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Vol. 18, Issue 6, 2296-2304, June 2007
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Departments of *Anatomy and Structural Biology and Obstetrics, Gynecology, and Women's Health, Albert Einstein College of Medicine, Bronx, NY 10461; and The Hospital for Sick Children, Toronto, ON M5G 1L7, Canada
Submitted February 20, 2007;
Accepted April 2, 2007
Monitoring Editor: A. Gregory Matera
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Clyman, 1963; Moricard and Moricard, 1964; Terzakis, 1965). It consists of several layers of tubular membrane cisternae in the nuclei of endometrial epithelial cells and is often associated with the nuclear envelope and nucleoli, hence the name (for a review see Spornitz, 1992). This organelle appears during a 3- to 4-d window in the midsecretory phase of the menstrual cycle when the endometrium is receptive to implantation of the fertilized egg (Clyman, 1963; Gordon, 1975). Oral contraceptives and intrauterine devices interfere with the timing and/or overall appearance of the NCS (Wynn, 1967; Feria-Velasco et al., 1972; Azadian-Boulanger et al., 1976; van Santen et al., 1988; Dockery et al., 1997). Several cases of unexplained primary infertility have been characterized by the absence or delayed development of NCSs as the only ultrastructural alteration in endometrial epithelial cells (Kohorn et al., 1972; Gore and Gordon, 1974; Dockery et al., 1996). This and other correlative evidence suggests a role for the NCS in the preparation of the uterine surface for embryo implantation and points to the NCS as a long sought after marker for human receptivity. Nevertheless, despite this tantalizing data and the detailed ultrastructural description of the NCS, it has resisted molecular characterization. Here we provide first insights as to the composition of this enigmatic organelle.
On the basis of their remarkable ultrastructural resemblance to the NCS, we previously characterized membrane systems, R-rings, that are induced in nuclei of tissue culture cells by overexpression of the nucleolar protein Nopp140 (Isaac et al., 1998; Isaac et al., 2001). Nopp140 functions as a chaperone for small nucleolar ribonucleoproteins (snoRNPs) in the nucleolus and Cajal bodies and has been implicated as a transcription factor (Meier and Blobel, 1992, 1994; Miau et al., 1997; Isaac et al., 1998; Chen et al., 1999; Yang et al., 2000; Chiu et al., 2002; Wang et al., 2002). Nevertheless, the presence of Nopp140 in R-rings suggests its physical involvement in their biogenesis. R-rings, like the NCS, consist of several layers of tubular membrane cisternae embedded in an electron-dense matrix and are often associated with nucleoli and the nuclear envelope. However, unlike the composition of the NCS, that of the R-rings is well defined. They consist of bona fide endoplasmic reticulum (ER), harboring integral membrane and luminal markers of both smooth and rough ER, but are devoid of nuclear envelope–specific structures, such as nuclear pore complexes and lamina. The electron-dense matrix of R-rings contains all markers tested from the dense fibrillar component and the fibrillar centers of nucleoli, in particular Nopp140, and its associated snoRNPs, but is devoid of proteins from the granular component, e.g., nucleolin and B23 (Isaac et al., 2001). Despite this detailed molecular definition, however, it is unclear how the soluble Nopp140 can induce membrane systems in the normally membrane-free nucleus. We now demonstrate how Nopp140, aided by calcium, induces R-rings and explore if and how this biogenesis extends to the NCS of human endometrium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), provided the endometrial biopsies for these studies. Biopsies were obtained at specified times within the menstrual cycle, based on previous cycle length (for follicular phase samples) and luteinizing hormone (LH) surge detection for postovulatory samples. A Pipelle catheter was inserted into the uterus and gently rotated as it was withdrawn under negative pressure. This method yielded intact tissue cylinders with preservation of tissue architecture. A portion of the tissue was fixed in 4% methanol-free paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) and processed for paraffin embedding. A small amount of formaldehyde-fixed tissue was stored in 2% paraformaldehyde at 4°C for use in cryosectioning.
Cryosectioning and Immunostaining of Endometrial Tissue
Endometrial tissue was fixed in 4% paraformaldehyde in neutral phosphate-buffered saline. Cryosectioning was done by the method of Tokuyasu (Tokuyasu, 1973; Griffiths et al., 1983b, 1984). Briefly, tissue was infiltrated after fixation using 2.3 M sucrose as a cryoprotectant. After mounting on cryopins and freezing in liquid nitrogen, 90-nm (ultrathin) cryosections were cut using a Leica UCT cryoultramicrotome and placed on nickel grids (Deerfield, IL). For immunostaining, sections were blocked in 1% powdered milk in phosphate-buffered saline and then stained by human Nopp140 antiserum RS8 at a dilution of 1:2 for 2 h followed by 10-nm gold-labeled secondary antibodies for 1 h. Human Nopp140 antibodies were raised in rabbits (Covance Research Products, Madison, WI) against bacterially expressed antigen (Isaac et al., 2001). Sections were contrasted in 0.2% uranyl acetate and dried in a film of 2% methylcellulose before being observed by transmission electron microscopy.
