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BOR - Papers in Press, published online ahead of print June 6, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062190
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BIOLOGY OF REPRODUCTION 77, 533–542 (2007)
DOI: 10.1095/biolreprod.107.062190
© 2007 by the Society for the Study of Reproduction, Inc.

Dysferlin Is Expressed in Human Placenta But Does Not Associate with Caveolin1

Dale D Vandré 3, William E Ackerman, IV 4 , Douglas A Kniss 4, Arun K Tewari 3, Miki Mori 5, Toshihiro Takizawa 5, and John M Robinson 2 3

Departments of Physiology and Cell Biology3 and Obstetrics and Gynecology,4 The Ohio State University, Columbus, Ohio 43210 Department of Molecular Anatomy,5 Nippon Medical School, Tokyo 113-8602, Japan

ABSTRACT

A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained ~4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed ~3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.

caveolin, dysferlin, placenta, syncytiotrophoblast, trophoblast

INTRODUCTION

The human placenta mediates the exchange of materials between the mother and developing fetus throughout gestation. In addition, the placenta serves as a barrier to the maternal immune system and as a source of hormones that support the endocrine, metabolic, and growth factor needs of pregnancy. Disorders in placental structure and/or function underpin many severe complications of pregnancy, including preeclampsia, fetal growth restriction, and alloimmunization disease such as rhesus isoimmunization.

By virtue of its highly invasive hemochorial arrangement, maternal blood enters the human placenta and bathes its villous surface, the syncytiotrophoblast (STB). This epithelium-like syncytium, derived from and maintained by fusion of underlying mononuclear cytotrophoblasts (CTB), comprises a single cell layer in thickness and has a large surface area due to amplification by microvilli on its apical surface [1]. The apical plasma membrane of the STB is the initial site for the uptake of small molecules (e.g., amino acids, glucose), water, metabolic gases, ions, vitamins, and lipids as well as macromolecules [2–7]. In spite of its importance, a detailed understanding of this plasma membrane, at the molecular level, is not available currently.

To enhance our knowledge of the membrane biology of the apical surface of the placenta, we have embarked on a directed series of studies aimed at obtaining high-quality proteomics data on the apical plasma membrane of the STB. Key features of our approach have been the development of methods for (A) enrichment of microvilli from the STB and (B) depletion of non-plasma membrane proteins from the microvilli, thereby further enriching the plasma membrane proteins (our unpublished data).

In the course of these studies, we have begun an analysis of the proteome of microvilli and the apical plasma membrane of the STB. When a subset of proteins from these fractions was submitted for proteomics analysis, a number of predicted microvillar and plasma membrane proteins were identified; several unpredicted proteins were identified as well. Among the proteins of the latter group was dysferlin (DYSF), a 230-kDa transmembrane protein with homology to fer-1 in Caenorhabditis elegans. In humans, mutations in the DYSF gene have been associated with the development of limb girdle muscular dystrophy type 2B and Myoshi myopathy [8, 9], and an important role for DYSF in the repair of the damaged plasma membrane (sarcolemma) in skeletal muscle fibers has been elucidated [10, 11]. Published evidence for the expression of DYSF in cells other than skeletal muscle is scant; therefore, identification of DYSF in human placenta is noteworthy.

A functional role for caveolins in trafficking DYSF to the plasma membrane has been proposed [12]. Caveolins are small membrane proteins that are critical components of plasma membrane microdomains known as caveolae. Caveolin 1 (CAV1) and caveolin 2 (CAV2) can form hetero-oligomeric complexes [13] that are found in many cell types in mammals, whereas caveolin 3 (CAV3) is muscle specific [14]. Like DYSF, mutations in CAV3 have been associated with a number of muscle pathologies [15–17], and interactions between DYSF and CAV3 have been reported [18]. A missence mutation in CAV3 (CAV3P104L) leads to abnormally low levels of DYSF in the sarcolemma [18]. When CAV3P104L is expressed in nonmuscle cells, this mutant form of CAV3 is largely retained in the Golgi complex and does not reach the cell surface [19]. A recent report shows that the mutant CAV3P104L or another mutant (CAV3R26Q), when cotransfected with epitope-tagged DYSF, results in retention of epitope-tagged DYSF in the Golgi, with little DYSF reaching the plasma membrane. A similar effect was observed with the heterologous expression of DYSF and a CAV1 mutant (CAV1P132L) [12].

In the present study, we document for the first time the presence of DYSF protein in the human placenta, describe its localization within the organ, compare its distribution and expression levels during placental development, and provide initial insights into the study of placental DYSF in an in vitro setting. In addition, we present data indicating that DYSF traffics to the STB plasma membrane independently of caveolin, suggesting a mechanism differing from the situation in skeletal muscle. Although the role of DYSF in the human placenta is unknown presently, this study provides the underpinnings for future functional analyses of placental DYSF.

