A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation

Abstract

The syncytiotrophoblast (STB) epithelial covering of the human placenta is a unique terminally differentiated, multi-nucleated syncytium. No mitotic bodies are observed in the STB, which is sustained by continuous fusion of underlying cytotrophoblast cells (CTB). As a result, STB nuclei are of different ages. Morphologically, they display varying degrees of chromatin compaction, suggesting progressive maturational changes. Until recently, it was thought that STB nuclei were transcriptionally inactive, with all the mRNAs required by the syncytium being incorporated upon fusion of CTB. However, recent research has shown the presence of the active form of RNA polymerase II (RNA Pol II) in some STB nuclei. In this study, we confirm the presence of transcriptional activity in STB nuclei by demonstrating immunoreactivity for a transcription factor and an RNA polymerase I (RNA Pol I) co-factor, phospho-cAMP response element-binding protein and phospho-upstream binding factor, respectively. We also show, through immunoco-localisation studies, that a proportion of STB nuclei are both RNA Pol I and II transcriptionally active. Finally, we quantify the numerical densities of nuclei immunopositive and immunonegative for RNA Pol II in the STB of normal placentas of 11–39 weeks gestational age using an unbiased stereological counting tool, the physical disector. These data were combined with estimates of the volume of trophoblast to calculate total numbers of both types of nuclei at each gestational age. We found no correlation between gestational age and the numerical density of RNA Pol II-positive nuclei in the villous trophoblast (r = 0.39, P > 0.05). As the number of STB nuclei increases exponentially during gestation, we conclude that the number of transcriptionally active nuclei increases in proportion to trophoblast volume. The ratio of active to inactive nuclei remains constant at 3.9 : 1. These findings confirm that the majority of STB nuclei have intrinsic transcriptional activity, and that the STB is not dependent on CTB fusion for the provision of transcripts.

Introduction

The trophoblast is the epithelial covering of the human placental villous tree, forming the interface with the maternal circulation. It is a highly dynamic tissue, functioning in the active transport of nutrients between the maternal and foetal circulations, as well as producing and secreting hormones throughout gestation. The trophoblast also protects the foetus from attack by the maternal immune system. The correct functioning of the trophoblast at all stages of gestation is therefore vital for a healthy pregnancy outcome.

The villous trophoblast consists of two compartments: the proliferating cytotrophoblast cells (CTB); and the terminally differentiated syncytiotrophoblast (STB). The latter is unique in that it is a syncytium, sustained by continuous fusion of differentiating CTB cells throughout gestation. It has been shown repeatedly that STB nuclei lack proliferative capacity (Galton, 1962; Kar et al. 2007). Fusion of differentiating CTB cells incorporates new nuclei, organelles, cytoplasm and mRNAs into the syncytium.

The nuclei within the two trophoblast compartments display contrasting morphologies. CTB cells generally have a rounded nucleus, with a morphologically diffuse chromatin structure and a distinct nucleolus. In contrast, a range of nuclear morphologies is seen within the syncytium. Most STB nuclei have a smaller volume, a more convoluted nuclear envelope and contain greater amounts of condensed chromatin than CTB nuclei. During early pregnancy, approximately 50% of STB nuclei display a nucleolus, but as gestation advances this proportion falls (Martin & Spicer, 1973). Equally, in later pregnancy, aggregations of STB nuclei occur. These are uncommon in normal pregnancies before 20 weeks, and their incidence increases until term (Loukeris et al. 2010). Many of the aggregates are artefacts caused by tangential sectioning through villous branch points (Cantle et al. 1987), but some represent collections of nuclei with heavily-condensed chromatin. The latter are termed syncytial knots, and are uncommon before 32 weeks of gestation (Fox, 1965). This range of appearances within the STB nuclei suggests a process of ageing, with effete nuclei being shepherded into knots away from areas of diffusional exchange.

