Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases

Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase–metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I–MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI–MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI.
Introduction
In vitro oocyte maturation offers immense potential for treatment of infertility, however, current systems are relatively inefficient (Tan et al., 2007). Additionally, mammalian oocytes are notorious for high rates of chromosomal abnormalities (Hassold and Hunt, 2001), resulting in subsequent embryonic aneuploidy, infertility and congenital defects. Unfortunately, components of successful oocyte maturation and regulation of these events remains enigmatic. Therefore, understanding regulatory mechanisms involved in oocyte meiotic maturation, especially those controlling chromatin remodeling, is imperative to establish therapies to improve current assisted reproductive technologies and circumvent oocyte-derived infertility and aneuploidy-induced congenital defects.
Remodeling of chromosomes during oocyte meiosis begins when homologues initially pair and condense via actions of the synaptonemal complex during initiation of prophase I, accompanied by homologous recombination and crossing-over events (Vallente et al., 2006). Chromatin subsequently decondenses as oocytes enter a phase of quiescence prior to completing prophase I. In response to the pre-ovulatory gonadotropin surge, follicle-enclosed oocytes resume meiosis and homologues condense in preparation for a reductional division (Mehlmann, 2005). A bipolar meiotic spindle forms, consisting of polymerized microtubules, and attaches to homologues at their centromeres. Subsequently, physical contact between homologous pairs at chiasmata counteract forces pulling apart homologues, resulting in alignment of chromosomes along the metaphase plate, signaling completion of metaphase I (MI). The meiotic spindle then facilitates separation and segregation as homologues are pulled toward opposite spindle poles at the beginning of anaphase. Oocytes progress through telophase, resulting in disproportionate cytokinesis and extrusion of the first polar body signaling completion of meiosis I (Wang and Sun, 2006). Subsequently, oocytes forego DNA replication and re-arrest at MII.
Reversible phosphorylation, controlled via actions of protein kinases and phosphatases, is an extremely important regulator of oocyte meiosis, and well-suited to the rapid changes required during the cell cycle (see reviews, Swain, 2007; Swain and Smith, 2007). Extensive research has been conducted on the role of kinases such as cyclin-dependant kinase (CDK1) and mitogen-activated protein kinase (MAPK) in mammalian oocytes (Motlik et al., 1998; Abrieu et al., 2001). Additionally, roles for protein phosphatases (PPP) in control of oocyte germinal vesicle breakdown (GVBD) (Alexandre et al., 1991; Gavin et al., 1991, 1994; Swain et al., 2003), spindle dynamics (Alexandre et al., 1991; Lu et al., 2002) and chromatin remodeling have been identified (Mailhes et al., 2003; Swain et al., 2007). However, relatively little attention has been paid to aurora kinases (AURK) in regard to mammalian oocyte maturation.
AURKs are a family of serine/threonine protein kinases responsible for regulating several mitotic cell cycle events important to maintaining proper cellular ploidy, including chromosome condensation, spindle dynamics and cytokinesis (see review, Carmena and Earnshaw, 2003). Thus, considering high rates of aneuploidy associated with oocyte meiosis, AURKs may play an instrumental role in the female gamete was well. Based primarily on research from lower eukaryotes and mitotically dividing mammalian cells, three AURKs exist categorized by sequence differences and subcellular localization; AURKA, AURKB and AURKC (Nigg, 2001). Aurora-A has been identified in mammalian oocytes localized to nuclei in GV-intact oocytes; to the spindle, spindle poles and condensing chromatin during MI; and to spindle poles at MII (Yao and Sun, 2005). Neutralization of oocyte AURKA activity delays GVBD and distorts MI spindle organization (Yao et al., 2004). Aurora-B kinase is known as the equatorial kinase and localizes to condensed chromatin during mitosis and meiosis in lower eukaryotes, where it is thought to regulate chromosome condensation (Hsu et al., 2000; Murnion et al., 2001) and homologue separation (Kaitna et al., 2002; Rogers et al., 2002). Aurora-C kinase is the least studied AURK isoform, previously reported to be testes-specific with localization to centrosomes (Tseng et al., 1998; Kimura et al., 1999; Hu et al., 2000); though transcript for AurkC has now been identified in human cumulus–oocyte complexes (Assou et al., 2006) and reports indicate that it binds to chromosomal passenger proteins during mitosis (Li et al., 2004; Sasai et al., 2004; Yan et al., 2005).
Objectives of this study were to determine which Aurk isoform transcripts are present in oocytes; examine functional roles of AURK activity during various meiotic stages of mouse oocyte maturation, and identify AURK phosphoprotein substrates and targets of actions, focusing primarily on regulation of chromatin remodeling during the first meiosis.
Materials and Methods
All procedures described within were reviewed and approved by The University Committee on Use and Care of Animals at the University of Michigan and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Mouse stimulation and oocyte collection
Meiotically incompetent GV-intact oocytes were collected from 11-day-old female CF1 mice (Harlan, Indianapolis, IN). Meiotically competent GV-intact oocytes were collected from 20–23-day-old CF1 female mice, 42–44 h following injection with 10 IU eCG (Sigma, St Louis, MO). Oocytes were isolated by manual rupturing of antral ovarian follicles in Hepes-buffered human tubal fluid medium (HTFH; Irvine Scientific, Santa Ana, CA) supplemented with 0.3% w/v polyvinylpyrrolidone (Sigma).
RNA isolation, reverse transcription and real-time PCR
Oocyte total RNA was extracted from 50 oocytes at each development stage using Picopure RNA isolation kit (Arcturus Bioscience, Mountain View, CA) following manufacturer’s instructions. Oocyte cDNA was synthesized using 125 pmol random hexamer, 500 µM dNTP, 20 U RNase inhibitor and 62.5 U MultiScribeTM reverse transcriptase (ABI systems) in a final volume 50 µl. Primers for mouse AurkA, AurkB and AurkC were designed with no sequence overlap between isoforms (AurkA—forward primer: 5′ cactagcaaagagccaacca 3′, reverse primer: 5′ ggtggcttcaatagggtgtt 3′; AurkB—forward primer: 5′ cctgacctactgccacaaga 3′, reverse primer: 5′ gccaaagtctgcaatcttca 3′; AurkC—forward primer: 5′ ctgccatgagaagaaggtga 3′, reverse primer: 5′ gtccagagtcccacacattg 3′). Real-time PCR was performed on Applied Biosystems 7300 Real-Time PCR system. Each PCR was performed with 1.5 oocyte equivalents of cDNA added to SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA). In addition, control reactions were conducted consisting of no template with primers and master mix. Real-time PCR reactions were carried out for 40 cycles (95°C for 15 s, 60°C for 1 min) after initial 10 min incubation at 95°C. Following PCR, products were isolated and run on a 2% agarose gel for 60 min at 100 V to verify size of the amplified product. Additionally, DNA was isolated from gels using QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA) and subjected to DNA sequencing to verify identity of the product. Standard curve method was used to compare relative abundance of a single Aurk isoform between oocyte meiotic stages using normalization of β-actin levels. Data were collected over three replicates, with triplicate samples for each isoform and fold increases were based on meiotically incompetent GV-intact oocytes levels, which were normalized to 1. Statistical significance was determined using unpaired Student’s t-test, P < 0.05). To examine relative abundance of all three isoforms within a single time point, we ensured primer efficiency of all samples were within a 5% range of an internal β-actin control. We then analyzed data using comparative Ct method.
Oocyte culture and AURK inhibition
Aurora kinase inhibitor ZM447439 (ZM, Astra Zeneca, Wilmington, DE) was dissolved in dimethylsulphoxide (DMSO) to obtain a 10 mM stock. Stock solution was dissolved in HTF to obtain final concentrations of 0.625, 1.25, 2.5, 5, 10 and 20 µM. Control treatments contained DMSO.