Electron Microscopy
For ultrastructural analysis, transfected tissue culture cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, and dehydrated through a graded series of ethanol, and the monolayer was scraped up and embedded in Epon resin. Ultrathin sections (90 nm) were cut on a Reichert Ultracut E microtome (Vienna, Austria), stained with uranyl acetate followed by lead citrate and viewed on a JEOL 100CXII transmission electron microscope (Peabody, MA) at 80 kV. Endometrial tissue was fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and processed as above. For electron spectroscopic imaging, specimens were prepared without using heavy metals such as osmium tetroxide and embedded in the resin Quetol 651 (Electron Microscopy Sciences). Electron spectroscopic imaging was performed as previously described (Dellaire et al., 2004) by using a Tecnai 20 transmission electron microscope (FEI, Eindhoven, The Netherlands) equipped with an electron imaging spectrometer (Gatan, Pleasanton, CA).
Glucose-6-Phosphatase Assay
The histochemical localization of glucose-6-phosphatase was adapted from published protocols (Wachstein and Meisel, 1956; Griffiths et al., 1983a). Briefly, 2-mm pieces of secretory endometrial tissue were fixed in 0.5% glutaraldehyde in 100 mM PIPES buffer, pH 7.0, containing 5% sucrose. After fixation, tissues were placed in an incubating medium made by dissolving 0.19 g of glucose-6-phosphate in 10 ml of 80 mM Tris-maleate buffer, pH 6.5, and slowly adding 80 µl of a 12% lead nitrate solution. After incubation for 4–12 h at room temperature, tissues were washed in Tris-maleate buffer, dehydrated, and processed for Epon resin embedding for transmission electron microscopy. The assay was done identically on monolayers of COS-1 cells that were transfected with the Nopp140 repeat domain to induce R-ring formation, with an incubation time of 2 h at room temperature.
Tissue Culture, Transfection, and Calcium Overlay
COS-1 cells were cultured in high-glucose DMEM (Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen). U2OS cells were cultured in low-glucose DMEM containing 10% FBS. Cells were grown on 12-mm coverslips and transiently transfected using FuGene 6 (Roche, Indianapolis, IN) for 30 h according to manufacturer's protocol. The following constructs were used: HA-hNopp140 (pNK59, full-length human Nopp140 [Isaac et al., 2001] was cloned into pSVM [Isaac et al., 1998]); mGFP-Sec61
(Snapp et al., 2003); and HA-hNopp
N (pNK53, human Nopp140 lacking its 59 amino terminal residues was amplified and cloned into pSVM). For BAPTA-AM treatment, cells were treated with 10 µM BAPTA-AM (Sigma, 10 mM stock solution in DMSO) for 30 h. For calcium overlay assays, proteins were separated by standard SDS-PAGE, transferred to nitrocellulose membranes, and probed with 45Ca as described (Maruyama et al., 1984). After exposure for autoradiography, the membranes were stained with amido black. The calcium-binding protein TRAP (formerly SSR) was a kind gift from G. Migliaccio (Instituto di Ricerce Biologia Moleculare, Rome, Italy) (Migliaccio et al., 1992).
Immunofluorescence and Antibodies
Cells were fixed, permeabilized, and processed for double immunofluorescence as previously described (Isaac et al., 1998). The following primary antibodies were used at the dilutions indicated in parentheses: anti-HA ascites fluid (12CA5 at 1:200; Wilson et al., 1984) and anti-calnexin polyclonal serum (SPA860 at 1:200; StressGen, San Diego, CA). Imaging was performed at room temperature using a 60x/1.4 NA planapo objective on an inverted microscope (1x 81; Olympus, Melville, NY) containing automatic excitation and emission filter wheels connected to an air-cooled charge-coupled device camera (Sensicam QE; Roper Scientific, Tucson, AZ) run by IPLab Spectrum software (Scanalytics, Billerica, MA). Images were processed for contrast and brightness using Photoshop CS2 (Adobe, San Jose, CA). When indicated, images were deconvolved using Hazebuster software (VayTek, Fairfield, IA) quick deconvolution method.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Interestingly, the apical plasma membranes of some of the epithelial cells have lost their microvilli and are bulging into the lumen, reminiscent of pinopodes (Figure 1A, double asterisks). Pinopodes have been proposed as ultrastructural markers for receptivity and have been observed near or at the site of blastocyst attachment (Martel, 1981; Nikas et al., 1995; Bentin-Ley et al., 1999; Nikas, 1999a,b). Therefore, NCSs appear at the right time and place to play a role in endometrial receptivity, although it should be noted that recently pinopodes have been questioned as markers for the implantation window (Petersen et al., 2005; Quinn et al., 2007).