Cytotrophoblast cells undergo a process of differentiation before fusing with the STB. As part of this process, the nuclei show greater irregularity in shape and progressively more chromatin condensation than undifferentiated CTB (Mayhew et al. 1999). Nuclei in CTB cells about to fuse are heterochromatic, resemble resident STB nuclei, and still display a nucleolus. These appearances suggest that the nuclei are likely to still be transcriptionally active at the time of incorporation. Fusion is a complex event that is thought to involve the formation of gap junctions, and expression of the endogenous retroviral fusogenic proteins syncytin 1 and 2 (Rote et al. 2010).

In addition, it has been proposed that CTB differentiation and fusion involves activation of the apoptotic cascade, and that as a result the STB nuclei are transcriptionally inactive (Huppertz et al. 2006; Gauster et al. 2010). Apoptosis initiated in CTB nuclei through the proteolytic actions of capsase-8 is thought to be held in a latent state by anti-apoptotic proteins, such as BCL2 and MCL2, which are also incorporated upon fusion (Huppertz et al. 1999a). Syncytial knots are speculated to be the end-point of the cascade, with a currently unknown stimulus causing the re-entry of STB nuclei to the effector stages of apoptosis. This hypothesis suggests that the increasing nuclear condensation observed in syncytial knots as differentiation proceeds is a feature of apoptosis, and that the knots are eventually shed and deported into the maternal circulation.

Further support for the idea that the STB nuclei are transcriptionally inactive came from experiments demonstrating reduced incorporation of the radionucleotide [³H]- uridine into STB nuclei in villous explants, as compared with their CTB counterparts (Huppertz et al. 1999b). However, these experiments involved culturing explants for 1 h under 95% oxygen, 5% carbon dioxide at 2.5 atmosphere pressure. It has since been shown that these conditions are hyperoxic compared with conditions in vivo, and are likely to have caused STB death (Jauniaux et al. 2000). These doubts prompted a re-investigation of the transcriptional status of STB nuclei, which demonstrated that a proportion of STB nuclei are immunopositive for the active form of RNA polymerase II (RNA Pol II), suggesting they are transcriptionally active (Ellery et al. 2009). Activity was confirmed by the incorporation of fluorouracil into a similar proportion of the STB nuclei, and the presence of histone modifications associated with active gene expression. The authors concluded that transcriptional activity is higher in early gestation than at term, but did not provide quantitative data.

The present study sought further confirmation that some STB nuclei are transcriptionally active by investigating other markers of transcription, namely cAMP response element-binding protein (CREB) and upstream binding factor (UBF). The presence of the nucleolar transcription factor UBF was used to confirm that STB nuclei are also capable of RNA polymerase I (RNA Pol I)-driven transcription. Proliferating cell nuclear antigen (PCNA) was used to distinguish recently incorporated nuclei, showing that transcriptional activity was not a relic of fusion. Furthermore, the numbers of transcriptionally active and inactive STB nuclei were quantified at different gestational ages using unbiased stereological tools.

Materials and methods

Sample preparation

Blocks from paraffin-embedded placentas (n = 22; Table 1), ranging from 11 to 39 weeks of gestation and used previously for the estimation of STB nuclear number, were obtained from an archive collected earlier in accordance with ethical protocols (Mayhew et al. 1999). For each placenta, adjacent 5-μm sections were cut from three blocks selected at random.

Table 1.

Gestational ages of placentas used in this study

Gestational age (weeks)	Number of placenta used in study
11	1
13	1
14	2
15	2
19	2
20	1
22	2
29	2
30	2
31	1
37	2
38	2
39	1

