Dragon protein was detected exclusively within oocytes (Fig. 3A, a, b, and d) and was more intense in oocytes from secondary follicles compared with antral follicles (Fig. 3A, a). In contrast, no Dragon staining was found in oocytes of primordial (Fig. 3A, b) or primary (Fig. 3A, c) follicles, nor in somatic cells of any follicles (Fig. 3A). In atretic follicles, oocytes showed weak Dragon staining (Fig. 3A, d). There was no staining of the ovarian surface epithelium (Fig. 3A, b–d). Oocyte staining was completely blocked by preincubating antiserum with competing immunizing peptide (Fig. 3A, e). In d 9 ovaries, Dragon staining was detected only in the oocytes of secondary follicles but not in the primordial or primary follicles (Fig. 3A, f). PMSG treatment had no effect on Dragon staining in oocytes (data not shown).
FIG. 3. Cellular localization of Dragon in the mouse ovary and uterus by immunohistochemistry and in situ hybridization. For immunohistochemistry, all sections were stained with DAB (brown) and counterstained with hematoxylin (blue). Images are shown at lower (x40) or higher (x100) magnification. A, Dragon immunostaining in ovaries at d 60 (D60, a–d) and d 9 (D9, f). The staining in oocytes of secondary follicle (arrow) is stronger than that in oocytes of antral (arrowhead) and atretic (curved arrow) follicles (a and d). Dragon is not expressed in oocytes of primordial (open arrow) and primary (triangle) follicles (b and c). For negative control, sections were incubated with Dragon antibody preincubated with competing immunizing peptide (e). B, Localization of Dragon mRNA in mouse adult ovaries by in situ hybridization. Dark (b and d) and bright (a and c) field images are shown. Signals are confined to oocytes, and signals are stronger in the secondary (arrow) than in antral (arrowhead) follicles (a and b). No signals are seen in primary follicles (triangle, c and d) and in corpus luteum (CL). C, immunohistochemistry of the mouse uterus showing protein expression of Dragon (a and b), and negative control (c), incubation with Dragon antibody preincubated with competing immunizing peptide. The solid arrow indicates luminal epithelial cells of endometrium, the arrowhead indicates glandular epithelial cells of endometrium, and the open arrow shows circular muscle.
Consistent with immunostaining, Dragon mRNA, as detected by in situ hybridization, was stronger in oocytes from secondary follicles (Fig. 3B, arrows) compared with antral (Fig. 3B, arrowheads) follicles and was undetectable in oocytes from primary follicles (Fig. 3B, triangle). Dragon mRNA was not detectable in ovarian somatic cells. These results suggest that Dragon may regulate the development of oocytes and follicles by influencing the interaction between the oocyte and granulosa cells.
In the uterus, Dragon protein was expressed in luminal and glandular epithelial cells of the endometrium (Fig. 3C, a and b). Weak staining was also found in circular muscle (Fig. 3C, a). Localization of Dragon in the luminal and glandular epithelial cells suggest Dragon may also be required for normal endometrial function.
Cellular localization of Dragon in the pituitary
In the pituitary, sporadic staining was observed in both the anterior and posterior lobes, whereas no staining was detected in the intermediate lobe (Fig. 4A). Because BMPs and their receptors are expressed in mouse pituitary gonadotropes, as well as in the L?T2 gonadotrope cell line (25, 26), and BMPs can stimulate FSH biosynthesis (27), we examined whether FSH-expressing gonadotropes also express Dragon. To this end, frozen pituitary sections were dual labeled with Dragon and FSH antibodies. FSH-expressing cells indeed overlapped extensively, albeit not completely, with Dragon-expressing cells (Fig. 4B). Interestingly, L?T2 cells also express Dragon (Fig. 5A). These results suggest that Dragon may influence BMP-mediated FSH biosynthesis in vivo and in vitro.
