Syncytial Knots (Tenney-Parker Changes) in the Human Placenta
2013; Elsevier BV; Volume: 183; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2013.03.016
ISSN1525-2191
AutoresNorah M. E. Fogarty, Anne C. Ferguson‐Smith, Graham J. Burton,
Tópico(s)Genetic Syndromes and Imprinting
ResumoSyncytiotrophoblast is the multinucleated epithelium of the placenta. Although many nuclei are dispersed within the syncytioplasm, others are aggregated into specializations referred to as true and false syncytial knots, and syncytial sprouts. Nuclei within true knots display highly condensed chromatin and are thought to be aged and effete. True knots increase in frequency with gestational age. Excessive formation (Tenney-Parker change) is associated with placental pathology, and a knotting index is used to assess severity. However, this index is potentially confounded by the creation of artifactual appearances (false knots) through tangential sectioning. In addition, knots must be distinguished from syncytial sprouts, which are markers of trophoblast proliferation. Here, we distinguish between sprouts, true knots, and false knots using serial sections and perform IHC for proliferating cell nuclear antigen, upstream binding factor, RNA polymerase II, and 8-oxo-deoxyguanosine as markers of recent incorporation, transcriptional activity, and oxidative damage. Villous explants were exposed to hydrogen peroxide to test the relationship between transcriptional activity and oxidative damage. Sprouts and false knots were found to contain recently incorporated and transcriptionally active nuclei. By contrast, most nuclei within true knots are negative for transcriptional markers but positive for 8-oxo-deoxyguanosine. In vitro, we observed a negative correlation between transcriptional activity and oxidative damage. These findings demonstrate that true knots contain effete damaged nuclei and provide IHC markers for their identification. Syncytiotrophoblast is the multinucleated epithelium of the placenta. Although many nuclei are dispersed within the syncytioplasm, others are aggregated into specializations referred to as true and false syncytial knots, and syncytial sprouts. Nuclei within true knots display highly condensed chromatin and are thought to be aged and effete. True knots increase in frequency with gestational age. Excessive formation (Tenney-Parker change) is associated with placental pathology, and a knotting index is used to assess severity. However, this index is potentially confounded by the creation of artifactual appearances (false knots) through tangential sectioning. In addition, knots must be distinguished from syncytial sprouts, which are markers of trophoblast proliferation. Here, we distinguish between sprouts, true knots, and false knots using serial sections and perform IHC for proliferating cell nuclear antigen, upstream binding factor, RNA polymerase II, and 8-oxo-deoxyguanosine as markers of recent incorporation, transcriptional activity, and oxidative damage. Villous explants were exposed to hydrogen peroxide to test the relationship between transcriptional activity and oxidative damage. Sprouts and false knots were found to contain recently incorporated and transcriptionally active nuclei. By contrast, most nuclei within true knots are negative for transcriptional markers but positive for 8-oxo-deoxyguanosine. In vitro, we observed a negative correlation between transcriptional activity and oxidative damage. These findings demonstrate that true knots contain effete damaged nuclei and provide IHC markers for their identification. The epithelial covering of the placental villous tree, the syncytiotrophoblast (STB), is a multinucleated, terminally differentiated syncytium. No mitotic figures are observed in the STB,1Galton M. DNA content of placental nuclei.J Cell Biol. 1962; 13: 183-191Crossref PubMed Scopus (28) Google Scholar, 2Kar M. Ghosh D. Sengupta J. Histochemical and morphological examination of proliferation and apoptosis in human first trimester villous trophoblast.Hum Reprod. 2007; 22: 2814-2823Crossref PubMed Scopus (32) Google Scholar and instead, the syncytium is enlarged and sustained across gestation by the continuous fusion of underlying cytotrophoblast (CTB) cells. The number of STB nuclei increases ninefold from 13 weeks of gestation to term,3Simpson R.A. Mayhew T.M. Barnes P.R. From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: a study of nuclear number using the disector.Placenta. 1992; 13: 501-512Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 4Mayhew T.M. Simpson R.A. Quantitative evidence for the spatial dispersal of trophoblast nuclei in human placental villi during gestation.Placenta. 