Immunostaining

In preparation for immunofluorescence, sections were rehydrated in histoclear (2 × 5 min), graded ethanol (100, 90 and 70%, each for 5 min) and deionised water (10 min). Heat-induced antigen retrieval was performed by boiling sections in 0.1 m citric buffer (pH 6.0) in a pressure cooker. Sections were blocked for 30 min at room temperature in non-immune serum. Primary antibodies including phospho (p)-UBF (1 : 100; Santa Cruz), PCNA (Abcam, Cambridge, UK; 1 : 100), phospho (p)-CREB (1 : 100; Abcam), cytokeratin-7 (Dako, Ely, UK; 1 : 100) and RNA Pol II (1 : 200; 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 (TBS-TT; 3 × 5 min). Sections were incubated with species-specific Alexa-488 or -568 secondary fluorescent antibody for 1 h at room temperature (Molecular Probes, Invitrogen Detection Technologies, Leiden, the Netherlands). Sections were washed in TBS and water before mounting in Vectashield mounting medium, containing 4'-6-Diamidino-2-phenylindole (DAPI) (Vector labs, Peterborough, UK). Negative controls were performed by replacing primary antibody with equal volume of non-immune serum. Images were captured using a Leica confocal microscope. (LeicaTCS-NT; Leica Instruments GmbH, Germany).

To prepare samples for colourimetric immunohistochemistry, the same protocol was followed with the following additional steps. Endogenous peroxidases were quenched by incubating the sections in 3% H₂O₂ for 15 min. Biotin-labelled species-specific antibodies were added at a concentration of 1 : 200 and incubated for 1 h at room temperature. Vectastain Elite ABC kit (Vector Labs) and SigmaFast DAB (Sigma, Poole, UK) were used according to the manufacturers’ instructions. Sections were lightly counterstained with haematoxylin. Images were captured using a NanoZoomer slide scanner (NanoZoomer 2.0-RS; Hamamatsu Photonics, Hertsfordshire, UK).

Disector method of numerical density estimation

The numerical densities of RNA Pol II-positive and -negative STB nuclei were quantified using the physical disector method (Sterio, 1984; Mayhew & Gundersen, 1996). Pairs of adjacent sections of placental samples were cut at a thickness of 5 μm. Sections within the pair were designated as either ‘reference’ or ‘look-up’, whereby particles were selected in the reference section and counted in the look-up section. After immunohistochemistry staining for RNA Pol II, slides were scanned using a NanoZoomer. This software allowed the adjacent sections to be viewed simultaneously.

A counting frame of dimensions 50 × 50 μm, with 25 associated points was superimposed on each histological image. Because the area of the frame and the thickness of the section were known, the volume in which nuclei were counted could be calculated (area × thickness). One pair of contiguous vertical and horizontal sides of the frame was designated as the allowed lines, whereas the other pair, together with their extensions, was designated as the forbidden lines. Nuclei falling on the forbidden lines were not included in the count (Fig. 1).

Fig. 1.

Illustration of unbiased counting of nuclei in 3D space using the physical disector method. Pairs of adjacent sections are cut at a known thickness. A counting frame of known area is applied randomly to the reference section and the same region of trophoblast is located in the look-up section. As both the area of the frame and the thickness of section are known, counting is performed in a known volume of trophoblast. One set of perpendicular lines, together with their extensions, is designated the ‘forbidden line’ (bold lines) and the other are the ‘allowed lines’ (dashed lines). Nuclei are counted only if they fall within the allowed confines of the counting frame in the reference section but do not appear in the look-up section. In this example, the green nucleus is counted as it is within the allowed confines and only appears in the reference section. The nucleus annotated (1) is excluded as it falls on the forbidden line. The red nucleus annotated (2) is excluded as it also appears in the look-up section and so is not unique to the sampled volume.

Fields of view were identified on a systematic uniform random basis, and selected if the counting frame fell on an area of trophoblast. The same field of view was located on the look-up section. Nuclei in the reference section were counted only if they appeared in the reference section but not the look-up section, and were categorised according to their immunoreactivity. This allowed for the inclusion of nuclei that were located completely within the sampled volume. The number of frame-associated points falling on the trophoblast was also counted. This allowed us to estimate the proportion of the disector sampling volume that actually contained trophoblast.

Five counting frames were applied at random to each pair of sections. The numbers of nuclei and points were pooled, and the total volume of trophoblast sampled was calculated for the five frames. Counts were conducted blind.