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; Griffiths et al., 1983a). As expected, in tissue culture cells transfected with the Nopp140 repeat domain for R-ring induction, lead phosphate precipitates specifically in the cytoplasmic ER, the contiguous nuclear envelope (NE), and in R-rings (Figure 2A). A higher magnification micrograph reveals all precipitates in the lumen of these organelles consistent with the intraluminal active site of this membrane-bound enzyme (van Schaftingen and Gerin, 2002; Figure 2B). Similarly, in endometrial biopsies, lead phosphate precipitates in the cytoplasmic ER, the nuclear envelope, and, importantly, the NCS (Figure 2C). Note the specificity of the labeling of only the ER but not mitochondria (mito), which are surrounded by a single layer of ER, a hallmark of the epithelial cells of this stage of the cycle (Verma, 1983). The presence of lead phosphate precipitates, and therefore glucose-6-phosphatase, in the lumen of the NCS cisternae identifies this organelle as ER derived (Figure 2D). Significantly, glucose-6-phosphatase is the first unequivocally identified molecular component of the NCS.
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). These membrane cisternae are generated in nuclei of tissue culture cells by exogenous expression of the repeat domain of Nopp140. Additionally, full-length rat Nopp140 induces intranuclear ring-shaped structures that are membranous based on their staining with the lipophilic dye DiOC6 at the light microscopic level (Isaac et al., 2001). It came therefore as a surprise, when these structures proved to be mostly electron-dense aggregates devoid of any discernable membranes at the ultrastructural level (not shown, but see Figure 3B, asterisks). Because the NCS is human specific and absent from all other species tested, we asked if full-length human Nopp140 would behave differently. Full-length human Nopp140, like its rat counterpart, accumulated in intranuclear ring-shaped structures when overexpressed (Figure 3A, I). Some of these contained the ER integral membrane marker calnexin (Figure 3A, II). Indeed, on an ultrastructural level, some of these structures are bona fide R-rings adjacent to or engulfed by nucleoli (Figure 3B, arrow), whereas some appear as electron-dense aggregates like those induced by rat Nopp140 (Figure 3B, asterisks). Identical results were obtained with the human Nopp140 alpha and beta isoforms, which differ by the inclusion of a 10-amino acid exon in the repeat region of Nopp140 alpha (Pai and Yeh, 1996). Therefore, full-length human Nopp140 induces R-rings consisting of intranuclear membrane cisternae reminiscent of the human-specific endometrial NCSs.
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). Therefore we investigated more directly if and how the soluble Nopp140 causes the formation of R-rings from the inner nuclear membrane. In addition to forming R-rings, transfected Nopp140 frequently accumulates in patches at the nuclear envelope suggestive of R-ring initiation at the nuclear periphery (Figure 4A). To monitor if R-rings indeed bud from the nuclear envelope, we performed live imaging experiments on cells whose ER was labeled by transient transfection of an integral ER membrane marker tagged with monomeric green fluorescent protein (GFP), mGFP-Sec61. When these cells were concomitantly transfected with the Nopp140 repeat domain to induce R-rings, membranous extensions developed from the nuclear envelope resulting in a globular ER structure inside the nucleus (Figure 4B). Over time these structures grew (Figure 4B, compare insets in II and III). These R-rings often remained attached to the nuclear envelope through a stalk (Figure 4B, arrows). In fact, similar stalks coming off the nuclear envelope with attached R-rings were identified in electron micrographs of cells transfected with full-length human Nopp140 (Figure 4C). These findings, supported by previously published data (Isaac et al., 2001), suggest that R-rings are a direct derivative of the inner nuclear membrane and that Nopp140, through its presence, plays a physical role in their initiation.