To assess effects of AURK inhibition on oocyte maturation, meiotically competent GV-intact oocytes (prophase I) were placed into culture in presence or absence of varying doses of ZM. Oocytes were assessed for GVBD and MII development at 2 and 16 h, respectively. Experiments were performed in triplicate and statistical differences in development were assessed using chi-square analysis with differences considered significant if P < 0.05.
To determine effects of AURK on chromosome condensation and spindle formation during the prophase I to MI transition, prophase I oocytes were matured in vitro to MI (7 h) in the presence or absence of ZM (10 µM) then subjected to immunocytochemistry (ICC) or processed for chromosome spreading. Prophase-I oocytes were also cultured to a time point consistent with MII to assess spindle and chromatin characteristics following extended AURK inhibition.
To assess effects of AURK inhibition on oocyte meiosis during the MI–MII transition, oocytes were matured for 7 or 9 h in the absence of any chemical manipulation to allow normal spindle formation and chromatin remodeling. Oocytes were then cultured to MII (an additional 9 or 7 h) in presence or absence of 10 µM ZM, followed by assessment of chromatin positioning and spindle configuration. All experiments were performed in triplicate and nonparametric parameters were analyzed for significant differences by Chi-square.
Finally, to begin to determine substrates for oocyte AURK, histone-H3 phosphorylation at ser10 and ser28 was assessed following AURK inhibition at various time points utilizing ICC and western blot analysis.
Immunocytochemistry
To examine effects of AURK inhibition on spindle formation and metaphase chromosome positioning, MI and MII oocytes were attached to poly-lysine coated coverslips, and fixed in 2% (w/v) paraformaldehyde with 0.05% (v/v) Triton X-100 in phosphate-buffered saline (PBS) (pH = 7.3) for 30 min. Oocytes were then blocked overnight with 2% (w/v) bovine serum albumin, 0.1 M glycine and 5% (w/v) dry milk in PBS at 4°C. Oocytes were incubated with β-tubulin antibody (Sigma, 1:200) and pericentrin antibody (Abcam, 1:1000). Negative controls included non-immune mouse serum in place of primary antibody. After three 5 min washes with blocking solution, samples were reacted with the appropriate Alexa 568 and 488 conjugated secondary antibodies (Molecular Probes) at a 1:750 dilution for 1 h at 37°C. Following washing, slides were incubated with Hoescht 33 342 (1 µg/ml) in PBS for 20 min at 37°C. Coverslips were then mounted on glass slides with 90% glycerol in PBS for fluorescence microscopic visualization under ×1000 on a confocal microscope.
Chromosomal spreading and analysis
Following culture to MI in ZM (10 µM), oocytes were collected and prepared for chromosomal spreading (Hodges and Hunt, 2002). Briefly, zona pellucida were removed by exposure to 1% pronase in HTFH. Zona-free oocytes were then washed and fixed by carefully placing them onto a microscope slide dipped in a solution of 1% paraformaldehyde in distilled water (pH 9.2) containing 0.15% Triton X and 3 mM dithiothreitol. Slides were then placed into a humidified chamber overnight before being subjected to triplicate 5 min washes in PBS and air-dried at room temperature. To analyze chromosomal condensation, slides were placed into a 1% solution of Hoescht 33 342 in PBS for 10 min and subjected to three more washes in PBS. Glycerol mounting solution and a coverslip were added and slides were sealed. Chromosomal spreads were analyzed blind to treatment at ×1000 on a Leica DMR microscope. Statistical differences between treatment groups were analyzed using chi-square analysis.
Electrophoresis and western blot analysis
To assess effects of AURK inhibition on oocyte histone-H3 phosphorylation, groups of oocytes (n = 100) were prepared for western blot analysis. Oocytes were placed in 2× sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) sample loading buffer [80 mM Tris–HCl (pH = 6.8), 20% glycerol, 4% SDS, 4% β-mercaptoethanol, 0.04% bromophenol blue], vortexed and placed on ice for 15 min. Following sonication on ice for 10 s, samples were denatured at 90°C for 10 min and loaded for electrophoresis. Total protein from equal numbers of mouse oocytes was loaded in each lane and separated by one-dimensional SDS–PAGE. Resolving gels were cast using 12% acrylamide; stacking gels contained 5% acrylamide. HeLa cell histone lysate was used as a positive control for recognizing phospho-ser10. Gels were equilibrated and transferred to Hybond-P PVDF transfer membrane (Amersham Life Science, Little Chalfont Buckinghamshire, UK) by Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Blots were blocked in 5% nonfat milk in Tris-buffered saline (TBS)+0.5% Tween (TBST) at room temperature for 1 h and incubated with the appropriate primary antibody diluted in TBST + 5% nonfat milk overnight at 4°C with agitation. Antibodies included anti-phospho-ser10-histone H3 (1:1000, Upstate) and anti-phospho-ser28-histone-H3 antibody (1:500, Upstate). After complete washing in TBST, blots were incubated with the appropriate horse-radish peroxidase-conjugated IgG secondary antibody (diluted 1:2000) at room temperature for 1 h, washed in TBST and developed with ECL Plus reagents (Amersham Life Sciences) according to the manufacturer’s instructions. To verify equal protein loading of lanes to allow densitometric analysis blots were stripped for 30 min in a 50°C water bath with agitation in a stripping buffer (62.5 mM Tris–HCl, pH 6.7, 100 mM β-mercaptoethanol and 2% SDS). Completely stripped blots were blocked in 5% nonfat milk in TBST for 1 h at room temperature, then incubated with histone-H3 antibody (diluted 1:1000, Chemicon) overnight at 4°C with agitation and processed further as described above. Band densities were assessed using NIH imaging software, Image J.
Results
AURK isoforms in mouse oocytes
To determine specific AURK isoforms present in mouse oocytes, isoform-specific primers were designed for AurkA, AurkB and AurkC. Transcripts were amplified utilizing real-time PCR for all three isoforms in meiotically incompetent GV-intact oocytes. Significant increases in transcript levels for all three Aurk isoforms were identified in meiotically competent GV-intact and meiotically maturing oocytes, compared with incompetent oocytes (Fig. 1). No differences in transcript levels were identified for any isoform between meiotically competent GV-intact, GVBD, MI or MII oocytes. Additionally, AurkA appeared to be the predominant isoform transcript, displaying an approximate 12-fold increase compared with AurkB and AurkC, which displayed comparable levels, in meiotically competent GV-intact oocytes (data not shown). Single bands were present on agarose gels for each isoform, corresponding to predicted sizes (AurkA—90 bp, AurkB—100 bp, AurkC—145 bp). Sequencing verified that amplified gene products shared 100% homology with mouse Aurk isoforms (data not shown).
Figure 1:
Graphical representation demonstrating fold-increases of aurora kinase (Aurk) isoform levels between various oocyte types obtained from real-time PCR.
Day 11 germinal vesicle intact (GVI) oocytes were used at controls and normalized to 1. (A) AurkA, (B) AurkB and (C) AurkC. Presence of an asterick represents statistical significance compared with groups without an asterick, P < 0.05.
AURKs and oocyte meiotic progression
To determine the influence of AURK on oocyte meiotic progression, meiotically competent GV-intact oocytes (prophase I) were matured in varying doses of the AURK inhibitor ZM and development was checked at 2 and 16 h. Though a significant reduction was observed with 2.5 μM, Inhibition of AURK had no effect on oocyte GVBD at 2 h at any other dose examined compared with control treatments (Table I). Furthermore, no differences were visually apparent at the light microscope level between ZM-treated and control oocytes at a time point consistent with MI development (7 h; Fig. 2, inset). However, concentrations of ZM at 2.5, 5, 10 and 20 µM prevented all oocytes from progressing to MII at 16 h (Fig. 3). Concentrations of 1.25 and 0.675 µM allowed a small portion of oocytes to extrude the first polar body at 16 h, which was significantly less than the percentage of MII oocytes obtained from control treatments, P < 0.01 (Table I). Additionally, culturing GV-intact oocytes for 7 h to MI in presence of 5 or 10 µM ZM, followed by thorough washing and 9 h of culture in the absence of the AURK inhibitor, indicated that, although ZM may be washed out from blocking the ATP binding pockets, defects caused by ZM treatment were not reversible, as oocytes were unable to complete meiosis and extrude the first polar body (data not shown). Based on these data, a dose of 10 µM ZM was selected for future experiments to ensure all AURK isoforms were inhibited. This dose has been used in other studies on mammalian oocytes (Jelinkova and Kulbeka, 2006), and is lower than the 20 µM used in other studies (Gadea and Ruderman, 2005).