1994; 15: 837-844Abstract Full Text PDF PubMed Scopus (41) Google Scholar and consequently nuclei within the syncytium are of different ages, depending on their time of incorporation. A range of nuclear morphologies is observed within the syncytiotrophoblastic compartment, with varying degrees of chromatin condensation. In addition, although most nuclei are dispersed within the syncytioplasm, others form aggregates that have been variously referred to as syncytial knots and sprouts, sometimes synonymously. The appearances of the nuclei within these aggregates differ, however, suggesting the aggregates have different functional and pathophysiologic significance. The nuclei within true syncytial knots display highly condensed chromatin, either dispersed throughout the nucleus or in the form of a dense peripheral ring. The nuclei are often closely juxtaposed, with smooth outlines and little intervening cytoplasm. Euchromatin is restricted to areas near nuclear pores or a central island,5Jones C.J. Fox H. Syncytial knots and intervillous bridges in the human placenta: an ultrastructural study.J Anat. 1977; 124: 275-286PubMed Google Scholar and few nucleoli are observed.6Mayhew T.M. Leach L. McGee R. Ismail W.W. Myklebust R. Lammiman M.J. Proliferation, differentiation and apoptosis in villous trophoblast at 13-41 weeks of gestation (including observations on annulate lamellae and nuclear pore complexes).Placenta. 1999; 20: 407-422Abstract Full Text PDF PubMed Scopus (135) Google Scholar In normal pregnancies, knots are rarely seen before 20 weeks of gestation, and their frequency increases as gestation proceeds.7Loukeris K. Sela R. Baergen R.N. Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks’ gestational age.Pediatr Dev Pathol. 2010; 13: 305-309Crossref PubMed Scopus (73) Google Scholar They are particularly pronounced in postmature placentas.8Fox H. The significance of villous syncytial knots in the human placenta.J Obstet Gynaecol Br Commonw. 1965; 72: 347-355Crossref PubMed Scopus (52) Google Scholar Because of these findings, true knots are generally considered a mechanism by which effete STB nuclei can be sequestered to areas of the villus surface where they do not impinge on diffusional exchange. In the past, the nuclear appearances have been interpreted as apoptotic, although nuclear fragmentation is never observed. The formation of syncytial knots is increased in placentas from complicated pregnancies,9Khalid M.E. Ali M.E. Ali K.Z. Full-term birth weight and placental morphology at high and low altitude.Int J Gynaecol Obstet. 1997; 57: 259-265Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 10Narasimha A. Vasudeva D.S. Spectrum of changes in placenta in toxemia of pregnancy.Indian J Pathol Microbiol. 2011; 54: 15-20Crossref PubMed Scopus (19) Google Scholar, 11Tomas S.Z. Prusac I.K. Roje D. Tadin I. Trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia.Gynecol Obstet Invest. 2011; 71: 250-255Crossref PubMed Scopus (58) Google Scholar a finding widely referred to as Tenney-Parker changes in recognition of the first description by Tenney and Parker.12Tenney B. Parker F. The placenta in toxemia of pregnancy.Am J Obstet Gynecol. 1940; 39: 1000-1005Scopus (70) Google Scholar These authors found that knots were present on nearly all terminal villi in preeclamptic placentas, whereas they were only seen on 10% to 15% in normal placentas. The increase in knotting has been attributed to premature aging of the placenta as part of the pathophysiology of preeclampsia, and Tenney-Parker changes are widely used as an index of placental well-being.13Benirschke K. Burton G.J. Baergen R.N. Pathology of the human placenta. Springer-Verlag, Berlin2012Crossref Scopus (52) Google Scholar However, the original descriptions of Tenney and Parker were based on examination of two-dimensional histologic sections. Subsequently, it has been demonstrated repeatedly that viewing a single section can give a false impression of the incidence of true syncytial knots because of the extensive branching and tortuosity of the villous tree. The appearance of nuclear aggregations resembling knots can be created artifactually if the plane of section passes tangentially through the STB.14Küstermann W. [Syncytial sprouts and intervillous bridges in the human placenta (author's transl)] German.Anat Anz. 1981; 150: 144-157PubMed Google Scholar, 15Burton G.J. Intervillous connections in the mature human placenta: instances of syncytial fusion or section artifacts?.J Anat. 1986; 145: 13-23PubMed Google Scholar, 16Cantle S.J. Kaufmann P. Luckhardt M. Schweikhart G. Interpretation of syncytial sprouts and bridges in the human placenta.Placenta. 