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), is a highly charged domain consisting of alternating positively and negatively charged repeats. Positively charged stretches of amino acids can bind directly to the head groups of phospholipids as in the case of myosin and other proteins with polybasic clusters (Doberstein and Pollard, 1992; Reizes et al., 1994; Areas et al., 1998; Heo et al., 2006). Alternatively or in addition, negatively charged amino acids, such as in the highly phosphorylated Nopp140 repeats, can interact with phospholipids via calcium bridges as in the case of synaptogamin I, PKC
, and dextran sulfate (Huster and Arnold, 1998; Zhang et al., 1998; Huster et al., 1999; Nalefski et al., 2001). Because the lumen of the ER and the nuclear envelope is highly enriched in calcium and because R-rings contain calcium channels, such as the IP3 receptor (Isaac et al., 2001), we tested if Nopp140 is a calcium-binding protein by using a blot overlay technique (Maruyama et al., 1984). For this purpose, we prepared Western blots from supernatants and pellets of immunoprecipitations with Nopp140 peptide antibodies in the presence and absence of free competing Nopp140 peptide, using low salt rat liver nuclear extracts (Figure 5A). Under these conditions, Nopp140 is precipitated and partially removed from the supernatant only in the absence of competing peptide (Figure 5A, lanes 1/2 and 6/7) but not in its presence (lanes 3/4 and 8/9). For visualization of calcium-binding proteins, the nitrocellulose membranes were first probed with 45Ca followed by autoradiography (Figure 5A, lanes 1–5) and subsequently for general visualization of proteins with amido black (lanes 6–10). The major calcium-binding protein in these extracts is a band of 140 kDa (Figure 5A, lane 3) that is identified as Nopp140 (arrowhead) by its precipitation (lane 2) and partial removal from the supernatant (lane 1) in the absence but not presence of competing peptide (lanes 3 and 4, respectively). In addition to Nopp140, only one or two minor, lower molecular weight calcium-binding proteins are detected in the extract (Figure 5A, lane 3, asterisk). Purified translocon-associated protein (TRAP) was included as a positive control on the blots, because only the alpha but not the beta form binds calcium (Wada et al., 1991; Hartmann et al., 1993; Figure 5A, compare lanes 5 and 10). To test if phosphorylation of Nopp140 was required for its calcium binding, the calcium overlay assay was performed on phosphorylated Nopp140 purified from rat liver and unphosphorylated recombinant Nopp140. Indeed, phosphorylated, but not unphosphorylated, Nopp140 bound calcium avidly (Figure 5B). We conclude that Nopp140 binds calcium in a phosphorylation-dependent manner.
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Complexed Calcium in the Matrix of R-Rings and NCSs
Calcium should be complexed between the phosphate groups of Nopp140 and those of the phospholipids in the membrane if it is involved in membrane invagination in this manner. Such enriched calcium phosphate complexes, unlike the soluble calcium stores within the ER, can be detected using electron spectroscopic imaging. This electron microscopic technique is based on the principle that each element in a sample absorbs characteristic amounts of energy from the incident electron beam. The loss of energy of the incident electrons can be analyzed by an electron spectrometer, which is also capable of forming an image with electrons of a particular energy loss. Thereby, the spectrometer produces an element-specific image (Dellaire et al., 2004). For this purpose, cells transfected with the repeat domain of Nopp140 for R-ring induction were embedded in Quetol resin, and ultrathin unstained sections were observed using an electron microscope equipped with an electron spectrometer capable of electron spectroscopic imaging. In the resulting images, R-rings were most highly enriched in complexed calcium when compared with any surrounding structure, e.g., heterochromatin or the nuclear envelope (Figure 6A, I). However, phosphorus was more enriched in heterochromatin than in R-rings, validating the specificity of calcium imaging (Figure 6A, II). When viewed at higher magnification, complexed calcium was enriched in the electron-dense matrix between the membrane tubules of the R-rings (Figure 6A, III) where Nopp140 is concentrated (Isaac et al., 2001). Similarly, phosphorus was enriched at the same location (Figure 6A, IV). This was not surprising given the accumulation of the highly phosphorylated Nopp140 and its associated small nucleolar ribonucleoproteins (Isaac et al., 2001). In summary, these data support a calcium-mediated Nopp140-membrane association.