Figure 2:
Representative micrographs demonstrating effect of aurora kinase (AURK) inhibition on oocyte meiotic spindle formation.
Inhibition of mouse oocyte AURKs with 10 µM ZM447439 (ZM) for 7 h had no observable effect on oocyte development at the light microscope level (inset images). However, immunocytochemical examination demonstrated that morphology of the meiotic spindle of ZM-treated oocytes appeared abnormal, with polymerized microtubules (β-tubulin: green), microtubule organizing centers MTOCS (pericentrin: red/orange) and chromatin (blue) was scattered around the metaphase plate, compared with vehicle treated controls. Furthermore, 16 h treatment of oocytes with ZM resulted in arrest of oocytes at a MI-like stage (inset). Immunocytochemical examination demonstrated aberrant polymerized microtubules, MTOCs and condensed chromatin (blue) and MTOCs (red/orange). Polar body of control MII oocytes is indicated by PB.
Figure 3:
Representative micrographs of oocyte chromosome spreads demonstrating negative impact of AURK inhibition on oocyte chromatin remodeling.
Treatment of oocytes with ZM447439 (ZM-10 µM) for 7 h resulted in metaphase I oocytes (MI) with significantly greater defects in bivalent formation, compared with controls (P < 0.0001).
Table I.
Development of mouse oocytes following treatment in varying doses of Aurora kinase inhibitor ZM447439 (ZM).
ZM concentration (µM)
2 h GVBD
16 h MII
0
115/121 (95%)a
38/52 (73%)a
0.625
29/30 (97%)
8/30 (27%)b
1.25
30/35 (86%)
8/26 (31%)b
2.5
84/99 (85%)b
0/54 (0%)c
5
59/66 (89%)
0/28 (0%)c
10
55/64 (86%)
0/28 (0%)c
20
55/64 (85%)
0/28 (0%)c
Significant differences in development between treatments within a time point are indicated by different superscripts, P < 0.01.
AURKs and oocyte spindle morphology and chromatin remodeling
Prophase–MI transition
To begin to determine a temporal window when AURK inhibition may be conveying observed phenotypes, experiments examined the effects of AURK inhibition during the prophase I–MI transition. Aberrant spindle morphology and improper positioning of chromatin were observed in oocytes cultured for 7 h in presence of 10 µM ZM (19% normal, n = 32) compared with controls (91% normal, n = 35; P < 0.0001; Fig. 2). Similar patterns were also obtained from treatments containing 2.5, 5 and 20 µM ZM (data not shown).
To examine effects of AURK inhibition on oocyte chromosome condensation in greater detail, chromosomal spreads of MI oocytes were examined following 7 h of ZM (10 µM) treatment from prophase I. Inhibition of AURKs resulted in oocytes with a significant reduction in normal chromosome condensation (0%, n = 21) compared with control treatments (95%, n = 22), as evidenced by the inability to resolve bivalents, P < 0.001 (Fig. 3).
Prophase I–MII transition
Culture of oocytes for 16 h in 10 µM ZM during the prophase I–MII transition indicated that arrest of oocytes was not due to inability of microtubule polymerization as β-tubulin staining indicated microtubules polymerized around chromatin (Fig. 2). Additionally, pericentrin staining indicated apparent microtubule organizing centers (MTOC) assembly. However, spindle formation and MTOC localization were disrupted compared with untreated controls. Furthermore, chromatin was scattered throughout the meiotic spindle, with the pattern of normal chromatin remodeling significantly reduced following ZM treatment (0%, n = 44) compared with controls (81%, n = 32), P < 0.001.
MI–MII transition
To determine effect of AURK inhibition following normal chromatin remodeling and spindle formation, oocytes were cultured for 7 or 9 h in the absence of ZM (cells typically at MI or AI, respectively). Subsequently, these oocytes were cultured to a time point consistent with MII (an additional 9 or 7 h) in presence or absence of ZM. Treatment of oocytes for 9 h with ZM resulted in significantly less cells progressing to MII (53%, n = 117) compared with 80% of control oocytes (n = 121), P < 0.05. Treatments of oocytes for 7 h with ZM also resulted in significantly fewer cells progressing to MII (58%, n = 88), compared with controls (73%, n = 78; P < 0.05). All MII oocytes obtained following ZM treatment displayed scattered chromatin around the metaphase plate and slightly irregular shaped spindles (Fig. 4).
Figure 4:
Representative micrographs of oocytes cultured to a time point consistent with MII in presence or absence of 10 µM AURK inhibitor ZM447439 during the MI-MII transition.
Control MI oocytes developed normally to MII, extruding the first polar body and displaying condensed chromatin on the metaphase plate (blue) within the meiotic spindle with normally condensed β-tubulin (red) (A). However, following AURK inhibition, a portion of oocytes arrested prior to MII, displaying scattered chromatin within a malformed meiotic spindle (B). Those oocytes that did complete MII under AURK inhibition displayed disorganized chromatin within the meiotic spindle (C). Similar patterns of chromatin disorganization and inability to complete cytokinesis and progress to MII were also observed following culture of oocytes to MII (7 h) following AURK inhibition from a time point where cells are beginning to enter anaphase (E and F) compared with controls (D).
AURKs and oocyte histone phosphorylation
Because of reported roles for histone-H3 phosphorylation in chromatin remodeling, phosphorylation of histone-H3 following AURK inhibition was examined as a possible cause of aberrant condensation during the prophase I to MI transition. Western blot analysis and ICC utilizing phospho-ser10 histone-H3 antibody was performed. Inhibition of AURK for 2 or 7 h with 10 µM ZM resulted in a total lack of histone-H3 ser10 phosphorylation (Fig. 5A and B). Treatment of oocytes with ZM also completely inhibited phosphorylation of histone-H3 at ser28, as evidenced by ICC and western blot (Fig. 6A and B). These data raise the possibility that a ZM-sensitive AURK is an oocyte histone-H3 kinase and that histone-H3 phosphorylation may be influencing normality of oocyte metaphase chromatin condensation and subsequent separation and segregation of homologues.
Figure 5:
Representative micrographs and western blot demonstrating inhibition of oocyte aurora kinases (AURK) results in ser10-histone-H3 hypophosphorylation.
(A) Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown(GVBD) in presence of 10 µM ZM447439 resulted in a total lack of ser10-Histone-H3 phosphorylation (d), compared with controls (a). A similar reduction in ser10 phosphorylation was observed following 7 h (MI) of culture in the presence of ZM (j) compared with controls (g). Chromatin was stained with Hoescht and is pictured in blue (b,e,h,k). Overlays are also indicated (c,f,i,l). (B) Western blot analysis confirmed that ZM treatment inhibits ser10 phosphorylation (n = 100 oocytes/lane).
Figure 6:
Representative micrographs and western blot demonstrating inhibition of oocyte aurora kinases (AURK) results in ser28-histone-H3 hypophosphorylation.
Chromatin is stained in blue, while phospho-ser28-histone-H3 is stained in red. (A) Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown(GVBD) in presence of 10 µM ZM447439 (ZM) resulted in a total lack of ser28 phosphorylation (b), compared with controls (a). A similar reduction in ser28 phosphorylation was observed following 7 h (MI) of culture in the presence of ZM (d) compared with controls (c). (B) Western blot analysis confirmed that ZM treatment inhibits ser28 phosphorylation (n = 100 oocytes/lane).