MATERIALS AND METHODS

Reagents and Supplies

A murine monoclonal antibody to DYSF was purchased from Vector Laboratories (Burlingame, CA). A rabbit polyclonal antibody to CAV1 and a monoclonal antibody to CAV3 were obtained from BD Transduction Laboratories (San Jose, CA). A monoclonal antibody to placental alkaline phosphatase (PLAP) was purchased from Sigma-Aldrich (St. Louis, MO). An antipeptide antibody to caveolin1{alpha} (CAV1{alpha}) was produced in chicken and has been characterized previously [20–23]. Fluorochrome-labeled goat secondary antibodies were obtained from Molecular Probes (Eugene, OR). Horseradish peroxidase-labeled donkey secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).

Ludox CL colloidal silica, poly (acrylic acid-co-maleic acid), sodium salt, MES buffer, sorbital, gelatin, Medium 199, protease inhibitor cocktail (cat. #P8340), Histodenz, and Percoll were obtained from Sigma-Aldrich. Dispase, penicillin/streptomycin, and fetal calf serum were obtained from Invitrogen (Carlsbad, CA). BCA protein determination and SuperSignal chemiluminescent kits were from Pierce Biotechnology (Rockford, IL). Criterion precast polyacrylamide gradient gels were from Bio-Rad Laboratories (Hercules, CA). Fuji Super RX x-ray film and tissue freezing medium for cryosectioning were obtained from Fisher Scientific (Pittsburgh, PA). Other reagents and supplies were as we have described previously [20, 24].

Tissue Collection

Human term (39–41 wk) placentas were obtained with informed consent according to a protocol approved by the Biomedical Sciences Institutional Review Board at Ohio State University, Columbus, OH. Tissue samples from uncomplicated cesarean deliveries were used. In addition, first-trimester as well as term placental tissues were obtained according to a protocol approved by the Nippon Medical School Hospital Ethics Committee, Tokyo, Japan. In the latter setting, placental tissues were obtained from elective termination of pregnancy and uncomplicated Cesarean deliveries. In all cases, tissue was processed as soon as possible following collection (within 20 min). Placental tissue was either fixed or lysed immediately (see below) or flash frozen in liquid nitrogen and stored at –80°C until used. The number of placentas used in this study was as follows: (A) Columbus = 9 and Tokyo = 4 (term) and (B) Columbus = 0 and Tokyo = 4 (first-trimester).

Skeletal muscle (gastrocnemius) was obtained from male Sprague-Dawley rats (~250 gm) (Harlan, Indianopolis, IN) following decapitation. The use of these animals was approved by the Institutional Animal Care and Use Committee of the Ohio State University. The excised tissue was cut into small pieces, flash frozen in liquid nitrogen, and stored at –80°C until used. Alternatively, small pieces of tissue were fixed and prepared for immunocytochemical analysis (see below).

Preparation of Placental Microvilli

The cationic colloidal silica method for isolation of plasma membranes from cultured cells [25, 26] was modified and applied to the placenta. Fresh term placentas were cut into segments (~12 cm x 8 cm) and the basal plate was then dissected away from these segments while the chorionic plate was retained. After washing in several changes of cold PBS, the tissue was washed in two changes of cold MES buffer (20 mM MES [2-(4-morpholino)-ethane-sulfonic acid], 150 mM NaCl, 280 mM sorbitol; pH 5.25) and then incubated in cold MES buffer containing 1% Ludox CL cationic colloidal silica for 3 min. Following coating, the tissue was washed in two changes of MES buffer and then incubated in cold MES buffer containing 1 mg/ml of polyacrylic acid for 3 min to cross link the silica particles. After washing in MES buffer, small pieces of tissue were dissected (60–80 g) and then homogenized with a Fisher PowerGen 700 homogenizer (Fisher Scientific) in homogenization buffer (25 mM Tris-HCl, 125 mM NaCl, 5 mM MgCl2; pH 7.4) containing protease inhibitor cocktail. The homogenate was filtered through Nitex mesh prior to centrifugation at 900 x g at 4°C to pellet nuclei and silica-coated microvilli. The pellet was resuspended in homogenization buffer and mixed with an equal amount of 100% Histodenz. A step gradient was set up as follows: (A) a 65% Histodenz cushion, (B) an overlay of 60% Histodenz, (C) another overlay of 55% Histodenz, (D) the homogenate in 50% Histodenz, and (E) the homogenization buffer. The tubes were centrifuged at 65 000 x g for 22 min at 4°C in an SW 28.1 rotor in a Beckman ultracentrifuge (Beckman Instruments, Palo Alto, CA). The silica-coated microvilli pellet was resuspened in homogenization buffer and centrifuged again as described above. The resulting pellets were washed three times in homogenization buffer and the final pellet was flash frozen in liquid nitrogen and stored at –80°C until used.

Proteomics Analysis

Detailed proteomics analyses of placenta microvilli and the apical plasma membrane of the STB will be reported in subsequent studies. The proteomics analysis presented in this study focuses on DYSF and was carried out on pellets of enriched microvilli (see above) that had been extracted in sodium carbonate (pH 11) for 30 min at 4°C to reduce non-plasma membrane proteins in the sample [27]. Membrane proteins remaining associated with the pH 11-treated membranes were extracted with 1% SDS and then separated on 1-D SDS-PAGE gradient gels (Bio-Rad). Selected regions of the stained gels were excised and subjected to automated overnight tryptic digestion using a Proteome Works MassPrep Station (Perkin-Elmer). The tryptic digests were analyzed by tandem mass spectrometry at the Keck Proteomics facility at Yale University.