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Nopp140 in the NCS
Using immunogold labeling on LR White–embedded tissue culture cells, we previously demonstrated the abundance of Nopp140 in the electron-dense matrix of the R-rings (Isaac et al., 2001). However, immunogold labeling of endometrial tissue sections proved more challenging. Only the combination of mild fixation conditions (4% paraformaldehyde) and ultrathin cryosectioning preserved the Nopp140 epitope sufficiently for detection by immunogold labeling. Although Nopp140 was readily detected in the dense fibrillar component of nucleoli (Figure 7, A and B), the NCS was only labeled to a minor extent (Figure 7, A and C) or not at all (Figure 7B). Therefore, despite their striking ultrastructural resemblance, the NCS differs from the R-rings in molecular composition. In particular, its content of complexed calcium and Nopp140 is lower, indicating that NCS induction, unlike that of R-rings, is not a mere consequence of increased Nopp140 expression but that additional factors are involved in NCS biogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Terzakis, 1965; Spornitz, 1992). We now identify glucose-6-phosphatase as a first molecular marker for these unusual organelles, suggesting that they are ER derived. This finding adds to a number of unique features of these organelles, e.g., they are membrane tubules in the otherwise membrane-free nucleus, they are human-specific and apparently absent even from baboon endometrium (MacLennan et al., 1971), they are transient structures appearing for only 3 to 4 d, their appearance correlates with the receptive phase of the menstrual cycle, and now they appear to represent intranuclear ER. Despite all these intriguing characteristics, the function of the NCS remains obscure. Nevertheless, the newly gained information may provide a reason for its existence. Thus, the ER in the nucleus may provide membrane surface in addition to the nuclear envelope for signaling from the nucleus to the ER and beyond. Or, these intranuclear membranes might be sequestering inhibitory molecules from the cell surface, thus facilitating implantation. Alternatively, these secretory cells are simply overproducing ER membranes (although this is not evident in the cytoplasm) that for some reason end up in the nucleus. It is also possible that the functionally important part of these structures is their electron-dense matrix, which we now show to contain some Nopp140 and complexed calcium. In that scenario, the membranes would merely provide a framework for the matrix. Which ever it is, the intriguing correlation of their appearance with the readiness of the endometrial surface for blastocyst attachment warrants their further scrutiny.
The Nopp140-induced R-rings, with their striking ultrastructural resemblance to the NCS, provided a model system for the generation of intranuclear and nucleolus-associated membrane systems. Five lines of evidence suggest that R-ring biogenesis is initiated at the inner nuclear membrane by a calcium-mediated Nopp140-membrane interaction. First, R-rings constitute simple invaginations of the inner nuclear membrane but not of the entire nuclear envelope (Isaac et al., 2001). Second, transfected Nopp140 often appears in patches at the nuclear envelope (Figure 4; Isaac et al., 2001). Third, Nopp140 is the most prominent calcium-binding protein in nuclear extracts (Figure 5A). A kinase and/or phosphatase may regulate calcium binding as only the phosphorylated form binds calcium (Figure 5B). In the case of the kinase it is likely casein kinase II, which is responsible for the extreme Nopp140 phosphorylation (Meier and Blobel, 1992; Meier, 1996; Li et al., 1997). Unfortunately, we have been unable to directly test this possibility because none of the available inhibitors of casein kinase II and other kinases yielded dephosphorylated Nopp140 in cell culture (data not shown). Fourth, complexed calcium is enriched between the membrane tubules where Nopp140 is concentrated (Figure 6A; Isaac et al., 2001). Finally and perhaps most importantly, calcium chelation interferes with R-ring formation consistent with a direct role for calcium in this process (Figure 5C). The fact that the inhibition by calcium chelation is only partial could have several explanations. For example, it is possible that calcium is not the only mediator for a Nopp140 membrane interaction. Thus, the positively charged stretches of the Nopp140 repeats could interact directly with the negatively charged head groups of the phospholipids as in the case of myosin and other proteins with polybasic clusters (Doberstein and Pollard, 1992; Reizes et al., 1994; Areas et al., 1998; Heo et al., 2006). Of course, lowering the calcium concentration of a cell also could have many indirect effects eventually resulting in reduced R-ring formation. However, during the limited time of BAPTA-AM exposure, we did not observe any general impact on cell viability or morphology of the cells. The simplest explanation for the only partial inhibition is that the cellular calcium concentration was lowered insufficiently, but higher concentrations of BAPTA-AM could not be used because they did have adverse effects on cell viability (data not shown).
The important question, of course, was how related the biogenesis of the R-rings was to that of the NCS. Despite the restricted experimental approaches possible with human endometrial biopsies, we succeeded in determining the presence of complexed calcium and Nopp140 in the NCS. Although they are both present in the electron-dense matrix, they are less enriched than in R-rings, particularly Nopp140, which is extremely concentrated in R-rings, but barely detectable in NCSs by immunoelectron microscopy. Therefore, it does not appear that NCS formation is a mere consequence of Nopp140 overexpression in the endometrium but that additional factors are involved. Such factors are likely specific to the human endometrial epithelial cells, as Nopp140 is a ubiquitously expressed protein. Indeed, transfection of Nopp140 into a human endometrial adenocarcinoma cell line, Ishikawa (Nishida et al., 1985) caused R-ring formation in much shorter time after transfection compared with transfection into nonendometrial cell lines (N. Kittur and U. T. Meier, unpublished observation). This suggests that endometrial epithelial cells may be specifically primed for NCS induction.