FIG. 4. Immunolocalization of Dragon in the pituitary and colocalization of Dragon and FSH. A, Immunostaining for Dragon (brown) in the mouse pituitary (paraffin sections). Sections were incubated with Dragon antibody (a–c) or with Dragon antibody preincubated with competing immunizing peptide (d–f). AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe. B, Colocalization of FSH? and Dragon in the mouse anterior pituitary (frozen sections). Dragon (green) and FSH? (red) were detected in the anterior pituitary. Double-labeled cells (yellow, arrows) indicate Dragon expression in the mouse pituitary gonadotrope. Note that some FSH?-positive cells are negative for Dragon staining and some Dragon-positive cells are negative for FSH staining.
FIG. 5. Expression of Dragon in cell lines derived from reproductive tissues and localization of Dragon into lipid rafts. A, RT-PCR analyses of Dragon mRNA in cell lines. ?-Actin was used as a control for cDNA quality. B, Immunochemical localization of Dragon in Ishikawa cells. The live unfixed cells were incubated with rabbit anti-Dragon serum on ice and then fixed in 2% paraformaldehyde. The bound antibodies were detected by incubating with FITC-conjugated donkey antirabbit IgG. C, Localization of Dragon in raft-enriched fractions prepared from Ishikawa cells. Cells were extracted using a buffer containing 1% Triton X-100. The lysate was mixed with 85% sucrose, sequentially layered with 35 and 5% sucrose, and centrifuged for 14 h at 150,000 x g. Nine fractions were collected and analyzed for Dragon, caveolin-1, and ?-actin by Western blot. The bands specific for Dragon are marked with asterisks.
Dragon expression in cell lines of the reproductive axis
In screening cell lines originating from reproductive organs (Fig. 5A), we found that Dragon was expressed in Hela (cervical carcinoma), MCF-7 (breast carcinoma), L?T2 (pituitary carcinoma), JEG3 (placenta carcinoma), and Ishikawa (endometrium adenocarcinoma) cells. In contrast, Dragon mRNA was undetectable in S4 spermatogonial cells (28) or KGN granulosa tumor cells (29).
Lipid raft localization of Dragon
We explored the location of Dragon on the cell surface using Ishikawa cells as a model. Live cells were incubated with Dragon antibody at 4 C and then fixed and processed for immunocytochemistry. Dragon appeared to have a punctate pattern on the cell membrane, which is typical of lipid raft proteins (Fig. 5B). To further characterize Dragon localization within membrane subdomains, cells were extracted on ice in the presence of 1% Triton X-100 and then subjected to sucrose gradient ultracentrifugation. As expected, Dragon was detectable primarily within the low-density fractions (Fig. 5C; fractions 2 and 3), along with caveolin-1, which is typically associated with lipid rafts. The high-density fractions, which include cellular and cytoskeletal proteins (fractions 6–9), contained ?-actin, some caveolin-1, and a small amount of Dragon. These results indicate that Dragon is indeed located within lipid rafts in Ishikawa cells.
Dragon enhances signaling of BMP2 and BMP4
Our results demonstrate that Dragon is expressed in gonadal germ cells and in reproductive tract epithelial cells. Moreover, we have previously shown that Dragon enhances the BMP2 response in the HepG2 liver cell and LLC-PK1 kidney cell lines (17). To examine whether Dragon has a similar role in reproductive cells, Ishikawa and KGN cells were transfected with Dragon cDNA together with BRE-Luc, a BMP-responsive luciferase reporter construct. Dragon dose-dependently increased BRE luciferase activity in both Ishikawa and KGN cells in the absence of added BMPs (Fig. 6A). Although Dragon induced similar reporter activity in both cell lines at lower doses (0.1–10 ng), it was more effective in Ishikawa cells at higher doses. Although Dragon protein was undetectable in Ishikawa cells before transfection, Dragon was detectable after transfection with 100 ng cDNA (Fig. 6A, inset), indicating that the effects of transfected Dragon are mediated by an increase in Dragon protein.