1987; 8: 221-234Abstract Full Text PDF PubMed Scopus (69) Google Scholar Only by following the structures in thick (20 μm) or serial sections can the correct identity of these false knots be revealed, although close inspection reveals that their nuclei show only peripheral heterochromatin, similar to that of the dispersed nuclei. Increased branching of the villous tree has been shown to occur in placentas affected by preeclampsia, intrauterine growth restriction, and high altitude, likely due to hypoxia.17Kaufmann P. Luckhardt M. Schweikhart G. Cantle S.J. Cross-sectional features and three-dimensional structure of human placental villi.Placenta. 1987; 8: 235-247Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 18Ali K.Z. Burton G.J. Morad N. Ali M.E. Does hypercapillarization influence the branching pattern of terminal villi in the human placenta at high altitude?.Placenta. 1996; 17: 677-682Abstract Full Text PDF PubMed Scopus (50) Google Scholar These conditions are associated with a developmental shift to a villous phenotype with richly capillarized and highly branched terminal villi, providing a mechanism to enhance the placenta’s ability to transfer oxygen to the fetus. However, the increased branching will lead to an increased incidence of tangential sectioning through the STB.18Ali K.Z. Burton G.J. Morad N. Ali M.E. Does hypercapillarization influence the branching pattern of terminal villi in the human placenta at high altitude?.Placenta. 1996; 17: 677-682Abstract Full Text PDF PubMed Scopus (50) Google Scholar Hence, unless the distinction between true and false knots is made, Tenney-Parker changes may reflect the topology of the villous tree as much as aging of the syncytial nuclei. Although both changes may be of interest to pathologists, they potentially have different origins and clinical significance. True knots must also be distinguished from a third type of nuclear aggregate, syncytial sprouts. Sprouts are outgrowths from the syncytium, which are associated with proliferation of the villous tree. They are most commonly observed in early gestation, when they form paddle-shaped protrusions with an expanded head and a long neck region.19Burton G.J. The fine structure of the human placental villus as revealed by scanning electron microscopy.Scanning Microsc. 1987; 1: 1811-1828PubMed Google Scholar They are also present in later stages of gestation in the more hypoxic regions of the lobule16Cantle S.J. Kaufmann P. Luckhardt M. Schweikhart G. Interpretation of syncytial sprouts and bridges in the human placenta.Placenta. 1987; 8: 221-234Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 20Castellucci M. Kosanke G. Verdenelli F. Huppertz B. Kaufmann P. Villous sprouting: fundamental mechanisms of human placental development.Hum Reprod Update. 2000; 6: 485-494Crossref PubMed Scopus (120) Google Scholar but are shorter and stubbier at this stage. Nuclei in sprouts are loosely packed relative to each other and are predominately euchromatic with a prominent nucleolus.21Burton G.J. Jones C.J. Syncytial knots, sprouts, apoptosis, and trophoblast deportation from the human placenta.Taiwan J Obstet Gynecol. 2009; 48: 28-37Abstract Full Text PDF PubMed Scopus (121) Google Scholar Because of the continual CTB cell fusion and expansion of the STB, nuclei in the STB differ in the length of time they have been resident in the syncytium. The spectrum of nuclear morphologies observed suggests progressive maturational changes, and it is thought that true knots may represent the end point of the life cycle of a syncytial nucleus. Tracking the progression of an STB nucleus after fusion is impossible in the human placenta in vivo for ethical reasons. Instead, proliferating cell nuclear antigen (PCNA) immunoreactivity can be used to distinguish between recently incorporated STB nuclei and those resident for longer. PCNA is not expressed in STB nuclei because they are nonproliferative, but due to its long half-life we hypothesize that it remains for a period in recently incorporated nuclei as a relic of CTB division and incorporation. We therefore hypothesize that nuclei in sprouts and true knots will differ in terms of their PCNA immunoreactivity, reflecting the different developmental states of these structures. Recently, it has been reported that a proportion of STB nuclei are transcriptionally active across all stages of gestation.22Ellery P.M. Cindrova-Davies T. Jauniaux E. Ferguson-Smith A.C. Burton G.J. Evidence for transcriptional activity in the syncytiotrophoblast of the human placenta.Placenta. 