The disector method was used to determine the number of RNA Pol II-positive STB nuclei in the defined sampling volume for each block. The numerical density was estimated from the following sequence of equations:

• Eq. 1: (counted frame points/total frame-associated points) = proportion of disector containing trophoblast;

• Eq. 2: volume of disector per sample = (50 × 50 × 5 μm) × 5 = 62 500 μm³;

• Eq. 3: volume of disector containing trophoblast = Eq. 1 × Eq. 2; and

• Eq. 4: numerical density of RNA Pol II-positive nuclei = total count/Eq. 3.

The numbers of nuclei and volume of trophoblast sampled across the three blocks were pooled to allow estimation of numerical density per placenta (expressed per cm³ of trophoblast). The numerical density was converted to number of nuclei by multiplying by the volume of trophoblast in each placenta. The placentas used had had trophoblast volumes calculated previously (Mayhew & Simpson, 1994).

Statistical analysis

The data were analysed using graphpad prism 5 (GraphPad Software). Relationships between gestational age, numerical density and absolute number were tested with Pearson's product moment coefficient. A significance level of 0.05 was used for all statistical tests.

Results

PCNA immunofluorescence

PCNA, which has a half-life of more than 20 h (Stewart & Dell'Orco 1992), was used to distinguish between recently incorporated nuclei and those more established in the syncytium. Double-immunofluoresecence for PCNA and RNA Pol II confirmed that all PCNA-positive STB nuclei were also positive for RNA Pol II, indicating that recently incorporated nuclei are transcriptionally active. It also confirmed that the majority of more established nuclei within the syncytium remain transcriptionally active (Fig. 2).

Fig. 2.

Immunofluorescence staining of proliferating cell nuclear antigen (PCNA; red) and RNA Polymerase II (RNA Pol II, green). Solid arrows indicate STB nuclei staining positive for RNA Pol II but not PCNA, as determined by their morphology and location in the syncytium. PCNA has a half life of > 20 h. Thus, it can be used to distinguish between recently fused nuclei and established, non-proliferating STB nuclei. Negative controls were performed by omission of primary antibody and showed that cross-staining did not occur. The intervillous space (IVS) and villous stroma (VS) are labelled.

pCREB immunohistochemistry

Ser-133 pCREB was selected as a marker for inducible RNA Pol II transcriptional activity. Double-immunohistochemical staining with pCREB and cytokeratin-7 was performed to confirm the location of the STB nuclei. Cytokeratin-7 is specific for CTB cells, thus allowing the two trophoblast nuclear populations to be distinguished (Fig. 3).

Fig. 3.

Detection of pCREB. (A, B) Immunohistochemistry for pCREB (brown) and cytokeratin-7 (red). Dashed arrows indicate immunopositive CTB nuclei, which are identified by the cellular staining for cytokeratin-7. Solid arrows indicate pCREB-immunoreactive STB nuclei, as determined by their morphology and location relative to the intervillous space (IVS) and villous stroma (VS). Asterisks show maternal red blood cells. Negative controls were performed by omission of primary antibody and showed non-specific binding was negligible. Scale bar 25 μm, unless otherwise stated. Gestational ages are shown in weeks.

Immunohistochemistry staining was performed on villi from five placentas, ranging from 15 weeks of gestation to term (15, 22, 22, 37 and 39 weeks gestational age). Immunoreactivity for pCREB was observed in a proportion of CTB, STB and stromal nuclei at all ages. The proportion of CTB nuclei staining for pCREB appeared to be higher than that of the STB nuclei, but this was not quantified. Non-specific binding of the secondary antibody was negligible, and background staining from maternal red blood cells in the intervillous space varied between samples.

pUBF immunohistochemistry

Serine 388 p-UBF was investigated as a marker for RNA Pol I-dependent transcription in another five placentas (14, 14, 19, 37 and 39 weeks gestational age). Dual-labelling with pUBF and cytokeratin-7 was used to locate pUBF to STB nuclei (Fig. 4). Stromal nuclei stained very intensely for pUBF in comparison to both STB and CTB nuclei, and CTB nuclei, in turn, stained more intensely than STB nuclei.

Fig. 4.