Treatment of Ishikawa cells with noggin-containing conditioned medium (100 ng/ml) resulted in partial inhibition of Dragon-dependent BRE-Luc activity and completely inhibited signaling by BMP2 (10 ng/ml) (Fig. 6B). Conditioned medium from mock-transfected HEK293 cells had no inhibitory activity (Fig. 6C), indicating that the BMP-inhibiting activity in the noggin-conditioned medium was caused by noggin itself. These results also suggest that the effect of Dragon on BMP reporter activity is dependent on endogenous ligand. We next examined the effect of Dragon on BMP signaling in the presence of added BMP ligands. Ishikawa cells were transfected with Dragon cDNA and treated with increasing doses of BMP ligands. Dragon significantly increased BMP2 signaling at 11–700 pM BMP2 doses (P < 0.05), but had no effect at 2800 pM (Fig. 6D). Thus, at lower BMP2 doses (i.e. 11 or 44 pM), Dragon transfection resulted in detectable reporter activity that is not seen in the absence of Dragon (Fig. 6D). Similar results were observed with BMP4 and in KGN cells (data not shown). These results indicate that Dragon acts to increase sensitivity of BMP-responsive cells to low concentrations of endogenous or exogenous BMP ligands.
Discussion
Although originally discovered as a GPI-anchored cell surface protein involved in neuronal differentiation and cell-cell contact in the developing nervous system (13), it is now evident that Dragon can also act as a coreceptor for BMP2 and BMP4 (17). In addition to previously reported sites of expression in neural tissues (13, 17), our studies demonstrate that Dragon is also expressed in many specific cell types throughout the reproductive system. In addition, we found that Dragon was expressed in numerous cell lines derived from reproductive tissues and that Dragon expression enhanced responsiveness of Ishikawa and KGN cells to BMP2 and BMP4. Thus, our results suggest that Dragon may have important roles in mediating BMP signaling in reproduction.
To define specific sites where Dragon-mediated BMP signaling might be important in reproduction, we explored cell-specific expression in the male and female reproductive tracts. In males, BMP8a and BMP8b are expressed in maturing spermatocytes, and BMP8b knockout males are infertile because of developmental arrest and degeneration of spermatocytes (11, 12), suggesting that these BMPs are critical for normal spermatocyte development. BMP receptors ALK3 and BMPR-II are localized in postnatal spermatogonia, and BMP4 is produced by Sertoli cells very early in postnatal development, consistent with an ongoing requirement for BMP signaling in the testis (30). In addition, BMP2 primarily stimulates spermatogonial proliferation, whereas BMP7 acts mainly on Sertoli cells in the testis from 7-d-old mice (31). Our results extend these findings to a novel BMP coreceptor that enhances BMP signaling, because in 3-d-old male mice, Dragon was highly expressed in gonocytes before and after they migrated from the tubule lumen to their basal position, with this immunoreactivity being maintained as spermatogonia in d 9 animals. By d 21, staining in spermatogonia was substantially diminished, but gonocytes remaining within the tubule lumen were still positive. In adults, Dragon staining appeared in maturing spermatocytes but not in other testicular cell types. The shift in expression from gonocytes and spermatogonia in juvenile animals to spermatocytes in mature males suggests that the role of BMPs may change as the testes mature to produce active sperm. Taken together, these results suggest a critical role for BMPs in regulating testis development and spermatogenesis and suggest that Dragon may be an important mediator of these processes.
BMP4, BMP7, and BMP8A are expressed in the epididymis, and knockout of each gene by itself resulted in degeneration of the epididymal epithelium (10, 11, 12). These results indicate a role for BMPs in the control of epididymal function. Interestingly, Dragon was strongly expressed on the apical surface of polarized epididymal epithelium in immature and mature males consistent with a role for Dragon in enhancing this essential BMP signaling.