The surprising finding that full-length human but not rat Nopp140 induces R-rings may constitute an interesting parallel between the presence of NCSs in human but not rodent endometrium. Thus, despite their high sequence conservation in their amino and carboxyl termini, the repeat domain responsible for R-ring induction exhibits the largest sequence differences between human and rat Nopp140 (Meier and Blobel, 1992; Pai et al., 1995; Meier, 1996). Therefore, the difference in Nopp140 could be directly responsible for the species differences in NCS formation. It was further surprising that nuclear structures induced by full-length rat Nopp140 stained with the lipophilic dye DiOC6 (Isaac et al., 2001), although, as judged by transmission electron microscopy, they were devoid of membranes (Figure 3B). It is thus possible that although lipids are present in these aggregates, they have not formed into visible membrane bilayers (compare R-rings and aggregates in Figure 3B). Unusually therefore, if these lipids could spontaneously form bilayers, R-rings could also form de novo.
Overall, our results suggest that despite their remarkable ultrastructural resemblance and their common derivation from the ER, NCSs differ from R-rings in their composition and biogenesis. Nevertheless, the true relationship between these two unusual organelles will not be known until technical advances allow a more complete picture of NCS composition to emerge, e.g., by detection and labeling at the light microscopic level.
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ACKNOWLEDGMENTS |
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plasmid, and to Elisa Guffanti for valuable comments on the manuscript. We thank the Albert Einstein College of Medicine Analytical Imaging Facility for services and use of microscopes. This work was supported by grants from the March of Dimes Birth Defects Foundation (U.T.M.), the National Institutes of Health HD-041978 (N.S.), and the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council (D.P.B.-J.). This article is dedicated to the memory of Giovanni Migliaccio. |
Footnotes |
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Address correspondence to: Thomas Meier (meier{at}aecom.yu.edu)
Abbreviations used: NCS, nucleolar channel system; ER, endoplasmic reticulum.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Azadian-Boulanger, G., Secchi, J., Laraque, F., Raynaud, J. P., Sakiz, E. (1976). Action of midcycle contraceptive (R 2323) on the human endometrium. Am. J. Obstet. Gynecol 125, 1049–1056.[Medline]
Bentin-Ley, U., Sjogren, A., Nilsson, L., Hamberger, L., Larsen, J. F., Horn, T. (1999). Presence of uterine pinopodes at the embryo-endometrial interface during human implantation in vitro. Hum. Reprod 14, 515–520.
Chen, H. K., Pai, C. Y., Huang, J. Y., Yeh, N. H. (1999). Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization. Mol. Cell. Biol 19, 8536–8546.
Chiu, C. M., Tsay, Y. G., Chang, C. J., Lee, S. C. (2002). Nopp140 is a mediator of the protein kinase A signaling pathway that activates the acute phase response alpha1-acid glycoprotein gene. J. Biol. Chem 277, 39102–39111.
Clyman, M. J. (1963). A new structure observed in the nucleolus of the human endometrial epithelial cell. Am. J. Obstet. Gynecol 86, 430–432.[Medline]
Dellaire, G., Nisman, R., Bazett-Jones, D. P. (2004). Correlative light and electron spectroscopic imaging of chromatin in situ. Methods Enzymol 375, 456–478.[CrossRef][Medline]
Doberstein, S. K. and Pollard, T. D. (1992). Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC. J. Cell Biol 117, 1241–1249.
Dockery, P., Ismail, R. M., Li, T. C., Warren, M. A., Cooke, I. D. (1997). The effect of a single dose of mifepristone (RU486) on the fine structure of the human endometrium during the early luteal phase. Hum. Reprod 12, 1778–1784.
Dockery, P., Pritchard, K., Warren, M. A., Li, T. C., Cooke, I. D. (1996). Changes in nuclear morphology in the human endometrial glandular epithelium in women with unexplained infertility. Hum. Reprod 11, 2251–2256.
Dubrauszky, V. and Pohlmann, G. (1960). Strukturveränderungen am Nukleolus von Korpusendometriumzellen während der Sekretionsphase. Naturwissenschaften 47, 523–524.[CrossRef]
Feria-Velasco, A., Aznar-Ramos, R., Gonzalez-Angulo, A. (1972). Ultrastructural changes found in the endometrium of women using megestrol acetate for contraception. Contraception 5, 187–201.[CrossRef][Medline]
Gordon, M. (1975). Cyclic changes in the fine structure of the epithelial cells of human endometrium. Int. Rev. Cytol 42, 127–172.[Medline]
Gore, B. Z. and Gordon, M. (1974). Fine structure of epithelial cell of secretory endometrium in unexplained primary infertility. Fertil. Steril 25, 103–107.[Medline]
Griffiths, G., McDowall, A., Back, R., Dubochet, J. (1984). On the preparation of cryosections for immunocytochemistry. J. Ultrastruct. Res 89, 65–78.[CrossRef][Medline]
Griffiths, G., Quinn, P., Warren, G. (1983a). Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J. Cell Biol 96, 835–850.