Isolation and Culture of Cytotrophoblasts

Cytotrophoblasts were isolated from term placenta with the trypsin/dispase/DNase digestion method of Kliman et al. [28] as modified by Nelson and colleagues [29, 30]. For immunofluorescence microscopy (IFM), cells were plated onto round glass coverslips that were previously coated with 0.1% gelatin in 24-well culture plates and cultured in Medium 199 supplemented with 10% fetal calf serum and penicillin/streptomycin. The cells were cultured at 37°C in 5% CO2; coverslips were collected at set times and prepared for immunocytochemical analysis (see below). For immunoblot analysis, cells were grown on 60-mm tissue culture dishes at a density of ~300 000 cells/cm2 in the same medium. In each case, the medium was changed 4 h after initial plating and daily thereafter.

Preparation of Tissue and Cell Lysates

Fresh placental tissue or placental tissue flash frozen in liquid nitrogen and stored at –80°C was cut into small pieces and lysed using one of three different lysis systems. These were (A) boiling in 1% SDS in 10 mM Tris buffer for 10 min; (B) octylglucoside lysis buffer (150 mM Na2PO4, 60 mM n-octyl ß-D-glucopyranoside, 10 mM D-gluconic acid lactone, 1mM EDTA, 0.02% NaN3) containing protease inhibitor cocktail; or (C) Tris lysis buffer (25 mM Tris-HCl, 125 mM NaCl, 5 mM MgCl2; pH 7.4) containing 1% Triton X-100, 0.1% SDS, 1 mM PMSF, and protease inhibitor cocktail; the latter two lysis procedures were carried out on ice for 20 min. Rat muscle tissue was lysed with either 1% SDS in Tris lysis buffer or octylglucoside lysis buffer in the same manner as placental tissue. Cytotrophoblast cell culture extracts were prepared with Tris lysis buffer. The lysates were centrifuged in a microfuge to remove cellular debris; the protein concentration was determined with the BCA method.

Immunoblot Analysis

Proteins from placenta, muscle, or CTB extracts were resolved by SDS-PAGE using continuous (7% or 15% acrylamide) or gradient (8%–16% acrylamide) gels. Proteins separated by gel electrophoresis were transferred electrophoretically to nitrocellulose membranes. Membranes were washed in Tris-buffered saline containing 0.2% Tween 20 (TBST), incubated for 2 h at 22°C with 5% nonfat milk in TBST, and then incubated overnight at 4°C with primary antibodies in TBST/milk. The antibody dilutions were mouse anti-DYSF, 1:8000; chicken anti-CAV1{alpha}, 1:5000; rabbit anti-CAV1, 1:5000; or mouse anti-CAV3, 1:1000. After washing in TBST, membranes were incubated with species-appropriate HRP-labeled secondary antibodies (diluted 1:5000) in TBST/milk for 1 h at 22°C. Antibody binding was detected using chemiluminescence and x-ray film.

Immunocytochemistry and Microscopy

Conventional IFM of paraformaldehyde-fixed tissue samples was carried out on cryosections (5 µm in thickness) as we have described previously [20, 31]. The sections were then incubated with anti-DYSF (1:200 or 1:400 dilution), anti-CAV3 (2.5 or 5.0 µg/ml), chicken anti-CAV1{alpha} (1:250 or 1:500 dilution), or rabbit anti-CAV1 (1:100, 1:250, 1:500, or 1:1000) for either 30 min at 37°C or overnight at 4°C. The sections were subsequently washed and then incubated with fluorochrome-labeled secondary antibodies diluted 1:200 for 30 min at 37°C or 60 min at 22°C. After washing in PBS, the nuclei were stained with DAPI for 10 min before mounting in the antiphotobleaching agent ProLong. Double-label IFM assays were conducted by simultaneously applying two different primary antibodies to the same sections and incubating as described above. Subsequently, species-specific fluorochrome-labeled secondary antibodies were also simultaneously applied to tissue sections. In control preparations, the primary antibodies were omitted, whereas the secondary antibody incubation was retained. For DYSF localization, cryosections were washed, incubated with 0.5% SDS in PBS for 5 min at 22°C, and rinsed six times in PBS as we have described previously [32, 33]. Also in some cases, sections were treated with 0.2% Triton X-100 in PBS for 5 min at 22°C and then rinsed six times in PBS.

Trophoblasts cultured on gelatin-coated glass coverslips were fixed in 4% paraformaldehyde (PFA) in PBS for 1 h at 22°C. The cells were then washed six times in PBS and permeabilized with either 0.2% Triton X-100 or 0.5% SDS in PBS for 5 min or left unpermeabilized. Cells were then incubated for the immunocytochemical localization of DYSF or PLAP as described above for tissue sections. The dilution for anti-DYSF was 1:400; for anti-PLAP, 1:1000. The control reaction was as described for tissue sections.