Confirmation of pUBF in STB nuclei. Immunohistochemistry for pUBF (brown) and cytokeratin-7 (red). Solid arrows indicate immunopositive STB nuclei. Dashed arrows indicate immunopositive CTB nuclei. Gestational age is shown in weeks. Villous stroma (VS) and intervillous space (IVS) are labelled. Scale bar 25 μm, unless otherwise stated.

Immunofluorescence co-localisation studies showed that a proportion of STB nuclei were positive for both RNA Pol II and pUBF. The presence of pUBF was observed in some RNA Pol II-positive STB nuclei (Fig. 5).

Fig. 5.

Co-localisation of phospho-upstream binding factor (pUBF) and RNA polymerase II (RNA Pol II). Immunofluorescence for pUBF (red) and RNA Pol II (green). Dashed lines indicate the syncytium and an underlying CTB nucleus. Villous stroma (VS) and the intervillous space (IVS) are labelled. Gestational ages are shown in weeks. (Scale bar: 8 μm.)

RNA Pol II immunohistochemistry

Serine-2 phosphorylated RNA Pol II was used to identify transcriptionally active nuclei in conjunction with their morphologies and locations within the trophoblast. Immunopositive CTB nuclei were more frequent than STB nuclei. There was more variation of staining within STB nuclear populations. Regions of entirely immunopositive and immunonegative nuclei were both observed. It was also noted that immunopositive and immunonegative nuclei could lie adjacent to each other within the syncytium (Fig. 6A–C). Immunopositive STB nuclei were commonly associated with an underlying immunopositive CTB nucleus (Fig. 6D). Negative CTB nuclei were generally associated with immunonegative STB nuclei (Fig. 6E). Negative controls showed no non-specific binding.

Fig. 6.

STB nuclei display different states of transcriptional activity. Immunohistochemistry for RNA Pol II was used to identify transcriptionally active nuclei. STB nuclei were identified on the basis of their relative size and location within the trophoblast. Black and red arrows indicate active and inactive nuclei, respectively. At all stages of gestation, both active and inactive STB nuclei were observed. Both transcriptional states were seen to lie adjacent to each other (A–C). Discrete stretches of inactive nuclei were also observed (not shown). Active STB nuclei seemed to correlate with the presence of an active underlying CTB nucleus, but inactive STB nuclei were seen adjacent to active CTB nuclei (D, E; dashed arrows indicate CTB nuclei).

Disector estimation of numerical density of RNA Pol II-positive and -negative STB nuclei

Pairs of adjacent sections were cut from three blocks per placenta for 22 placentas (range 11–39 weeks of gestation). Immunohistochemistry was performed to detect RNA Pol II-positive nuclei. Positive and negative nuclei were counted using the physical disector method, and estimates obtained of numbers of both types of nuclei per cm³ trophoblast (Fig. 7). The numerical density of RNA Pol II-positive nuclei was found to remain constant across gestation, with a mean of 47.27 × 10⁷ nuclei cm⁻³ (standard error = 2.044). There was no correlation between gestational age and numerical density (r = 0.39, P > 0.05).

Fig. 7.

The numerical density of RNA Pol II-positive STB nuclei per volume trophoblast remains constant across gestation. The disector method was used to calculate the number of RNA Pol II-positive STB nuclei in a sampled volume of trophoblast. Each data point represents the numerical density of each placenta (n = 22), which was calculated by pooling nuclear counts and sampled volumes from three blocks. There was no correlation between gestational age and numerical density (r = 0.39, P > 0.05). The mean was 47.27 × 10⁷ RNA Pol II-positive STB nuclei per cm³ trophoblast (standard error = 2.044).

The mean density of RNA Pol II- negative nuclei was 36.34 × 10⁷ nuclei cm⁻³ (standard error = 1.87). There was no correlation between numerical density and gestational age (r = 0.07, P > 0.05).