2009; 30: 329-334Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 23Fogarty N.M. Mayhew T.M. Ferguson-Smith A.C. Burton G.J. A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation.J Anat. 2011; 219: 601-610Crossref PubMed Scopus (31) Google Scholar Immunostaining for RNA polymerase II (RPol II) and phospho–upstream binding protein (pUBF) shows a range of transcriptional statuses among the syncytial nuclei. Stretches of active nuclei are seen to be punctuated by the presence of an inactive nucleus, suggesting that transcriptional activity is regulated at the level of individual nuclei. Here, we assess the transcriptional status of nuclei within syncytial sprouts and true and false knots, identified by following through serial sections, to correlate the contrasting morphologies with nuclear activity. We correlate these findings with evidence of DNA oxidative damage and use an in vitro explant model to test the relationship between oxidative damage and transcriptional activity. We hypothesize that true knots contain transcriptionally inactive nuclei and are oxidatively damaged, enabling them to be reliably distinguished in two-dimensional sections from artifactual false knots and syncytial sprouts. These molecular differences can be implemented to increase the specificity of assessments of placental pathology using Tenney-Parker changes. Blocks from formalin-fixed, paraffin-embedded placentas of two ranges of gestational age (14 to 22 weeks, n = 4, and 37 to 39 weeks, n = 5) were obtained from an archive collected in accordance with ethical protocols.4Mayhew T.M. Simpson R.A. Quantitative evidence for the spatial dispersal of trophoblast nuclei in human placental villi during gestation.Placenta. 1994; 15: 837-844Abstract Full Text PDF PubMed Scopus (41) Google Scholar Serial sections were cut from blocks at a thickness of 5 μm. Samples for explant culture were obtained from normal placentas delivered by elective caesarean section with approval from Cambridge Local Ethical Committee and informed patient consent. Villous tissue was collected from the maternal side of the placenta, midway between the chorionic and basal plates. The maternal aspect of the placenta was removed, and small pieces were collected from four to six separate lobules, all positioned around the center of the placenta. After a brief rinse in ice-cold phosphate-buffered saline, samples were placed into ice-cold transport medium (TCS large vessel endothelial cell basal medium; TCS CellWorks, Milton Keynes, UK) containing 2% fetal bovine serum, heparin, hydrocortisone, human epidermal growth factor, human basic fibroblast growth factor, 25 μg/mL of gentamicin, 50 ng/mL of amphotericin B, 1 mmol/L vitamin C, and 1 mmol/L Trolox that had been equilibrated to 5% O2/90% N2/5% CO2. Sections were rehydrated in Histoclear (Sigma, Poole, UK), graded ethanol, and deionized water. Heat-induced antigen retrieval was performed by boiling sections in 0.1 mol/L Tris-EDTA buffer (pH 9.0) in a pressure cooker. Sections were blocked in nonimmune serum for 30 minutes at room temperature. Endogenous peroxidases were quenched by incubating the sections in 3% H2O2 for 15 minutes. Primary antibodies, including PCNA (1:200; Abcam, Cambridge, UK), pUBF (1:100; Santa Cruz, Santa Cruz, CA), RPol II (1:200; Abcam), and 8-oxo-deoxyguanosine (8OHdG; 1:100; Abcam) were added and incubated overnight at 4°C. Sections were washed in Tris-buffered saline with 0.1% Tween 20 and 0.1% Triton X-100. Biotin-labeled species-specific secondary antibodies were added at a concentration of 1:200 and incubated at room temperature for 1 hour. Vectastain Elite ABC system (Vector Labs, Burlingame, CA) and SigmaFast DAB (Sigma) were used according to manufacturer instructions. Sections were lightly counterstained with hematoxylin, rinsed in deionized water, and dehydrated in increasing grades of alcohol and Histoclear. Coverslips were mounted with DPX (Sigma). Images were captured using a Nanozoomer slide scanner (Nanozoomer 2.0-RS; Hamamatsu Photonics, Hertfordshire, UK). Sections were rehydrated according to the previously described protocol. Sections were stained in hematoxylin for 10 minutes and briefly rinsed in deionized water before acid-alcohol differentiation (1% HCl and 70% ethanol; 5 minutes). Sections were then incubated in eosin for 20 minutes, rinsed in deionized water, dehydrated, and mounted as previously described. Serial sections (n = 30) were cut at 5 μm to minimize the superimposition of nuclei obscuring the images. Every third serial section was stained for the target antigen, whereas adjacent sections were stained with H&E. The antigenicity of nuclear aggregations was detected blind and subsequently the nature of the aggregation confirmed by viewing the H&E serial sections. Sprouts were identified as an aggregation that protruded from the villous surface, often with an expanded body that was connected to the main syncytium by a narrower stalk.19Burton G.J. The fine structure of the human placental villus as revealed by scanning electron microscopy.Scanning Microsc. 