Discussion
Maintaining integrity of chromatin remodeling is especially important in the oocyte considering its extreme susceptibility to aneuploidy, primarily during the first meiosis (Hassold and Hunt, 2001). AURKs are a family of serine/threonine kinases that regulate various structural elements and mechanistic events associated with the dynamic process of chromatin remodeling. Therefore, examination of AURKs during oocyte maturation is of interest when attempting to discern causative factors and molecular signaling pathways involved in aberrant oocyte chromosome modifications. We have determined that mouse oocytes contain transcripts for all three Aurk isoforms: AurkA, AurkB and AurkC and that levels of these transcripts increase significantly as oocytes gain meiotic competence, but do not change as oocytes progress through meiosis to MII. The predominant Aurk isoform transcript in meitocally competent and maturing oocytes appears to be AurkA. In agreement with these findings, during preparation of this manuscript, transcripts for AurkA, AurkB and AurkC were also reported in fully-grown immature bovine oocytes, with AurkA as the predominant isoform (Uzbekova et al., 2007).
To determine effects of AURK on oocyte maturation, we utilized the highly selective pharmacological AURK inhibitor, ZM. Inhibition of AURKs via ZM occurs through blockage of the ATP binding site at an adjacent cleft not present in other kinases (Ditchfield et al., 2003). Although ZM is a selective inhibitor of AURKs, it does inhibit other kinases, including CDK1, MAPK and CDC25. However, concentrations of ZM much higher than those utilized in the majority of our studies (20 µM) had no effect on CDK1, CDC25 or MAPK activities in Xenopus egg extracts, indicating ZM did not directly affect these kinases, or affect any upstream regulatory kinases involved in their activation (Gadea and Ruderman, 2005). Furthermore, cellular characteristics and phosphorylation patterns following inhibition of CDK1 (Marchal et al., 2001; Kubelka et al., 2002; Swain et al., 2003; Bui et al., 2004) and MAPK (Tong et al., 2003; Yu et al., 2007) in mammalian oocytes are dramatically different than those observed following ZM treatment in this study; suggesting observed effects are indeed the result of AURK inhibition. It should be mentioned that ZM displays differential inhibitory action toward different AURK isoforms, demonstrating inhibition of AURKB ∼20 times more potently than AURKA (Girdler et al., 2006). Thus, differential phenotypes observed in our study using lower concentrations of ZM may indicate AURKB-specific functions. Future experiments will attempt to determine if lower doses of ZM, as well as utilization of other AURK inhibitors with differential inhibitory actions, can verify this and determine isoform-specific functions within the mammalian oocyte.
AURK do not appear to play a role in oocyte meiotic resumption or regulation of oocyte nuclear envelope (NE) integrity during GVBD. Although ZM appeared to cause a slight delay in NE disassembly, no significant differences were apparent at any dose of ZM examined other than 2.5 μM. This is in agreement with findings that inhibition of bovine oocyte AURKs with VX680 has no effect on GVBD (Uzbekova et al., 2007). However, these findings are in contradiction to a study utilizing pig oocytes, which demonstrated lack of GVBD following exposure to elevated doses of ZM (7–10 µM; Jelinkova and Kubelka, 2006). Difference may be the result of varying experimental conditions or species-specific differences, as Jelinkova and Kulbelka (2006) utilized porcine cumulus–oocyte complexes from abattoir ovaries, whereas the current study utilized denuded oocytes obtained from gonadotropin stimulated mice.
In our study, inhibition of AURKs during 16 h of culture inhibited progression to MII and polar body extrusion, similar to developmental observations in clam oocytes following treatment with another AURK inhibitor, Hesperadin (George et al., 2006), and bovine oocytes treated with VX680 (Uzbekova et al., 2007). Failure to complete cytokinesis has also been observed following AURK inhibition during mitosis (Ditchfield et al., 2003). Evidence exists suggesting AURKB may be the specific isoform responsible for failure of oocytes to extrude the first polar body, as specific inhibition of AURKB in Drosophila cells prevented cytokinesis (Adams et al., 2001; Giet and Glover, 2001). Interestingly, AURKB was localized to the region of the contractile ring in bovine MII oocytes; (Uzbekova et al., 2007). However, AURKA and AURKC were also found in the vicinity of the contractile ring in bovine oocytes, confounding interpretation. Whether failure to complete cytokinesis and extrude the polar body in our study is a primary effect of AURK inhibition on cytokinesis, or a secondary effect due to chromosome remodeling or spindle defects remains to be elucidated.
To begin to determine possible causes for oocyte arrest prior to MII, we examined effects of AURK inhibition on chromatin remodeling and spindle formation during the prophase I–MI transition. Inhibition of AURKs during this time point resulted in improper positioning of chromatin, as evidenced by scattering throughout the meiotic spindle. This is in agreement with recent studies of AURK inhibition in mouse (Wang et al., 2006), pig (Jelinkova and Kubelka, 2006) and bovine oocytes (Uzbekova et al., 2007) reporting abnormal chromosome positioning at MI. Additionally, chromosomal spreading in our study indicates AURK inhibition negatively affects ability of MI oocyte chromosomes to condense properly and resolve bivalents. This may be due to premature decondensation, similar to that observed following AURK inhibition in Xenopus egg extracts (Gadea and Ruderman, 2005). It has been reported that AURKB inhibition via RNAi is responsible for chromosome misalignment in Drosophila cultured cells and results in amorphous chromatin (Adams et al., 2001) and only partial condensation (Giet and Glover, 2001). Thus, AURKB may be the AURK isoform responsible for chromatin defects observed in mammalian oocytes in this study. Indeed, a recent report demonstrates AURKB localized to condensed chromatin in MI and MII bovine oocytes (Uzbekova et al., 2007).
Failure of ZM-treated oocytes to progress to MII does not appear to be the result of inability to polymerize microtubules or form MTOCs, as indicated by β-tubulin and pericentrin staining. This is in contrast to AURK inhibition studies in Xenopus egg extracts, where ZM treatment resulted in failure to form the mitotic spindle (Gadea and Ruderman, 2005). However, fidelity of spindle function in our study remains in question, as spindle morphology and MTOC localization was disrupted following AURK inhibition. AURKs, such as AURKA, regulate several components of the spindle apparatus and spindle poles (see review, Ducat and Zheng, 2004). Indeed, AURKA localized to spindle poles in mouse (Yao et al., 2004) and pig oocytes (Yao and Sun, 2005), but not bovine oocytes (Uzbekova et al., 2007) and specific neutralization of the kinase resulted in disorganization of the meiotic spindle. This disorganization is in agreement with initial reports of ZM influences on intact somatic cells (Ditchfield et al., 2003). Thus ZM inhibition of oocyte AURKA may account for observed defective spindle phenotypes. Alternatively, condensed chromosomes direct formation of the spindle apparatus via nucleation/stabilization of microtubules (Merdes and Cleveland, 1997; Khodjakov et al., 2000). Therefore, defects in meiotic spindle morphology and scattering of chromatin observed in these experiments may be the result of aberrant chromatin condensation, possibly controlled by AURKB (see review, Shannon and Salmon, 2002). Reports in Xenopus mitotic cell-free extracts indicate AURK inhibition does interfere with chromatin driven microtubules assembly (Gadea and Ruderman, 2005).