Ultrahigh-resolution IFM was carried out on ultrathin cryosections as we have described previously [21, 24, 34]. Ultrathin cryosections (70–100 nm in thickness) were cut with a Leica ultramicrotome EM UC6b equipped with an FC6 cryounit (Leica, Wetzlar, Germany). Ultrathin cryosections were incubated with primary and secondary antibodies and DAPI as described for conventional cryostat sections (see above). Control incubations were also as described above.

Conventional fluorescence and differential interference contrast (DIC) images were collected with a Nikon Optiphot equipped with a Photometrics Cool Snap fx CCD camera (Roper Scientific) or an Olympus BX60 microscope equipped with a Spot RT SE6 CCD (Diagnostic Instruments, Sterling Heights, MI). In each case, images were captured with the MetaMorph image analysis system (Universal Imaging, Dowingtown, PA). All images were collected within the linear response range of the CCD camera. A Zeiss 510 META laser scanning confocal microscope (Carl Zeiss, Inc., Thornwood, NY) was also used to examine sections of placental tissue.

Fluorescence intensity measurements comparing DYSF labeling in first-trimester and term placentas were made using the MetaMorph software system. A total of 65 images from first-trimester and 117 images from term placentas were used in this analysis; sectioned profiles of multiple villi were present in each image. Terminal, intermediate, and stem villi were analyzed in term placentas. Fluorescence intensity measurements comparing the DYSF labeling in apical and basal plasma membranes of the STB were made using confocal images and the Zeiss LSM 5 software. A total of 25 images containing several villi in each image were used in this analysis. Measurements for each comparison group were done together to minimize variation in excitation illumination.

RESULTS

Documentation of Dysferlin in the Human Placenta by Proteomics Analysis

Cationic colloidal silica-coated fractions derived from placental STB were separated by 1-D SDS PAGE gradient gels, and selected regions were excised, digested with trypsin, and analyzed by tandem mass spectrometry. Five different peptides, widely distributed within the cytoplasmic domain of DYSF, were identified. These peptides and their location within the DYSF polypeptide chain are given in Table 1. These mass spectrometry results were validated using immunoblotting. Bands of apparently identical mobility (~230 kDa) were observed in both placental and skeletal muscle extracts when probed with anti-DYSF antibody (Fig. 1).


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  		TABLE 1 DYSF peptides identified by tandem mass spectrometry.


Figure 01
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  		FIG. 1 Expression of DYSF in human placenta. Immunoblot analysis of lysates from term placenta and from rat skeletal muscle shows that a major band of ~230 kDa was detected in each lysate when probed with a monoclonal antibody to DYSF. The skeletal muscle lysate served as a positive control for the placental sample.

Distribution of Dysferlin in the Human Placenta

To characterize the expression of DYSF in human placental cells, IFM was applied to conventional cryostat sections. In term specimens, we found that DYSF was highly expressed in the STB (Fig. 2A). Interestingly, DYSF was not detectable in most other cells of the placenta (i.e., smooth muscle, pericytes, or stromal cells [fibroblasts, Hofbauer cells]), with the exception of endothelial cells; endothelial labeling was essentially absent in some placental samples, whereas in others a low level of labeling in some blood vessels was observed. Quantitative fluorescence intensity measurements showed that DYSF labeling in the STB exhibited no regional variability, being equally prominent among all villous subdivisions (terminal, intermediate, and stem villi; Fig. 2B). In positive control sections, the anticipated localization of DYSF to the sarcolemma of muscle fibers was observed (Fig. 2C).


Figure 02
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  		FIG. 2 DYSF localizes primarily to the apical plasma membrane of the STB in placenta. A) IFM of cryostat sections of term human placenta probed with a monoclonal antibody to DYSF demonstrates that the major site for DYSF localization is the apical plasma membrane of the STB (arrows). Other cell types present in the tissue section were not labeled with anti-DYSF. B) Fluorescence intensity measurements of immunolabeled STB apical plasma membranes in terminal, intermediate, and stem villi show that DYSF is expressed at equivalent levels in all regions of the term placenta. C) IFM of cryostat sections of rat skeletal muscle probed with the same monoclonal antibody to DYSF that was used with placental tissue. Positive fluorescence is associated with the sarcolemma of the muscle fibers (arrows). Skeletal muscle served as a positive control for testing the specificity of the antibody. Bars = 20 µm.

Within the STB, DYSF appeared to be prominent within the apical plasma membrane, with lesser amounts in the basal plasma membranes. To quantify this difference, we applied fluorescence intensity measurements to sections of immunolabeled placental tissue visualized by confocal microscopy (Fig. 3). A total of 100 separate measurements for the apical membranes and 100 corresponding measurements for the basal membranes were used. Based on this analysis, we found that DYSF expression was 3.9-fold higher in the apical plasma membrane relative to the basal surface. These results suggest that DYSF undergoes polarized trafficking with the STB, with preference given to apical distribution.