1987; 1: 1811-1828PubMed Google Scholar The nuclei were euchromatic and of similar size to dispersed STB nuclei (Figure 1A). As expected, sprouts were most common in the early second trimester samples. False knots were those nuclear aggregations that on tracing through the series were revealed to be a bridge between adjacent villi or a tangential section of the STB at a branch point.15Burton G.J. Intervillous connections in the mature human placenta: instances of syncytial fusion or section artifacts?.J Anat. 1986; 145: 13-23PubMed Google Scholar, 16Cantle S.J. Kaufmann P. Luckhardt M. Schweikhart G. Interpretation of syncytial sprouts and bridges in the human placenta.Placenta. 1987; 8: 221-234Abstract Full Text PDF PubMed Scopus (69) Google Scholar Nuclei were of similar size to those dispersed in the syncytium (Figure 1B). True knots were defined as sessile aggregations of nuclei, which gently protruded from the syncytium and were observed appearing and disappearing through the serial sections.21Burton G.J. Jones C.J. Syncytial knots, sprouts, apoptosis, and trophoblast deportation from the human placenta.Taiwan J Obstet Gynecol. 2009; 48: 28-37Abstract Full Text PDF PubMed Scopus (121) Google Scholar Nuclei were smaller in diameter than the dispersed nuclei and often demonstrated a peripheral ring of chromatin, which stained darkly (Figure 1C). As expected, true knots were only observed in the samples from 37 to 39 weeks of gestational age. Explants from placentas (n = 6) were dissected into pieces 5 mm3 in size in ice-cold culture medium in a glove box under 10% O2/85% N2/5% CO2. Samples were transferred to Netwell mesh inserts in 6-well plates containing large endothelial vessel medium (TCS Cellworks). Equilibrated medium was supplemented with 2 mmol/L ascorbic acid and 1 mmol/L Trolox. H2O2 was added at 0, 100, or 1000 μmol/L because these concentrations have been previously demonstrated to increase syncytial knot formation in vitro.24Heazell A.E. Moll S.J. Jones C.J. Baker P.N. Crocker I.P. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.Placenta. 2007; 28: S33-S40Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Plates were incubated at 10% O2/85% N2/5% CO2 over 48 hours because this represents the ambient placental environment at term and maintains viability of explants in culture.25Miller R.K. Genbacev O. Turner M.A. Aplin J.D. Caniggia I. Huppertz B. Human placental explants in culture: approaches and assessments.Placenta. 2005; 26: 439-448Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 26Huppertz B. Kingdom J. Caniggia I. Desoye G. Black S. Korr H. Kaufmann P. Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation.Placenta. 2003; 24: 181-190Abstract Full Text PDF PubMed Scopus (247) Google Scholar Samples were fixed at 0, 24, and 48 hours in paraformaldehyde before paraffin embedding and sectioning in preparation for IHC for the detection of 8OHdG and RPol II. After the 48-hour culture, explants were transferred to medium containing 0.5 mg/mL of MTT (Sigma) for 20 minutes at 37°C in the dark. Explants were frozen in Tissue-Tek OCT and sectioned in a cryotome and mounted onto slides. Viability of the STB of the explants was confirmed by demonstrating mitochondrial activity, as detected by the appearance of the blue formazan product. To quantify the percentage of RPol II–immunopositive STB nuclei in the explants, 50 counting frames were applied in a systematic, random manner to the sections. Nuclei falling on the designated forbidden lines were excluded to avoid double counting. The percentages of positive RPol II and 8OHdG STB nuclei were calculated with the following equation:PercentageofSTB+Nuclei=No.STB+Nuclei(No.STB+Nuclei+No.STB−Nuclei)×100(1) Data were analyzed using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA). Proportions of RPol II–positive and 8OHdG-positive STB nuclei in explants were compared to controls using the unpaired t-test. P < 0.05 was used for all tests. PCNA, which has a half-life of >20 hours,27Stewart C.A. Dell’Orco R.T. Expression of proliferating cell nuclear antigen during the cell cycle of human diploid fibroblasts.In Vitro Cell Dev Biol. 1992; 28A: 211-214Crossref PubMed Scopus (15) Google Scholar was used to distinguish between nuclei that had been recently incorporated into the STB nuclei and those that had been resident for longer. Occasional PCNA-positive nuclei were observed