To begin to determine if the inability of ZM-treated oocytes to reach MII following AURK inhibition was due only to defects incurred during the prophase–MI transition, or if AURKs had roles at other meiotic transition time points, we matured oocytes in vitro to time points consistent with MI and AI oocytes, thus allowing normal spindle formation and chromatin remodeling to occur. Subsequently, we then cultured oocytes in presence of ZM to a time point consistent with MII. Inhibition of AURKs during the MI-MII transition resulted in a portion of oocytes failing to segregate chromosomes and extrude the first polar body, while those that did complete MII displayed severely scattered chromatin. Thus, it appears as if AURKs may not only control spindle formation and chromatin condensation during early meiotic events, but also regulate separation and/or segregation of oocyte chromosomes during later meiosis. Effects may be directly on meiotic spindle components, possibly regulated by AURKA. Alternatively, defective chromosome remodeling could also be due to ZM inhibition of AURKB. Aurora B regulates kinetochores and their interactions with microtubules (Kaitna et al., 2002; Cimini et al., 2006). Another possible explanation for aberrant separation/segregation following AURK inhibition during late oocyte meiosis may be interferences with regulation of cohesion. Aurora B regulates release of chromosome cohesion during meiosis in Caenorhabditis elegans, apparently via phosphorylation of REC-8 (Rogers et al., 2002). Future studies will attempt to determine if defects include premature separation of sister chromatids due to premature release of cohesion, or if aberrant phenotypes are the result of failure to separate homologous chromosomes.
To begin to determine possible targets of oocyte AURKs responsible for observed defects in chromosomal remodeling, we examined the phoshorylation state of histone-H3 at ser10 and ser28. It is thought that phosphorylation of histone-H3 may cause the histone to act as a receptor or recruitment factor for condensation factors (Hirano, 2000), or possibly reduce the affinity of histone-H3 for DNA and make the relatively compact chromatin fiber more readily accessible to remodeling factors (Hirano, 2000), such as the condensin complex. Condensin is a multi-subunit protein complex that play a central role in chromosome compaction and condensation and is reported to co-localize and bind with phosphorylated histone-H3 (Schmiesing et al., 2000; Ball et al., 2002). Interestingly, AURKB controls association of condensin with chromosomes during mitosis (Lipp et al., 2007; Takemoto et al., 2007), and may be functioning in a similar manner during mammalian oocyte meiosis, though this remains unknown. It should be mentioned that reports for the requirements of histone-H3 phosphorylation in chromatin condensation varies greatly (Van Hooser et al., 1998; Wei et al., 1998, 1999; Goto et al., 1999; Kaszas and Cande, 2000; Schmitt et al., 2002). These contradictions also appear to hold true in regard to oocyte meiosis, with differing reports on temporal and spatial localization and correlation with condensation (Jelinkova and Kubelka, 2006; Wang et al., 2006; Swain et al., 2007). However, contradictions may be explained by differences in experimental conditions or perhaps species-specific differences. Regardless, contradictions in the literature make it increasingly evident that differences in the role of histone phosphorylation exist depending on the organism and/or type of cellular division examined (Fuchs et al., 2006), and that the specific histone-H3 AURK within mammalian oocytes remains unknown.
In summary, these studies indicate AURK plays a significant role in mouse oocyte maturation involving progression to MII, acting in both early (prophase–MI transition) and late meiotic events (MI–MII transition). When AURK is inhibited in early meiosis, microtubules polymerize and MTOCs form, but spindle morphology and MTOC localization is disrupted. Furthermore, metaphase chromatin does not condense or position normally. Inhibition of oocyte AURKs during late meiosis (MI–MII transition), following chromatin condensation and spindle formation, negatively affects separation and segregation of chromosomes. This aberrant chromatin remodeling following AURK inhibition in oocytes appears to be due, in part, to hypophosphorylation of histone-H3 at both ser10 and ser28. We report amplification and sequencing of all three Aurk isoforms transcripts: AurkA, AurkB and AurkC. Levels of these transcripts increase as oocytes achieve meiotic competence, with AurkA being the predominant isoform. Future studies will focus on determining protein expression levels of AURK isoforms, as well as specific functional roles and intracellular targets.
Funding
Funding for research by Gary D. Smith provided by NIH RO1 grant #HD046768-01A2. Support for Jason E. Swain provided by an NIH T32 grant. Partial support for Jingwen Wu provided from the Lyle C. Roll Research Fund to Professor James O. Woolliscroft, Dean of Medical School, University of Michigan.

oocyte
kinase
meiosis
chromosomal disorders
signal transduction


Epithelioid sarcoma in the thoracic spine

Epithelioid sarcoma is a rare and highly malignant soft tissue tumor that is commonly found in the extremities and rarely in the trunk area. This malignant tumor often mimics granuloma or nodular fasciitis, which causes a delay in establishing the diagnosis. This type of cancer has a high recurrence rate. Surgical treatment requires wide radical resection. The objective of this case report is to highlight the unique location of a rare neoplasm and to illustrate the relentless course of epithelioid sarcoma despite initial radical resection. A 14-year-old boy was admitted to our facility with a soft tissue mass on the right lower thoracic spine. The large tumor mass had deeply penetrated into the muscles, infiltrated the neuroforamen of T9–T10 level, and compressed the dural sac. Immunohistological study of the biopsy was highly consistent with an epithelioid sarcoma. Wide excision of the mass, laminectomy and spine fusion with instrumentation was performed. The patient received chemotherapy and irradiation. The first recurrence of the neoplasm was seen as a contralateral metastasis 21 months after the resection. On the last follow-up, 3 years postoperatively, the patient was in a good general condition. However, further progression of the sarcoma had to be recognized. Our case encompasses multiple features that represent negative prognostic factors. Initial wide excision of the neoplasm and adjuvant therapy including chemotherapy and irradiation seem to slow down the relentless course of epithelioid sarcoma in the trunk.
Introduction
Epithelioid sarcoma is a rare type of a fibrohistiocytic tumor, which is not extensively documented in the literature. It is common at the extremities, and rare at the trunk area. The incidence of epithelioid sarcoma in the trunk is 4% with a range from 0 to 44% [2, 12, 14]. It affects mainly adolescent males [1–3, 11, 13, 14, 17]. To our knowledge there are only two cases reported of localization in the spine (lumbosacral junction and sacrum) [7, 14], and there are no reports on epithelioid sarcoma at the thoracic spinal level. Laskowski [9] has first described the unique histological appearance in 1961. It resembles a chronic inflammatory process, necrotizing granuloma, squamous cell carcinoma or other fibrohistiocytic tumors. Electron microscopic appearance suggests origin from synovial structures [6, 10]. There is 85% local recurrence rate and 30–50% likelihood for metastases predominately lymphatic or in the lungs [2, 4, 11]. The 5-year survival rate varies from 65 to 100% [1, 12, 13]. Factors adversely affecting the survival rate are [2, 12]:Size of the tumor;Infiltration of the tumor into muscular, neural or vascular structures;Primary location in the trunk;Pulmonary metastasis.Wide surgical resection is indicated, since epithelioid sarcoma frequently follows a relentless course with multiple recurrences [4, 17]. Chemotherapy and irradiation are recommended to decrease recurrence rate [2, 8].
Case report
A 14-year-old overweight boy presented at an orthopaedist’s private practice with a painless, bulging mass on his back that was incidentally discovered by his grandmother. He was 183 cm tall and weighted 107 kg. Besides obesity no other medical problems were reported. There was no family history of a soft tissue mass. On the physical examination the tumor had a soft consistence and could not be shifted from the underlying tissue. Neurological examination did not reveal any deficit. Magnetic resonance imaging (MRI) with gadolinium of his thoracolumbar spine revealed a right paravertebral cystic mass. The soft tissue tumor extended from T7 to T11 level and measured 9.5×3×3 cm. The mass infiltrated via the neuroforamen at T9–T10 levels compressing one quarter of the spinal cord (Fig. 1). There was no osseous involvement. Open biopsy of the mass showed necrobiotic granuloma including epithelioid and fusiform cells. Immunohistochemical studies revealed a co-expression of vimentin and cytokeratin, with pathological diagnosis of epithelioid sarcoma grade III. During the tumor staging a craniolateral metastasis was depicted on the PET scan (Fig. 2). Consecutive MRI confirmed a soft tissue mass of 1 cm diameter, located about 10 cm distant from the main mass. Wide mass resection, including the locoregional metastasis, led to a sacrifice of large parts of the paravertebral thoracic muscles. In addition a hemilaminectomy of T9–T10 vertebra with resection of the adjacent pedicles and careful dissection from the dural sac was performed. There was no sparing of right ninth nerve root. The tumor resection was intralesional. Posterior transpedicular instrumentation from T6 to L1 was carried out. After surgery no new motor or sensory deficits were noted. Chemotherapy was initiated on the 6th postoperative week and irradiation on 10th postoperative week. The patient received nine courses of chemotherapy within 25 weeks (Table 1) and a total dose of 3-Gy irradiation over 4 weeks. He developed a wound dehiscence 2 weeks after initiation of the irradiation therapy. Under daily dressing no infection was observed. The wound was finally closed with a pedicle musculocutaneous flap using the Latissimus dorsi muscle 4 weeks after the completing the course of irradiation. Routine follow-up consisted of a physical examination, plain radiograms of the thoracic spine plus MRI Scans every 3 month (Fig. 3).