Calculation of absolute number of RNA Pol II-positive and -negative STB nuclei

The numerical densities of RNA Pol II-positive and -negative STB nuclei calculated for each sample were multiplied by the previously determined total trophoblast volume (Simpson et al. 1992) to obtain the absolute number of RNA Pol II-positive and -negative nuclei in each sample (Fig. 8). The results showed the absolute number of RNA Pol II-positive nuclei to increase exponentially as gestation progressed (R² = 0.89). Numbers of negative nuclei also increased across gestation (R² = 0.80). Despite these respective increases, the ratio of positive to negative nuclei within individual placentas remained constant across gestational age at 3.9 : 1.0.

Fig. 8.

The absolute number of RNA polymerase II (RNA Pol II)-positive and negative STB nuclei increases across gestation. Numerical densities of nuclei were converted to absolute numbers by using the previously calculated trophoblast volumes for each placenta (Simpson et al. 1992). Each data point represents the absolute number of each placenta. The absolute numbers were seen to increase exponentially across gestation (R² = 0.89058). Negative nuclei were indicated by open squares and positive nuclei were indicated by closed circles.

Discussion

Immunostaining for two markers of transcription, pCREB and pUBF, has provided further confirmation that a proportion of STB nuclei are transcriptionally active. In qualitative terms, pUBF-positive STB nuclei appeared to be more frequent in early gestation than at later stages. In contrast, pCREB appears to be present in more STB nuclei in later gestation than in early villous samples. These observations are based on 2D sectioning, and further quantitative methods are needed to confirm these results.

cAMP response element-binding protein is a ubiquitous transcription factor that acts in response to the secondary messenger cAMP. CREB drives the expression of target genes in response to a diverse range of signals, including hormones, growth factor and neuronal messengers (Shaywitz & Greenberg, 1999). Specific to placental gene expression, CREB has been shown to drive the expression of leptin and human chorionic gonadotropin (hCG) in trophoblast cell lines via mitogen-activated protein kinase (MAPK) and protein kinase A pathways, as well as regulating fusion during trophoblast differentiation (Maymo et al. 2010; Delidaki et al. 2011). First trimester explants have confirmed the role of CREB in human placental growth hormone expression (Depoix et al. 2011). In primary placenta cell culture cAMP has been shown to regulate corticotrophin-releasing hormone (Cheng et al. 2000).

cAMP response element-binding protein stimulates RNA Pol II-directed transcription via a constitutively active domain, but transcription may be enhanced by serine-133 phosphorylation in response to extracellular signalling (De Cesare et al. 1999; Kim et al. 2000). Because trophoblast development is largely driven by growth factor and hormone signalling, pCREB was investigated in this study (Mayr et al. 2001). The presence of Ser-133 pCREB in STB nuclei shown in this study adds to the current body of evidence that RNA Pol II-driven transcription, including inducible transcription, takes place in a subset of STB nuclei.

The presence of pUBF provided further evidence for transcriptional activity, and extends this to include RNA Pol I-driven transcription. UBF is a nucleolar transcription factor that is required for the activity of RNA Pol I. RNA Pol I drives transcription from 45S ribosomal genes to produce ribosomal RNAs that assemble into functional ribosomes (Glibetic et al. 1995). UBF activity is regulated by phosphorylation with Ser-388 phosphorylation being necessary for interactions with RNA Pol I. It has been shown that mutations at this residue are not able to initiate transcription (Voit & Grummt, 2001). These results show that a proportion of STB nuclei are RNA Pol I and RNA Pol II transcriptionally active.

Staining for RNA Pol II has shown that there is a range of transcriptionally active STB nuclei. Stretches of transcriptionally active STB nuclei, interrupted by a single inactive nucleus, suggest that transcription is tightly regulated at the nuclear level. Negative nuclei were found to make up between 20% and 40% of total nuclei. The continuous nature of the syncytium allows for the free movement of substances throughout the syncytium (Gaunt & Ockleford, 1986). It is possible that inactivation of transcriptional activity in STB nuclei may be in response to oxidative or other damage in the nucleus.