Figure 03
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  		FIG. 3 Comparison of DYSF labeling in the apical and basal plasma membranes of the STB. Confocal microscopy images of immunolabeled cryostat sections of placenta were used to determine the fluorescence intensity associated with the apical and basal plasma membranes of the STB. In this representative image, the apical plasma membrane (double arrows) and the basal membranes (single arrows) are indicated. Low-level labeling of endothelial cells was present in this section; the asterisk (*) indicates the vessel lumen. Bar = 10 µm.

Distribution of Dysferlin in the Human Placenta: First Trimester Versus Term

The issue of the temporal expression of DYSF was addressed by comparing first-trimester and term placentas in IFM assays. All procedures used for localization of DYSF and for collection of images were identical. Although DYSF was detected primarily at the apical surface of the STB in the first-trimester samples (Fig. 4A) as well as in the term samples (Fig. 4B), the intensity of the fluorescence signal was almost three times higher in the first-trimester sample than in the term samples (Fig. 4C).


Figure 04
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  		FIG. 4 Comparison of DYSF labeling in first-trimester and term placentas. A) IFM of a cryostat section of first-trimester human placenta probed with a monoclonal antibody to DYSF demonstrates that the major site for DYSF localization is the apical plasma membrane of the STB (arrows). B) IFM of a cryostat section of term placenta probed with the monoclonal anti-DYSF; the major site of DYSF localization was the apical plasma membrane of the STB (arrows). C) Comparison of fluorescence intensity measurements from immunolabeled sections of first-trimester and term placentas shows that first-trimester STB express about 3-fold more DYSF than does the STB from term placenta. Bar = 100 µm.

DYSF Traffics to STB Plasma Membranes in the Absence of Caveolin

It has been proposed that CAV3 participates in the trafficking of DYSF to the plasma membrane in skeletal muscle [12]. We therefore addressed whether caveolins are responsible for DYSF trafficking in the STB. By immunoblotting, we found that both the placenta and skeletal muscle express CAV1; in contrast, CAV3 expression was limited to skeletal muscle (Fig. 5).


Figure 05
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  		FIG. 5 Expression of caveolin in human placenta: immunoblot analysis. A) Immunoblot analysis of lysates from term placenta and from rat skeletal muscle probed with the antibody to CAV1{alpha}. A band of ~22 kDa is recognized in each of the lysates. B) Immunoblot analysis of lysates from term placenta and from rat skeletal muscle probed with the antibody to CAV3. Caveolin 3 is detected in muscle but not placental lysates. Three different placentas were used in this analysis (lanes 1–3).

By IFM, CAV1{alpha} in the placenta was present in endothelial cells, pericytes, smooth muscle cells, and stromal cells, but was not detected in the STB (Fig. 6A). In skeletal muscle, CAV1{alpha} was present in endothelial cells, but was not detected in muscle fibers (Fig. 6B). As expected, CAV3 was not detected in any cell type in the placenta (Fig. 6C), but was localized to the sarcolemma in skeletal muscle (Fig. 6D). The morphology of the tissue sections is also shown (Fig. 6, E and F). To ensure that neither the {alpha} nor ß isoforms of CAV1 were present in the STB, in addition to anti-CAV1{alpha} we utilized a second antibody recognizing both isoforms. Though these antibodies detected precisely the same structures in placenta sections in double-labeling experiments (not shown), neither of them labeled the STB (Fig. 6G). To test for low levels of expression of CAV1 in the STB, images were collected using exposure times that were five times longer than the optimal exposure; CAV1 was still undetectable in the STB even in these prolonged exposures (Fig. 6H). These results indicate that DYSF can reach the plasma membrane, both apical and basal, of the STB through a caveolin-independent mechanism.


Figure 06
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  		FIG. 6 Expression of caveolin in human placenta. A) IFM localization of CAV1{alpha} in term placenta. The fluorescence signal is found in endothelial cells (arrows) and stromal cells but was not detectable in the STB (double arrows). Lumens of some blood vessels are indicated with asterisks (*). B) IFM localization of CAV1{alpha} in skeletal muscle; the fluorescence signal was primarily in capillary endothelial cells (arrows) and was not detected in the sarcolemma of muscle fibers. C) The same section as shown in A that was incubated for the IFM detection of CAV-3 in placenta. No fluorescence signal specific for CAV3 localization was detectable. Arrows and asterisks serve as reference points. D) The same section as shown in B was incubated for the IFM detection of CAV3 in skeletal muscle. CAV3 was readily detected in the sarcolemma of all muscle fibers (double arrows). E) The same section as shown in A and C with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the tissue morphology. Arrows and asterisks serve as reference points. F) The same section as shown in B and D with the DIC image merged with the fluorescence image of DAPI-stained nuclei. Arrows serve as reference points. G) IFM detection of CAV1 using a rabbit antibody that detects both the {alpha} and ß isoforms of CAV1; an optimal exposure time was used to collect the image. In this section, CAV1 was detected in endothelial cells (single arrows) but not in the STB (double arrows). H) The same section as shown in G except that the image was purposefully overexposed (five times longer exposure than in G to test for low levels of expression of CAV1 in the STB. Even under these conditions, CAV1 was not detected in the STB (double arrows). The fluorescence signal from the endothelial cells was saturated (single arrows). Bars = 20 µm.