Griffiths, G., Simons, K., Warren, G., Tokuyasu, K. T. (1983b). Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins. Methods Enzymol 96, 466–485.[Medline]
Hartmann, E., Gorlich, D., Kostka, S., Otto, A., Kraft, R., Knespel, S., Burger, E., Rapoport, T. A., Prehn, S. (1993). A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur. J. Biochem 214, 375–381.[Medline]
Heo, W. D., Inoue, T., Park, W. S., Kim, M. L., Park, B. O., Wandless, T. J., Meyer, T. (2006). PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461.
Huster, D. and Arnold, K. (1998). Ca2+-mediated interaction between dextran sulfate and dimyristoyl-sn-glycero-3-phosphocholine surfaces studied by 2H nuclear magnetic resonance. Biophys. J 75, 909–916.[Medline]
Huster, D., Paasche, G., Dietrich, U., Zschornig, O., Gutberlet, T., Gawrisch, K., Arnold, K. (1999). Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction. Biophys. J 77, 879–887.[Medline]
Isaac, C., Pollard, J. W., Meier, U. T. (2001). Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium. J. Cell Sci 114, 4253–4264.
Isaac, C., Yang, Y., Meier, U. T. (1998). Nopp140 functions as a molecular link between the nucleolus and the coiled bodies. J. Cell Biol 142, 319–329.
Kohorn, E. I., Rice, S. I., Hemperly, S., Gordon, M. (1972). The relation of the structure of progestational steroids to nucleolar differentiation in human endometrium. J. Clin. Endocrinol. Metab 34, 257–264.[Medline]
Li, D., Meier, U. T., Dobrowolska, G., Krebs, E. G. (1997). Specific interaction between casein kinase 2 and the nucleolar protein Nopp140. J. Biol. Chem 272, 3773–3779.
MacLennan, A. H., Harris, J. A., Wynn, R. M. (1971). Menstrual cycle of the baboon. II. Endometrial ultrastructure. Obstet. Gynecol 38, 359–374.
Martel, D. (1981). Surface changes of the luminal uterine epithelium during the human menstrual cycle, a scanning microscopic study. In: The Endometrium: Hormonal Implants, ed. J. Brux and J. P. Gantry. New York: Plenum, 15–29.
Maruyama, K., Mikawa, T., Ebashi, S. (1984). Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J. Biochem 95, 511–519.
Meier, U. T. (1996). Comparison of the rat nucleolar protein Nopp140 to its yeast homolog SRP40. Differential phosphorylation in vertebrates and yeast. J. Biol. Chem 271, 19376–19384.
Meier, U. T. and Blobel, G. (1992). Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell 70, 127–138.[CrossRef][Medline]
Meier, U. T. and Blobel, G. (1994). NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J. Cell Biol 127, 1505–1514 [correction appeared in 140, 447].
Miau, L.-H., Chang, C.-J., Tsai, W.-H., Lee, S.-C. (1997). Identification and characterization of a nucleolar phosphoprotein Nopp140, as a transcription factor. Mol. Cell. Biol 17, 230–239.[Abstract]
Migliaccio, G., Nicchitta, C. V., Blobel, G. (1992). The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation. J. Cell Biol 117, 15–25.
More, I. A. and McSeveney, D. (1980). The three dimensional structure of the nucleolar channel system in the endometrial glandular cell: serial sectioning and high voltage electron microscopic studies. J. Anat 130, 673–682.[Medline]
Moricard, R. and Moricard, F. (1964). Modifications cytoplasmiques et nucléaires ultrastructurales utérines au cours de l'état follico-lutéinique à glycogène massif. Gynecol. Obstet 63, 203–219.
Nalefski, E. A., Wisner, M. A., Chen, J. Z., Sprang, S. R., Fukuda, M., Mikoshiba, K., Falke, J. J. (2001). C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity. Biochemistry 40, 3089–3100.[CrossRef][Medline]
Nikas, G. (1999a). Cell-surface morphological events relevant to human implantation. Hum. Reprod 14, Suppl 2, 37–44.[Medline]
Nikas, G. (1999b). Pinopodes as markers of endometrial receptivity in clinical practice. Hum. Reprod 14, Suppl 2, 99–106.[Medline]
Nikas, G., Drakakis, P., Loutradis, D., Mara-Skoufari, C., Koumantakis, E., Michalas, S., Psychoyos, A. (1995). Uterine pinopodes as markers of the ‘nidation window’ in cycling women receiving exogenous oestradiol and progesterone. Hum. Reprod 10, 1208–1213.