The marker used to determine transcriptional activity was the Ser-2 phosphorylated form of RNA Pol II. This is the active, elongating form of the enzyme, and is found only in actively transcribing nuclei (Palancade & Bensaude, 2003). The numerical densities of both transcriptionally active and inactive STB nuclei per unit of volume trophoblast were found to remain constant across gestation. This suggests that numbers of transcriptionally active nuclei are regulated tightly, both as the volume of trophoblast grows by CTB fusion and also as the syncytium thins to reduce the diffusion barrier in late gestation (Mayhew & Simpson, 1994). The numerical density of nuclei may be regulated at the level of CTB differentiation and fusion, through aggregation of condensed STB nuclei into syncytial knots or as a combination of both processes.

The absolute numbers of transcriptionally active and inactive nuclei in the syncytium were found to increase exponentially across gestation. This is consistent with a constant numerical density, but an increasing volume of trophoblast. These results highlight the importance of conducting studies in 3D when the trophoblast is concerned, as observations based merely on individual sections cannot accurately reflect the extensive changes in volume and branching patterns of the villous tree as pregnancy advances nor account for regional differences.

These results show that the syncytium is capable of producing its own transcripts and is therefore not wholly dependent upon the CTB for incorporation of mRNAs. Indeed, fusion may be revealed to be required for the incorporation of regulatory molecules or perhaps microRNAs that direct the transcriptional status of individual nuclei. Further research is needed to address this question. These current findings have major implications for our understanding of trophoblast biology, as they allow for tighter, more rapid regulation of transcription within the syncytium. The syncytium is located at a highly dynamic interface between maternal and foetal circulations. Changes in maternal blood concentrations, accumulation of wastes and growing foetal demands require appropriate responses from the syncytium.

If both the numerical density of transcriptional active STB nuclei, and the basal level at which they transcribe, remain constant across gestation, we can speculate that the transcriptional capacity of the syncytium can increase across gestation. This feature would allow for the syncytium to produce gene products such as hormones in the large quantities that are needed as term approaches. Support for this hypothesis comes from the fact that at term the STB produces 1–4 g human placental lactogen per day (Strauss & Barbeiri, 2009).

This assumption does not preclude the ability of STB nuclei to upregulate the level at which they transcribe specific genes. The fluctuations in hCG concentrations produced by the syncytium across gestation reflect this possibility. Median levels of hCG production are low at initial stages of gestation, but increase substantially at about 8 weeks of gestation. By the end of the first trimester, the levels begin to decrease but still remain higher than those of early gestation (Cole, 2010). It has been shown that the beta subunit of hCG is located specifically in the syncytium, and both alpha and beta subunits are required for hCG secretion (Gaspard et al. 1980). That the STB-specific gene expression is so temporally regulated would suggest that regulation is intrinsically controlled by the syncytium itself, and not by CTB incorporation.

This study did not quantify levels of transcription, or identify the transcripts being produced by the syncytium. Additionally, further studies are needed to determine signals regulating transcription in STB nuclei. Immunohistochemical staining reveals that there are regional differences of activity, with stretches of inactive nuclei being observed. The regulation of transcription may act on the level of individual nuclei as active nuclei were seen located adjacent to inactive nuclei. It would be interesting to investigate if this inactivity was due to nuclear damage and the syncytium had a mechanism of shutting them down in order to prevent aberrant gene expression and further complications.

In conclusion, we have provided further evidence for RNA Pol II-driven transcription in a proportion of STB nuclei. It has also been confirmed that active transcription by RNA Pol I takes place in some nuclei. The numerical density of active STB per volume trophoblast remains constant across gestation, but the absolute numbers of active nuclei increase exponentially. More research is needed to understand the regulation of transcription in STB nuclei and to determine what transcripts are produced. This study focused on placentas from normal pregnancies, and it would be of interest to determine if any difference in transcriptional activity can be identified in complicated pregnancies such as pre-eclampsia or intrauterine growth restriction.

Author contributions

NMEF performed the experimental work and data analysis, and drafted the manuscript. TMM contributed the placental blocks and advised over the stereological methodology. ACFS and GJB conceived of the study, and advised over the immunohistochemistry and presentation of the results.