In females, both BMPs and their receptors have been identified in numerous ovarian cell types, including oocytes and granulosa cells (8). In vitro studies have demonstrated that BMP2, 4, 6, 7, and 15 regulate granulosa cell functions, and BMP4 and 7 promote the primordial-to-primary follicle transition during follicle maturation (reviewed in Ref. 8). The significance of BMP signaling in ovarian function is also underscored by the altered ovulation rates in Inverdale sheep with a natural point mutation in the BMP15 gene (32) and in Booroola sheep with a point mutation in the BMPRIB gene (33). Our results demonstrate that in the ovary, Dragon is expressed exclusively in oocytes and most prominently in oocytes within secondary follicles. This is a time of oocyte growth and cytoplasmic maturation (34), suggesting that BMP signaling in general, and Dragon enhancement of this signaling in particular, may be important for growth and maturation of oocytes.
BMP signaling components are expressed in a variety of cells within the rat uterus (9). BMP2 mRNA is restricted to periluminal stroma, and BMP7 is expressed in periluminal stroma and glandular epithelial cells, whereas BMP4 and BMP6 are expressed in blood vessels in the uterus. BMPRIA, BMPRIB, and BMPRII are expressed in a number of cell types in the uterus including luminal and glandular epithelial cells. We observed Dragon expression in luminal and glandular epithelial cells of the mouse endometrium, suggesting that Dragon may enhance BMP signals involved in regulating uterine maturation in preparation for implantation.
BMP2, 4, 6, 7, and 15 are expressed in mouse pituitary, and BMP6, 7, and 15 have been shown to stimulate FSH synthesis and secretion (25, 27, 35). BMP6 and BMP7 can also stimulate FSH mRNA biosynthesis in L?T2 mouse pituitary cells in culture (27). We observed Dragon expression in L?T2 cells as well as in numerous cells within the mouse pituitary, some of which also stained for FSH. These results suggest that BMPs may act in an autocrine manner to modulate FSH biosynthesis and that Dragon may enhance this process.
In cell culture studies using cell lines from the reproductive tract, we found that Dragon expression enhanced the response to endogenous BMP ligand as well as low doses of exogenous BMP2 and BMP4, results that agree with our earlier observations in nonreproductive cell lines (17). Although the precise mechanism for this signaling enhancement has not yet been fully elucidated, our immunocytochemical analysis indicates that Dragon is located on the plasma membrane in discrete patches, consistent with its belonging to the class of GPI-anchored proteins that are known to localize in lipid rafts (36). Moreover, our earlier studies indicate that Dragon can interact directly with BMPRII, ActRII, and the Alk3 and Alk6 type I receptors (17). Taken together, these results suggest a model for enhanced BMP signaling in which Dragon acts as a BMP coreceptor in collecting type II and type I receptors into lipid rafts where they are optimized to respond to low doses of BMP ligands. Because Dragon can bind BMP2 and BMP4 directly, it is also possible that Dragon acts to stabilize the ligand-receptor complex in lipid rafts, thereby facilitating endocytosis and signaling. Of course, these two possibilities are not mutually exclusive. Based on the localized expression of Dragon in developing and maturing germ cells, as well as specific epithelial cells within the reproductive tract that are known to be BMP responsive, our results support the concept that BMPs play an important role in regulating reproduction in mammals and that this role may be regulated by Dragon.
Acknowledgments
Dr. Ernestina Schipani and Janet Saxton provided expertise and access to critical facilities for the immunocytochemical and in situ hybridization analyses. Dr. Carla Boitani provided helpful suggestions for germ cell classifications in the testis from newborn mice. Cell lines were generously provided by Dr. Martin Dym (S4 spermatogonial cells), Dr. Pamela Mellon (L?T2 cells), and Dr. Hajime Nawata (KGN cells). Dr. Peter ten Dijke provided the BRE-Luc reporter.
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