Fig. 1Preoperative antero-posterior (a) and lateral (b) plain X-rays of the middle thoracic spine. On the ap view no irregular formation of the pedicles can be depicted—the lateral view of the thoracic spine show apophyseal endplate changes as a feature of Scheuermans disease. No signs of osteolytic lesion can be detected. Preoperative transverse (c) and sagittal (d) MRI scan (T2 sequences: TR=3,070, TE=120) demonstrating the tumor mass in the region of the paravertebral muscles, reaching and compressing the dural sac via the right neuroforamen Th9/10Fig. 2On the PET scan a craniolateral metastasis was detected (a) and the paravertebral tumor mass was confirmed (b). No further satellite lesions were foundTable 1Agents and dosage of the quadruple chemotherapyAgent (trade name)Total dose (mg)Ifosfamid (Holoxan®)100,440Vincristin (Onkocristin®)20Actinomycin D (Lyovac®)12Doxorubicin (Ribodoxo®)494Fig. 3Postoperative antero-posterior (a) and lateral (b) plain radiograph of the thoracic spine at final follow-up. Postoperative transverse (c) and sagittal (d) MRI scan (T2 sequences: TR=3,682, TE=120) at 1.5 year showing no sign of recurrence
A metastasis at the fifth intercostal space of the chest contralateral to the initial tumor location was observed at 21 months follow-up. At this stage the patient denied further intervention. Two years postoperatively pulmonary metastases were detected on CT scans in addition to a local recurrence on the right thoracic spine. There was no significant progression of the tumor growth at the last follow-up 37 month after surgical treatment. The young male was in good general condition fully participating in social life.
Discussion
Epithelioid sarcoma is a rare soft tissue neoplasm with a high local recurrence rate. Epithelioid sarcoma presents histologic characteristics similar to inflammatory processes and other benign soft tissue tumors, which frequently delays the diagnosis and adequate treatment. Typical for this soft tissue sarcoma is an immunohistochemical co-expression of cytokeratin and/or epithelial membrane antigen (EMA) and vimentin, and in half of the cases an immunoreactivity for CD34, an occasional reactivity for smooth muscle actin (SMA), desmin, neuron specific enolase and S100 protein [2, 5]. In our specimen a strong positive immunoreaction with antibodies to cytokeratin, vimentin and EMA was registered (Fig. 4). Histomorphology and immunohistochemical pattern are typical and separates this tumor entity from other sarcomas. To differentiate from osteogenic sarcoma, which may also co-express cytokeratin and vimentin, and may demonstrate a immunoreactivity with SMA and CD99, epithelioid sarcoma, however, does not produce tumor osteoid, which is the key finding in osteogenic sarcoma.
Fig. 4a Histomorphologic aspect of epithelioid sarcoma with solid and nodular growth pattern, H&E (10×). b The tumor cells are roundish, admixed with spindle shaped cells and are featuring an eosinophilic cytoplasm with vesicular, irregular nuclei (20×). c Immunohistochemical investigation with antibodies against cytokeratin shows a strong positive immunoreaction (20×). d The immunoreaction against epithelial membrane antigen (EMA) demonstrates a comparable picture (20×). e The immunoreactivity against smooth muscle actin (SMA) features a weak staining (20×). f The immunoreaction against CD 99 is negative (20×)
Wide local resection is the recommended treatment of epithelioid sarcoma [1, 8, 11, 14]. In the spine however, total eradication of the sarcoma by an enbloc excision is compromised by the presence of neural structures [16]. Thus the concept of intralesional resection was followed both for the main tumor mass and the metastasis. Nerve sheaths may serve as a pathway for the spread of the sarcoma [1, 2, 8, 11, 17]. Enzinger [4] reported six cases with a mass infiltration along nerve sheaths or vascular structures. In our case there was involvement of the right ninth nerve root. The mass expansion into the right T9–T10 neuroforamen with compression of the dural sac necessitated a wide hemilaminectomy with resection of the facet joints and the adjacent pedicles. The wide paraspinal muscle resection required six levels segmental instrumentation and fusion to avoid scoliotic deformity due to muscular imbalance [15]. Pulmonary metastasis and local recurrence of the sarcoma were first diagnosed 24 month after the resection. In comparison, a survival period of 22 month after the initial surgery was described in a case report of epithelioid sarcoma in the lumbosacral spine [14]. Moreover, on first diagnosis, our case embodied many factors that promote recurrence of this neoplasm: large size, localization in the trunk and infiltration in both muscle and neural structures. Recurrence as the typical feature of epithelioid sarcoma cannot be prevented even in extremities, where a radical surgical regimen can be pursued [8]. In conclusion, epithelioid sarcoma of the spine is an extremely rare entity, which differs from other sarcoma subtypes in propensity for local recurrence and spread along the lymphatic pathways. Initial radical resection followed by adjuvant chemotherapy and irradiation can retard the relentless course of this malignancy.

epithelioid sarcoma
thoracic spine
survival


Dual-source computed tomography in patients with acute chest pain: feasibility and image quality

The aim of this study was to determine the feasibility and image quality of dual-source computed tomography angiography (DSCTA) in patients with acute chest pain for the assessment of the lung, thoracic aorta, and for pulmonary and coronary arteries. Sixty consecutive patients (32 female, 28 male, mean age 58.1±16.3 years) with acute chest pain underwent contrast-enhanced electrocardiography-gated DSCTA without prior beta-blocker administration. Vessel attenuation of different thoracic vascular territories was measured, and image quality was semi-quantitatively analyzed by two independent readers. Image quality of the thoracic aorta was diagnostic in all 60 patients, image quality of pulmonary arteries was diagnostic in 59, and image quality of coronary arteries was diagnostic in 58 patients. Pairwise intraindividual comparisons of attenuation values were small and ranged between 1±6 HU comparing right and left coronary artery and 56±9 HU comparing the pulmonary trunk and left ventricle. Mean attenuation was 291±65 HU in the ascending aorta, 334±93 HU in the pulmonary trunk, and 285±66 HU and 268±67 HU in the right and left coronary artery, respectively. DSCTA is feasible and provides diagnostic image quality of the thoracic aorta, pulmonary and coronary arteries in patients with acute chest pain.
Introduction
Acute chest pain represents one of the most difficult diagnostic challenges in emergency medicine. Chest pain history alone often cannot identify a group of patients who could be treated without further diagnostic testing [1], and triage decisions based on initial cardiac enzyme levels [2] and electrocardiography (ECG) [3] are often insufficient. Imaging may improve patient triage by decreasing delay in diagnosis and treatment and thus morbidity and mortality [4, 5]. Most studies [6–8] have focused on patients being suspected of having acute coronary syndrome, but did not include additional differential diagnoses such as aortic dissection or pulmonary embolism, conditions that may clinically mimic coronary syndromes [9].