within the bodies of syncytial sprouts and false knots but never within a true knot, although occasional PCNA-immunopositive nuclei were observed adjacent to a knot (Figure 2). Serine-388 pUBF was investigated as a marker for RPol I–dependent transcription. Immunopositivity was observed in CTB and stromal cell nuclei and in a proportion of dispersed STB nuclei. Sprouts also contained a similar proportion of pUBF-positive nuclei that were uniformly distributed throughout the sprout, including the stalk and the head. False knots also contained a high proportion of pUBF-positive nuclei. By contrast, pUBF-positive nuclei were restricted to the periphery of the nuclear aggregates in true knots and were not observed in the bulk of the knot. Serine-2 phosphorylated RPol II was used as a marker of transcriptionally active nuclei. Sprouts contained a proportion of RPol II–positive nuclei that were uniformly distributed throughout the body of the sprout (Figure 3, A and B). False knots also contained a high proportion of RPol II–positive nuclei (Figure 3C). True knots did not contain immunopositive nuclei. Occasionally, RPol II–positive nuclei were seen adjacent to a true knot but never within the nuclear aggregation (Figure 3, D–I). False knots contained a heterogenous population of 8OHdG-positive nuclei (Figure 4A) and 8OHdG-negative nuclei (Figure 4B). True knots contained a high proportion of 8OHdG-positive nuclei (Figure 4C). These nuclei also displayed a highly condensed nuclear morphology compared with 8OHdG-negative nuclei and dispersed nuclei (Figure 4C). Sprouts contained mostly 8OHdG-negative nuclei but occasional 8OHdG-positive nuclei were observed in sprouts, the proportion of which was similar to that observed dispersed throughout the syncytium (Figure 4E). To confirm that our model induced oxidative stress and caused an increase in the proportion of 8OHdG-positive nuclei, we quantified the percentage of positive STB nuclei after exposure to 0, 100, and 1000 μmol/L hydrogen peroxide for 24 and 48 hours. Explants exposed to 100 μmol/L and 1000 μmol/L demonstrated significant increases in the percentage of 8OHdG-positive STB nuclei after 24 hours compared with the 0 μmol/L 0 hours control (100 μmol/L, 64.8% ± 7.7%; 1000 μmol/L, 64.7% ± 12.7%) (Figure 5). Similarly, proportions of 8OHdG-positive STB nuclei were increased with H2O2 treatment after 48 hours compared with 0 μmol/L 0 hours control (100 μmol/L, 70% ± 0.7%; 1000 μmol/L, 77.7% ± 7.8%). There was no increase in the percentage in 0 μmol/L control explants in the absence of H2O2 during the 48-hour period (42.1% ± 10% and 41.5% ± 14.8%, respectively). The percentages of RPol II–positive STB nuclei in explants exposed to 0, 100, and 1000 μmol/L H2O2 for 24 and 48 hours were quantified (Figure 6). Explants exposed to 0 μmol/L H2O2 showed a slight increase in the percentage of RPol II–positive STB nuclei during the 48-hour culture period. After 24 hours in 100 and 1000 μmol/L H2O2, there was a significant reduction in RPol II–positive STB nuclei compared with the 0 μmol/L control at the same time point (42.5% ± 9% and 49.4% ± 13% compared with 59.3% ± 11% ; P = 0.023 and 0.012; means ± SD). Similarly, explants cultured in 100 and 1000 μmol/L H2O2 for 48 hours had reduced proportions compared with those in 0 μmol/L for 48 hours (48.4% ± 6.3% and 46.5% ± 8.8% compared with 70.1% ± 16.5%; P = 0.013 and P = 0.012). In this study, we aim to distinguish between sprouts, true knots, and false knots on the basis of their relative nuclear ages, transcriptional activity, and oxidative damage. We first investigated the relative ages of nuclei in sprouts and knots. STB nuclei have no proliferative capabilities,1Galton M. DNA content of placental nuclei.J Cell Biol. 1962; 13: 183-191Crossref PubMed Scopus (28) Google Scholar and the STB is expanded by the continual fusion of the underlying progenitor CTB cells. Tracer studies to follow the life history of the STB nuclei are not possible in vivo for obvious ethical reasons. IHC for PCNA was therefore used as a marker of recent incorporation. PCNA is a co-factor of DNA polymerase and is required for the processivity of leading strand synthesis in DNA replication, as well as coupling postreplication processes to the cell cycle.28Tsurimoto T. Molecular structures in DNA replication.Tanpakushitsu Kakusan Koso. 1999; 44: 475-484PubMed Google Scholar It has a long half-life of approximately 20 hours.27Stewart C.A. Dell’Orco R.T. Expression of proliferating cell nuclear antigen during the cell cycle of human diploid fibroblasts.In Vitro Cell Dev Biol. 1992; 28A: 211-214Crossref PubMed Scopus (15) Google Scholar PCNA is present in CTB nuclei