DYSF Is Not Expressed in Villous Cytotrophoblasts: High-Resolution IFM

Although IFM studies using conventional cryostat sections allowed us to determine that DYSF was expressed in the STB, these were of insufficient resolution to enable a definitive conclusion regarding DYSF expression in mononuclear villous CTB. To gain a more refined understanding of the distribution of DYSF in first-trimester and term placentas, we employed ultrathin cryosections (70–100 nm in thickness) as the immunolabeling substrate for high-resolution IFM. In first-trimester placentas, DYSF was localized predominantly to the apical plasma membrane of the STB, but was not detected in the underlying CTB or in stromal cells located interior to the CTB (Fig. 7A). Caveolin 1{alpha}, on the other hand, was not detected in the STB or in the CTB, but was present in an intracellular compartment in the stromal cells (Fig. 7B). Indeed, only low levels of CAV1{alpha} were detected in first-trimester placenta compared to term. Merged images show the tissue morphology (Fig. 7, C and D).


Figure 07
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  		FIG. 7 DYSF and caveolin in the placenta: analysis of ultrathin cryosections. A) First-trimester placental tissue sample labeled with antibodies to DYSF and CAV1{alpha}. DYSF was localized primarily to the apical plasma membrane of the STB (double arrows). Nuclei of the STB are indicated with asterisks (*). DYSF was not detected in CTBs (arrowheads) or in stromal cells (single arrows). B) The same section shown in A in which CAV1{alpha} was detected in perinuclear regions of stromal cells (single arrows) but not in CTBs (arrowheads) nor in the STB (double arrows). C) The same section shown in A and B with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the tissue morphology and to identify the CTBs. Arrows and asterisks serve as reference points. D) Merger of the green dysferlin signal (A) with the red CAV1{alpha} signal (B). There was no overlap of the red and green signals. Arrows and asterisks serve as reference points. E) Term placental tissue sample labeled with antibodies to DYSF and CAV1{alpha}. DYSF was localized primarily to the apical plasma membrane of the STB (double arrows). Nuclei of the STB are indicated with asterisks (*). Positive DYSF labeling is also observed in some endothelial cells (single arrows) but not in adjacent pericytes (p) or in CTBs (arrowhead). The lumens of some blood vessels are indicated (#). F) The same section shown in E in which CAV1{alpha} was detected in endothelial cells (single arrows) and pericytes (p) but not in CTBs (arrowhead) or the STB (double arrows). G) The same section shown in A and B with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the tissue morphology and to identify the CTBs. Arrows and asterisks serve as reference points. H) Merger of the green DYSF signal (A) with the red CAV1{alpha} signal (B). There was no overlap of the red and green signals. Arrows and asterisks serve as reference points. Bar = 10 µm.

In term placentas, DYSF predominantly localized to the apical plasma membrane of the STB, with lower levels in the basal plasma membrane (Fig. 7E), and once again DYSF-positive endothelial cells were observed in some cases. Importantly, DYSF was not observed in CTB of term placentas. As in the conventional sections, CAV1 was expressed at high levels in endothelial cells but was not detectable in the STB or CTB in ultrathin cryosections (Fig. 7F). Merged images show the tissue morphology (Fig. 7, G and H).

Restricted Expression of Dysferlin in Cultured Cytotrophoblasts

Cytotrophoblasts isolated from term placentas were cultured for various times then fixed in 4% PFA and subsequently probed by IFM for detection of PLAP or DYSF. As expected, isolated mononuclear CTB spontaneously fused to form syncytial structures [28, 35]. These structures typically ranged from small syncytia, containing from 4 to 6 nuclei, up to relatively large syncytia, containing from 60 to 70 nuclei, and all syncytia expressed the unique STB marker-enzyme PLAP, as determined by IFM (Fig. 8A). In addition to PLAP, DYSF was also detected in the cultured syncytia by IFM. In contrast, individual cells that had not fused with the syncytial structures did not label positively for the presence of dysferlin (Fig. 8C). The morphology of the cultured cells is shown (Fig. 8, B and D). By immunoblot analysis, DYSF was also detected in both cultured CTB and skeletal muscle (Fig. 8E), whereas skeletal muscle extracts contained CAV3, as expected, and this protein was not detectable in the CTB extracts (Fig. 8F).