Multi-detector row computed tomography (CT) angiography is widely accepted and routinely used as a primary tool in the emergency assessment of pulmonary embolism [10] and aortic dissection [11]. In addition, 64-slice CT coronary angiography has demonstrated the capability to diagnose and to rule-out coronary artery disease (CAD) [12, 13]. Some recent studies have shown that ECG-gated multi-detector row CT is logistically feasible [14] and yields promising results as a modality for evaluating chest pain patients with cardiac and non-cardiac disease in the emergency setting [15, 16]. Even though using 64-slice CT scanner technology, however, patients with elevated heart rates require medical heart rate control prior to CT coronary angiography by administering beta-blockers and/or benzodiazepines [8, 13, 17].
Dual-source CT (DSCT) represents the most recent scanner technology and is characterized by two tubes and two detectors that are mounted in orthogonal orientation onto the gantry. As compared to 64-slice CT, this scanner configuration maintains a consistently high spatial resolution while enabling ECG-gated imaging with an increased temporal resolution of 83 ms [18]. First studies have shown robust results of DSCT angiography (DSCTA) regarding image quality of cardiac structures even at high heart rates [19–21].
The purpose of this study was to investigate the feasibility and image quality of ECG-gated DSCTA of the chest as a tool to evaluate cardiac and non-cardiac causes of acute chest pain in patients presenting to the emergency department.
Materials and methods
Patient population
Sixty consecutive patients (32 females, 28 males, mean age 58.1±16.3 years, age range 26–84 years) were prospectively included in this study. Intake was performed on weekdays from 7 am to 7 pm from August to October 2006. All patients suffered from acute chest pain and were referred to our department to diagnose or to rule out pulmonary embolism (n=56) or aortic dissection (n=4). Inclusion criteria were acute chest pain >5 min within the previous 24 h and/or elevated serum D-dimer levels. Dyspnea and hemodynamic instability were not considered exclusion criteria. Similarly, all patients irrespective of their mean or regularity of heart rate and irrespective of their ability to perform breath-hold were included. Exclusion criteria included pregnancy, previous adverse reaction to iodinated contrast agent, nephropathy (serum creatinine >1.3 mg/dl), elevated troponine-I or creatine kinase-MB level in the initial blood sample, initial diagnostic ECG changes indicating an acute coronary syndrome (i.e., ST elevation or depression >1 mm, T-wave inversion >4 mm in >2 anatomically contagious leads), and interference with standard clinical care of patients. The study was approved by the local ethics committee; informed consent was obtained.
Scan protocol and data reconstruction
All CT examinations were performed on a DSCT scanner (Somatom Definition, Siemens Medical Solutions, Forchheim, Germany). First, a single non-enhanced low-dose scan at the level of the aortic root was obtained. In this slice, a region of interest (ROI) was set in the lumen of the aorta for monitoring intraluminal contrast enhancement. The delay from start of contrast material injection to start of scanning was planned using the bolus-tracking technique. A total of 110 ml iodinated contrast material (iodixanol, Visipaque 320; 320 mg/ml, GE Healthcare, Buckinghamshire, UK) was administered at a flow rate of 4 ml/s via an 18-gauge needle placed into a superficial vein in the left antecubital fossa, followed by 30 ml saline solution at the same flow rate (4 ml/s). After reaching the preset contrast enhancement level of 80 Hounsfield Units (HU) in the ROI, a breath-hold signal was given and the scan was initiated automatically after a delay of 6 s. A topogram was used for planning the examination and determining the scan range. Data acquisition was performed using a dedicated biphasic chest pain protocol. As illustrated in Fig. 1, the lower chest including the heart was scanned with a tube current time of 320 mAs while the upper chest was scanned with a tube current time product of 160 mAs. The borderline between upper and lower scan range was set at approximately 2 cm below the carina. Data acquisition was performed in a cranio-caudal direction. Acquisition parameters were as follows: detector collimation 2×32×0.6 mm by using a z-flying focal spot for the simultaneous acquisition of 2×64 overlapping 0.6-mm slices, gantry rotation time 330 ms, and tube potential 120 kV. The pitch varied according to the patient's heart rate and ranged from 0.2–0.43, with higher pitch at higher heart rates. ECG-pulsing for radiation dose reduction [22] was applied in all patients. At mean heart rates below 60 bpm, full tube current was applied from 60 to 70%, at 61–70 bpm from 50 to 80%, and at heart rates above 70 from 30 to 80% of the R-R interval.
Fig. 1Scan topogram illustrating planning of the chest pain protocol. The scan range covered the entire chest (red box). Premonitoring for bolus tracking was performed at the level of the aortic root (white line). The border for full tube current for the heart and half tube current for the upper lung is set approximately 2 cm below the tracheal bifurcation. It is delimited by a virtual horizontal line connecting the upper ends of the blue boxes on both sides
Retrospective ECG-gating for phase synchronization was used. For the heart, CT data sets were reconstructed at 70% of the R-R interval with a slice thickness of 0.75 mm (increment 0.5 mm) by using a medium soft-tissue convolution kernel (B26f) (mean field of view, FoV: 151±17 mm, image matrix 512×512). If considered necessary, additional images were reconstructed in 5% steps using the same parameters within the time window of full tube current. Images of the mediastinum (mean FoV: 293±43 mm) including the aorta and pulmonary arteries were reconstructed with a slice thickness of 1 mm (increment 0.8 mm) by using a medium soft-tissue convolution kernel (B30f), and images of the lung were reconstructed with a slice thickness of 2 mm (increment 1.5 mm) by using a sharp convolution kernel (B60f, same FoV as for the mediastinum). All images were transferred to a second Wizard (Siemens) equipped with cardiac post-processing software (Syngo Circulation, Siemens).
Data analysis
All data were qualitatively evaluated regarding image quality and artifacts of different thoracic structures by two independent readers who are both experienced in cardiovascular radiology. This evaluation was performed on transverse source images, multi-planar reformations (MPR), curved MPR, and thin-slab maximum intensity projections.
Image quality and artifacts-lung parenchyma
Image quality of lung parenchyma was independently rated using a two-point scale, adapted from a previous publication [23]. Lung parenchyma allowing diagnostic assessment due to distinct anatomic details of bronchial and parenchymal structures without significant artifacts and noise was rated with a score of 1 (diagnostic). Lung parenchyma with artifacts or noise causing reduction of image quality and diagnostic value was rated with a score of 2 (non-diagnostic).
Artifacts were rated to quantify the cranio-caudal distribution of artifacts within the lung parenchyma for the right and left lung separately in coronal MPR. Because of the different tube current at upper and lower parts along the z-axis (see Fig. 1), the following scores were separately applied for the apex and the basis of the lung, respectively: 1= no artifacts, 2= breathing artifacts (stair step artifacts), 3= ECG-gating (i.e., synchronization or interpolation artifacts), and 4= noise artifacts. If breathing and ECG-gating artifacts appeared at the same time in one patient, the artifact with the worst impact on image quality was noted.
Image quality and artifacts-vascular structures
Image quality of thoracic vascular structures was independently rated using the same two-point scale [23] as used for the rating of the lung parenchyma. Regarding the thoracic aorta, a score of 1 (diagnostic) indicated confident evaluation of the ascending aorta, the aortic arch, and descending aorta. Regarding pulmonary arteries, a score of 1 indicated confident evaluation of central, lobar, segmental, or subsegmental pulmonary arteries. Regarding coronary arteries, a score of 1 represented confident depiction (homogenous attenuation; no artifacts decreasing coronary analysis) of the right coronary artery (RCA), left main artery (LMA), left anterior descending artery (LAD), left circumflex artery (LCX), and their side branches. A score of 2 indicated decreased image quality of thoracic vascular structures with severe impairment of diagnostic value (non-diagnostic) due to breathing, motion, or ECG-gating artifacts.