but not in the nonreplicative STB nuclei. Any PCNA observed in STB nuclei may therefore be a relic of CTB cell division, differentiation, and fusion. This immunostaining assay thus allows for recently incorporated nuclei to be distinguished from those residing in the syncytium for longer periods. Most sprouts contain a proportion of PCNA-positive nuclei uniformly distributed throughout the body of the sprout. False knots occasionally contained PCNA-positive nuclei. By contrast, true knots as defined here do not contain PCNA-positive nuclei. Differential detection of PCNA-positive nuclei in syncytial sprouts and knots reveals that the nuclei in these structures differ in terms of time of their incorporation into the syncytium. pUBF and RPol II were selected as markers of active transcription. UBF is a nucleolar co-factor required for RPol I–dependent transcription of ribosomal RNAs for the assembly of ribosomes.29Glibetic M. Taylor L. Larson D. Hannan R. Sells B. Rothblum L. The RNA polymerase I transcription factor UBF is the product of a primary response gene.J Biol Chem. 1995; 270: 4209-4212Crossref PubMed Scopus (26) Google Scholar Ribosome biogenesis is up-regulated during cell growth and proliferation because these processes require massive quantities of translation. In prokaryotic and eukaryotic cells, biogenesis is tightly coupled to external conditions so as to prevent excessive energy consumption by ribosome production.30Mayer C. Grummt I. Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases.Oncogene. 2006; 25: 6384-6391Crossref PubMed Scopus (382) Google Scholar A high proportion of pUBF-positive nuclei were identified in both sprouts and false knots. Because sprouts are sites of villous proliferation and growth, it is appropriate that RPol I–active nuclei are present throughout their structure. Knots did not contain pUBF-positive nuclei, although positive nuclei were often observed directly adjacent to the knot. Recent work has found that 60% to 80% of STB nuclei are transcriptionally active across all stages of gestation.31Palancade B. Bensaude O. Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.Eur J Biochem. 2003; 270: 3859-3870Crossref PubMed Scopus (200) Google Scholar The marker used to assess this was the Ser-2 phosphorylated form of RPol II, which is required for active, elongating transcription. Sprouts contained a proportion of RPol II–positive STB nuclei uniformly distributed throughout the sprout. Any knots that appeared to contain RPol II–positive nuclei were revealed to be false knots on tracing. True knots did not contain any RPol II–positive nuclei within the nuclear aggregation. The absence of RPol II nuclei in true knots, coupled with electron microscopical data detailing the chromatin condensation, absence of nuclear pores, abnormal distribution of endoplasmic reticulum, and reduced mitochondrial numbers in these structures demonstrates that true knots are indeed transcriptionally inactive.5Jones C.J. Fox H. Syncytial knots and intervillous bridges in the human placenta: an ultrastructural study.J Anat. 1977; 124: 275-286PubMed Google Scholar Occasional pUBF- and RPol II–positive nuclei were observed adjacent to some true knots. Active nuclei may be located at these sites to provide gene products required for processing of the damaged nuclei and their aggregation. Alternatively, these nuclei may have no function pertaining to the knot and may simply be collaterals located here by the shunting of other nuclei into the knot. Finally, it has recently been reported that a proportion of CTB cells interdigitate into the STB, and hence their nuclei may be incorrectly assigned as being part of the latter.31Palancade B. Bensaude O. Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.Eur J Biochem. 2003; 270: 3859-3870Crossref PubMed Scopus (200) Google Scholar Confocal microscopy and dual immunolabeling with anti-pUBF and anti–E-cadherin would be required to confirm whether this may also occur in the vicinity of true knots. It has long been proposed that true knots are accumulations of aged or effete nuclei. Our demonstration that many of the contained nuclei stain strongly for 8OHdG provides the first evidence of DNA damage. 8OHdG is a biomarker for oxidative DNA damage that is formed by hydroxyl radicals on guanosine bases in DNA.32Shigenaga M.K. Ames B.N. Assays for 8-hydroxy-2′-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.Free Radic Biol Med. 1991; 10: 211-216Crossref PubMed Scopus (336) Google Scholar It was selected as a marker for DNA damage because it has a distinct nuclear localization that facilitates the identification of damaged nuclei. The negative association between oxidative damage and transcriptional activity was confirmed by the ex vivo manipulation after exposure of villous explants to the pro-oxidant hydrogen peroxide at concentrations that have previously been found to cause an increase in syncytial knot formation.24Heazell A.E. Moll S.J. Jones C.J. Baker P.N. Crocker I.P. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.Placenta. 2007; 28: S33-S40Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar The STB is vulnerable to oxidative damage in vivo because of its intimate relationship with the maternal blood but also because it contains low levels of the principal antioxidant enzymes.33Watson A.L. Skepper J.N. Jauniaux E. Burton G.J. Susceptibility of human placental syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age.J Clin Endocrinol Metab. 1998; 83: 1697-1705Crossref PubMed Scopus (128) Google Scholar, 34Palmer M.E. Watson A.L. Burton G.J. Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture.Hum Reprod. 1997; 12: 379-382Crossref PubMed Scopus (48) Google Scholar Many complications of pregnancy in which syncytial knotting is increased, such as preeclampsia, are associated with placental oxidative stress.35Myatt L. Cui X. Oxidative stress in the placenta.Histochem Cell Biol. 2004; 122: 369-382Crossref PubMed Scopus (600) Google Scholar Thus, we speculate that the increased knotting seen in these cases represents a mechanism of sequestering damaged nuclei into areas of the STB where they do not impede maternal-fetal exchange. However, it is also possible that the oxidative damage detected represents a mechanism by which the number of active nuclei within the STB is regulated. Given that the mRNAs transcribed can diffuse freely throughout the syncytioplasm, it is not necessary for all of the nuclei to be active. However, the benefits of controlling the number remain unclear at present. False knots contained a heterogenous population of pUBF- and RPol II–immunopositive and negative nuclei. In addition, some false knots contained some PCNA-positive nuclei, whereas others did not. These findings reflect the fact that the function of collecting nuclei at these branching points is currently not understood. Perhaps nuclei are shepherded indiscriminately to these branch points, either to provide structural support to the expanding villus or as a mechanism to accommodate the increasing nuclear number in the syncytium. This would result in a mixed population of nuclei in terms of age and transcriptional activity. We report for the first time, to our knowledge, the molecular differences between syncytial sprouts and false and true knots (Table 1). Sprouts contain a proportion of recently incorporated nuclei that appear to be transcriptionally active on the basis of RPol I and RPol II immunoreactivity. This finding is consistent with the presence of abundant ribosomes and endoplasmic reticulum in the surrounding cytoplasm, supporting the function of sprouts as regions of synthesis and proliferation.36Lyall F, Belfort M: Pre-eclampsia: etiology and clinical practice. Edited by Cambridge, Cambridge University Press, 2007. pp 138–151Google Scholar In false knots, the proportion of the nuclei displaying transcriptional activity is similar to that among the dispersed nuclei, consistent with their origin as tangential sections through the STB. By contrast, the nuclei within true knots appear transcriptionally inactive and display evidence of oxidative damage. These criteria provide a method by which pathologists observing Tenney-Parker changes in preeclampsia and other complications of pregnancy can distinguish between true knots and false knots. Further studies are required to test whether indices based on these criteria provide a more accurate assessment of the severity of the pathophysiologic process.Table 1Checklist for Identification of Syncytial Sprouts, True Knots, and False Knots Arising from Tangential SectioningPCNARPol IIpUBF8OHdGSprout++++True knots−−−+++False knots+++++Immunoreactivity of syncytial sprouts, true knots, and false knots was determined as present (+) or absent (−). Immunoreactivity of 8OHdG-positive nuclei is further quantified as (+) 0-20% positive nuclei, (++) 20-50% positive nuclei and (+++) > 50% positive nuclei. Open table in a new tab Immunoreactivity of syncytial sprouts, true knots, and false knots was determined as present (+) or absent (−). Immunoreactivity of 8OHdG-positive nuclei is further quantified as (+) 0-20% positive nuclei, (++) 20-50% positive nuclei and (+++) > 50% positive nuclei. We thank Prof. Terry M. Mayhew for access to placental blocks collected across gestational age.
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