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  		FIG. 8 Syncytial formation by cytotrophoblasts in vitro. A) Isolated CTB that were cultured for 4 days and then prepared for the IFM localization of PLAP. Mononuclear CTBs have fused together to form syncytial-like structures that express PLAP (green color). The fluorescence image DAPI-stained nuclei are indicated by blue color. Adjacent cells not yet fused with the syncytial-like structure do not express PLAP (arrows). B) The same cells shown in A with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the morphology of the syncytialized structure. Arrows serve as reference points. C) Isolated CTB were cultured for four days and then prepared for the IFM localization of DYSF. Mononuclear CTB have fused together to form syncytial-like structures that express DYSF (green color). The fluorescence image DAPI-stained nuclei are indicated by blue color. Adjacent cells not yet fused with the syncytial-like structure do not express DYSF (arrows). D) The same cells shown in C with the DIC image merged with the fluorescence image of DAPI-stained nuclei to show the morphology of the syncytialized structure. Arrows serve as reference points. E) Immunoblot analysis confirms the expression of dysferlin in cultured CTB. Lysates of cultured CTB (lane 1) and lysates of rat skeletal muscle (lane 2) were probed with the monoclonal anti-DYSF. Bands of the same apparent electrophoretic mobility were detected in cultured CTB and muscle. F) Immunoblot analysis failed to detect the expression of CAV3 in cultured CTB. Lysates of cultured CTB (lane 1) and lysates of rat skeletal muscle (lane 2) were probed with a monoclonal antibody to CAV3. Though present in skeletal muscle, CAV3 could not be detected in cultured CTB. Bars = 20 µm.

DISCUSSION

A positional cloning approach was originally used to identify the gene now known as DYSF [9]. Northern blot analysis using multiple human tissues revealed major transcripts in skeletal muscle, the heart, and the placenta; however, little or no signal was detected in the other tissues examined (lung, liver, kidney, and pancreas). Similar results were reported in another study [8]. The expression of DYSF protein in placenta was not determined in either of these studies, which focused on skeletal muscle; importantly, however, they indicate that DYSF is not expressed ubiquitously [8, 9].

For the first time, the expression of DYSF in the placenta has been examined in this report using proteomic, immunochemical, and immunolocalization methods. Through this analysis, we found that DYSF was exclusively located in the STB in the placenta, with the exception of occasional and variable labeling in the fetal placental endothelium. None of the other cell types in the placenta expressed detectable levels of DYSF. These results show that DYSF expression is cell-type specific in the placenta. Further, we showed that greater levels of DYSF expression are expressed in first-trimester placentas compared to term placentas.

A recent report using a cell-transfection strategy to study the interaction of DYSF and caveolin concluded that caveolin was necessary for the proper trafficking of DYSF to, or its retention at, the plasma membrane [12]. When mutated forms of CAV3 or CAV1 were transfected along with epitope-tagged DYSF into cultured cells, DYSF did not reach the plasma membrane efficiently but was retained in the Golgi complex. It was reported previously that though caveolins are expressed in the placenta, they are conspicuously absent from the STB [20, 21, 36]. Another report suggested that very low levels of CAV1 staining were present in the STB in immunohistochemical preparations of placenta, but morphological entities resembling caveolae were not detectable in the STB by electron microscopy [37]. An immunoelectron microscopy study reports that labeling for CAV1 in the STB was not statistically significantly different from labeling of the extracellular matrix, whereas endothelial labeling was significantly higher than either the STB or the matrix [38]. Taken together, all of these studies of the localization of CAV1 in human placenta indicate that there is very little, if any, CAV1 in the STB. However, the study reporting that caveolin functions to direct DYSF to the plasma membrane [12] prompted us to reexamine the distribution of caveolin in placenta using additional antibodies. The new results are consistent with our previous finding that caveolins are not expressed in the STB [20]. These results indicate that DYSF can reach the apical and basal plasma membrane of the STB through a caveolin-independent mechanism. Therefore, the recent proposal of Hernández-Deviez et al. [12], that proper trafficking of DYSF to the plasma membrane requires functional caveolin, does not appear to extend to the STB of the placenta.

The DYSF gene family contains the founding member fer-1 from C. elegans and the mammalian proteins DYSF, myoferlin, otoferlin, and Fer1L4. These proteins are characterized by having a single pass transmembrane domain, short extracellular domain, and a large intracellular domain. They are further characterized by having several C2 domains in the cytoplasmic portion of the protein. In response to calcium binding, the C2 domains can interact with negatively charged phospholipids and proteins [39]. It is this property that seems to be crucial in DYSF function in skeletal muscle. Two different studies concluded that DYSF function is crucial to repair damaged plasma membranes of skeletal muscle fibers in a calcium-dependent manner [10, 11], and each has proposed slightly differing models to explain DYSF function. One model envisions a pool of DYSF in muscle fiber plasma membranes (in association with annexin 1 and 2 and possibly other proteins) coexisting with a separate pool of DYSF-containing repair vesicles located beneath the plasma membrane. This model posits that following membrane injury, rapid entry of calcium into the muscle fiber cues the recruitment of DYSF-positive repair vesicles to the damage site where they fuse with the membrane forming a patch [40]. The second model differs in that it envisions DYSF being restricted to the plasma membrane, and DYSF-negative intracellular granules such as lysosomes serve to patch the damaged plasma membrane of the muscle fibers [11]. In support of this second model there is evidence from studies of nonmuscle cells that lysosomes can serve to patch damaged plasma membranes [41]. Though it is not yet known if DYSF performs a similar repair function in the placenta, our high-resolution IFM of ultrathin cryosections indicates that DYSF is localized only at the apical and basal plasma membrane of the STB; there is no evidence for an intracellular pool of DYSF-positive vesicles.