Nishida, M., Kasahara, K., Kaneko, M., Iwasaki, H., Hayashi, K. (1985). [Establishment of a new human endometrial adenocarcinoma cell line, Ishikawa cells, containing estrogen and progesterone receptors]. Nippon Sanka Fujinka Gakkai Zasshi 37, 1103–1111.[Medline]
Pai, C.-Y., Chen, H.-K., Sheu, H.-L., Yeh, N.-H. (1995). Cell cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis. J. Cell Sci 108, 1911–1920.[Abstract]
Pai, C.-Y. and Yeh, N.-H. (1996). Cell proliferation-dependent expression of two isoforms of the nucleolar phosphoprotein p130. Biochem. Biophys. Res. Commun 221, 581–587.[CrossRef][Medline]
Parsons, A. K. and Lense, J. J. (1993). Sonohysterography for endometrial abnormalities: preliminary results. J. Clin. Ultrasound 21, 87–95.[Medline]
Petersen, A., Bentin-Ley, U., Ravn, V., Qvortrup, K., Sorensen, S., Islin, H., Sjogren, A., Mosselmann, S., Hamberger, L. (2005). The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro. Fertil. Steril 83, Suppl 1, 1255–1263.[CrossRef][Medline]
Quinn, C., Ryan, E., Claessens, E. A., Greenblatt, E., Hawrylyshyn, P., Cruickshank, B., Hannam, T., Dunk, C., Casper, R. F. (2007). The presence of pinopodes in the human endometrium does not delineate the implantation window. Fertil. Steril Epub ahead of print. doi: 10.1016/j.fertnstert.2006.08.101.
Reizes, O., Barylko, B., Li, C., Sudhof, T. C., Albanesi, J. P. (1994). Domain structure of a mammalian myosin I beta. Proc. Natl. Acad. Sci. USA 91, 6349–6353.
Snapp, E. L., Hegde, R. S., Francolini, M., Lombardo, F., Colombo, S., Pedrazzini, E., Borgese, N., Lippincott-Schwartz, J. (2003). Formation of stacked ER cisternae by low affinity protein interactions. J. Cell Biol 163, 257–269.
Spornitz, U. M. (1992). The functional morphology of the human endometrium and decidua. Adv. Anat. Embryol. Cell Biol 124, 1–99.[Medline]
Terzakis, J. A. (1965). The nucleolar channel system of the human endometrium. J. Cell Biol 27, 293–304.
Tokuyasu, K. T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol 57, 551–565.
van Santen, M. R., Haspels, A. A., Heijnen, H. F., Rademakers, L. H. (1988). Interfering with implantation by postcoital estrogen administration. II. Endometrium epithelial cell ultrastructure. Contraception 38, 711–724.[CrossRef][Medline]
van Schaftingen, E. and Gerin, I. (2002). The glucose-6-phosphatase system. Biochem. J 362, 513–532.[CrossRef][Medline]
Verma, V. (1983). Ultrastructural changes in human endometrium at different phases of the menstrual cycle and their functional significance. Gynecol. Obstet. Investig 15, 193–212.[CrossRef][Medline]
Wachstein, M. and Meisel, E. (1956). On the histochemical demonstration of glucose-6-phosphatase. J. Histochem. Cytochem 4, 592.
Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J. J. d., Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., Bergeron, J. J. (1991). SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem 266, 19599–19610.
Wang, C., Query, C. C., Meier, U. T. (2002). Immunopurified small nucleolar ribonucleoprotein particles pseudouridylate rRNA independently of their association with phosphorylated Nopp140. Mol. Cell. Biol 22, 8457–8466.
Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., Lerner, R. A. (1984). The structure of an antigenic determinant in a protein. Cell 37, 767–778.[CrossRef][Medline]
Wynn, R. M. (1967). Intrauterine devices: effects on ultrastructure of human endometrium. Science 156, 1508–1510.
Yang, Y., Isaac, C., Wang, C., Dragon, F., Pogacic, V., Meier, U. T. (2000). Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140. Mol. Biol. Cell 11, 567–577.
Zhang, X., Rizo, J., Sudhof, T. C. (1998). Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I. Biochemistry 37, 12395–12403.[CrossRef][Medline]
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80 phosphates between the two Nopp140 forms. (C) Effect of the membrane permeable calcium chelator BAPTA-AM on R-ring formation. Cells were transfected with Nopp140 lacking its amino terminus as in 