Cells that give rise to the placenta (trophoectoderm) are the first cells to differentiate following fertilization. The resulting trophoblasts generate the CTBs and the STB. The CTBs are progenitors that divide throughout pregnancy, forming daughter cells that fuse with the overlying STB. Fusion of CTBs with the STB continues throughout pregnancy and is necessary for the maintenance of the STB [42]. The placenta grows rapidly during the first several weeks of pregnancy, and numerous CTBs are located immediately underneath the STB. The unusual biology of the STB, with its rapid growth during early pregnancy due to the continual fusion of CTBs, suggests a possible function for DYSF. The CTBs fuse with the STB and empty their contents, cytoplasm, organelles, and nucleus into the STB. This accumulation of cellular material may apply pressure to the apical plasma membrane of the STB, making it more fragile and susceptible to damage. In addition, maternal blood is constantly moving over the apical surface of the STB, which may also lead to mechanical damage of this membrane. It is thus tempting to speculate that DYSF is present in high levels at the apical plasma membrane of the STB to participate in repair of this membrane following damage that may routinely occur. It should be noted that fusion of CTBs with the STB continues throughout pregnancy; it is estimated that the number of CTBs is approximately six times greater than needed for STB growth [43]. Thus, the issue of increased cellular content in the STB applies to later stages of pregnancy as well. The STB has developed a means to eliminate much of this excess material derived from CTBs. Nuclei from CTBs begin the process of apotosis after entering the STB. Ultimately apototic nuclei become clustered together into structures called syncytial knots; these structures are shed from the apical surface of the STB [44, 45]. It is estimated that there is a 3–4 wk interval between entry of a CTB nucleus into the STB and sheding in a syncytial knot [42]. The number of nuclei incorporated into the STB is enormous because the STB occupies a very large area; the surface expansion of the STB reaches ~12.5 m2 at term [42]. It has been estimated that a placenta of 12-wk gestation sheds the equivalent of 1 x 105 CTBs in 4.7 x 104 syncytial knots per day, whereas the average placenta of 9-mo gestation sheds 1.8 x 106 CTBs in 8.5 x 105 syncytial knots per day [46]. Based on these estimates, at least 1 x 108 syncytial knots will be shed over the entire gestational period. The mechanism by which this shedding occurs, and the means by which the plasma membrane is repaired following shedding, is not known at present; perhaps DYSF functions to repair the plasma membrane at sites where syncytial knots leave the apical surface of the STB in a mechanism analogous to the proposed role of DYSF in the repair of muscle fiber plasma membranes.

Functional studies to determine the role of DYSF in the intact human placenta in vivo cannot be conducted for obvious ethical reasons. In an effort to bypass this problem, we have begun initial studies to test the feasibility of using cells isolated from the term placenta to address this issue. It is known from pioneering work that mononuclear CTBs can be isolated from term placentas and maintained in cell culture [28]. Moreover, these isolated CTBs will spontaneously fuse to form syncytia-like structures. Our initial goal was to determine if isolated CTBs express DYSF in cell culture. Isolated CTBs fused to form syncytia of varying sizes in our hands, and expressed PLAP, the enzyme routinely used as the marker for the apical plasma membrane of the STB [47]. Dysferlin was also expressed in cultured CTBs, as evidenced by immunoblotting data; however, the cultured CTBs did not express CAV3. Using IFM we found that DYSF was expressed in syncytial structures derived from CTBs, but was undetectable in mononuclear cells adjacent to the syncytia. This appears analogous to the situation in vivo where, in first-trimester placentas, CTBs did not label for DYSF, whereas the adjacent STB was positively labeled. It is interesting to note that DYSF was not expressed in isolated myoblasts, but was expressed after they fuse to form myotubes in culture [48]. This apparent similarity between cultured CTBs and cultured myoblasts and myotubes is intriguing and merits future analysis. We have established that the cultured CTB system provides a platform for study to address the functional role of DYSF in the human placenta.

ACKNOWLEDGMENTS

We are grateful to the Campus Microscopy and Imaging Facility and the Davis Heart-Lung Research Institute Proteomics Facility at Ohio State University for assistance with sample preparations. We also thank Drs. Gen Ishikawa and Toshiyuki Takeshita of Nippon Medical School for their technical support.

FOOTNOTES

1Supported in part by the National Institutes of Health grants HD38764 (J.M.R) and HD49628 (W.E.A.) and the Ministry of Education, Culture, Sports, and Science grants-in-aid 16390479 and 16659457 (T.T.). Back

Correspondence: 2John M. Robinson, Department of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. FAX: 614 292 4888; e-mail: robinson.21{at}osu.edu

Received: 11 April 2007.

First decision: 4 May 2007.

Accepted: 5 June 2007.

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