Image noise and attenuation-vascular structures
Image noise was determined as the standard deviation of attenuation in a ROI placed in the ascending aorta [24]. Contrast attenuation was measured in each patient in the ascending aorta, pulmonary trunk, LMA, proximal segment of the RCA, and right and left ventricle. Image noise and contrast attenuation were assessed using a circular ROI positioned exactly within the vessel or ventricular lumen while avoiding superimposition or partial volume effects from the vessel wall or myocardium. Measurements of the ascending aorta and pulmonary trunk as well as of the right and left ventricle were performed on the same transverse image, while the ROI in the coronary arteries was individually placed on separate transverse images.
Imaging findings
Imaging findings indicating the possible underlying cause of acute chest pain were documented in each patient by both readers in consensus.
Statistical analysis
Statistical analysis was performed using commercially available software (SPSS 11.5, SPSS Inc., Il). Quantitative variables are expressed as mean ± standard deviation (SD) including 95% confidence intervals (CI) or range when appropriate. Categorical data were expressed as frequencies or percentages. Inter-observer agreement (kappa statistics) for image quality ratings was calculated. Two-tailed Student's t test for paired samples was used to explore significant differences in vessel attenuation among the ascending aorta, RCA, LMA, pulmonary trunk, and right and left ventricle. Intra-individual differences regarding vessel attenuation among the different vascular territories were performed using pairwise comparisons. Bonferroni correction for multiple comparisons was made, and a P-value <0.003 was considered statistically significant.
Results
All CT scans were well tolerated and were successfully performed in all 60 patients without complications. All patients had a sinus rhythm, and the average heart rate during data acquisition was 74.9±19.0 beats per minute (bpm) (range 45-130 bpm). No beta receptor antagonists or benzodiazepines were administered prior to CT; 13 patients (22%) took oral beta blockers as part of their baseline medication at the time of the scan. Mean scan time was 12.2±2.3 s (range 9.7–17.5 s) and mean scan length was 24.1±2.1 cm (range 19.3–29.7 cm). The reconstruction interval used for image reading was 70% of the R-R interval. In seven patients it was considered necessary to reconstruct additional data sets in 5% intervals within the window of full tube current to obtain images with diagnostic quality.
Image quality and artifacts-lung parenchyma
Image quality of lung parenchyma was rated as being diagnostic (score 1) by both readers in all 60 patients (100%; excellent inter-observer agreement, kappa =1.0), and pathology both at upper and lower lung parts could be diagnosed or excluded in all 60 patients.
Lung parenchyma was rated by both readers as being artifact-free in 42 patients (70%). Breathing artifacts causing stair-step artifacts were encountered in nine patients (15%) by one reader and eight (13%) of these nine patients by the other reader (excellent inter-observer agreement, kappa =0.93). Breathing artifacts were exclusively found at the lung base, i.e., at the end of the breath-hold period. ECG-gating-related artifacts were found in ten patients (17%) by one reader, and in nine (15%) of these ten patients by the other reader (excellent inter-observer agreement, kappa =0.84). The two types of artifacts were not encountered in the same patient, and image quality was diagnostic despite the artifacts.
Image quality and artifacts-vascular structures
Image quality of the thoracic aorta was rated by both readers as being diagnostic (score 1) in all 60 patients (100%; excellent inter-observer agreement, kappa =1.0), and pathologies of the thoracic aorta could be diagnosed or excluded by both readers in all 60 patients. Both readers rated image quality of the pulmonary arteries in the one (i.e., the same) patient (2%) as being non-diagnostic (score 2) due to insufficient attenuation of segmental and subsegmental pulmonary arteries. In this patient, attenuation did not allow excluding segmental or subsegmental pulmonary embolism, while attenuation in the pulmonary trunk and right and left lobar artery was considered sufficient for diagnosis or exclusion of central and lobar pulmonary embolism. In this patient, image quality of the coronary arteries was considered as being diagnostic by both readers. Image quality of coronary arteries was rated by both readers as being diagnostic (score 1) in the same 58 patients (97%; excellent inter-observer agreement, kappa =1.0). In the remaining two patients (3%), image quality of the coronary arteries was considered by both readers as being non-diagnostic (score 2) due to ECG-gating-related artifacts (mean heart rate 71 bpm, ranging from 50 to 84 bpm during scanning) in one and severe image noise due to obesity (body mass index 40.4 kg/m2) in the other. In both patients, both readers rated image quality of the pulmonary arteries as being diagnostic.
Image noise and attenuation-vascular structures
Mean image noise in the ascending aorta (mean ROI size 3.6±0.2 cm2) was 29.8±5.6.
Mean attenuation in the ascending aorta (same ROI and same ROI size as for measurements of image noise) was 291±65 HU (95% CI: 274–308 HU), mean attenuation in the pulmonary trunk (mean ROI size 2.4±0.2 cm2) was 334±93 HU (95% CI: 310–358 HU), mean attenuation in the proximal RCA (mean ROI size 0.14±0.02 cm2) was 285±66 HU (95% CI: 268–302 HU), and mean attenuation in the LMA (mean ROI size 0.12 cm2±0.01) was 286±67 HU (95% CI: 269–304 HU). Mean attenuation in the right ventricle (mean ROI size 2.6±0.2 cm2) was 313±89 HU (95% CI: 290–336 HU) and mean attenuation in the left ventricle (mean ROI size 2.6±0.2 cm2) was 277±66 HU (95% CI: 260–294 HU) (Fig. 2).
Fig. 2Mean attenuation values (HU) within different vessels indicating a relatively homogenous contrast distribution between the different thoracic vascular territories. RCA = right coronary artery; LMA = left main coronary artery
No significant differences regarding attenuation were found between the RCA and LMA (P=n.s.), and between the ascending aorta and both coronary arteries (P=n.s.). No significant differences in attenuation were present between the right and left ventricle (P=n.s.), between the right ventricle and ascending aorta (P=n.s.), between the right ventricle and pulmonary trunk (P=n.s.), and between the right ventricle and the RCA (P=n.s.) and LMA (P=n.s.). Similarly, there were no significant differences in attenuation between the left ventricle and ascending aorta (P=n.s.), and between left ventricle and LMA (P=n.s.) and RCA (P=n.s.). Significant differences were only found between the pulmonary trunk and ascending aorta (P <0.003), between the pulmonary trunk and the RCA and LMA (P<0.003), respectively, and between the left ventricle and pulmonary trunk (P<0.003).
Pairwise comparisons of mean intra-individual differences regarding vessel attenuation were small and ranged between 1.18±5.86 HU comparing the right and left coronary artery and 56.45±8.96 HU comparing the pulmonary trunk and left ventricle (Table 1).
Table 1Pairwise intra-individual comparisons between different thoracic vascular territories Mean differenceStandard deviation95% confidence intervalLower boundaryUpper boundaryAortaPulmonary trunk-4311-75-11AortaRCA65-1022AortaLCA54-817AortaRight ventricle-2313-6318AortaLeft ventricle145-330Pulmonary trunkRCA49111682Pulmonary trunkLCA47121283Pulmonary trunkRight ventricle2012-1657Pulmonary trunkLeft ventricle5792984RCALCA-16-1917RCARight ventricle-2813-6913RCALeft ventricle85-824LCARight ventricle-2714-7015LCALeft ventricle96-1028RCA = right coronary artery; LCA = left coronary artery
Imaging findings
No pathologic findings were found in 33 patients (55%), whereas various pathologies of the aorta, pulmonary and coronary arteries, lung, and mediastinum were present in 27 patients (45%) (Table 2, Figs. 3, 4, 5).
Table 2Imaging findings in the study populationDiagnosisNumber of patients (n=60)No pathology33Pulmonary embolism11Aortic pathology, total5 Dissection type B3 Pseudoaneurysm of the aortic arch1 Plaque rupture1Coronary pathology, total3 Significant stenosis of the LAD2 Occlusion of the RCA1Pulmonary consolidation3Pericarditis/pericardial effusion2Seropneumothorax1Non-small cell lung cancer1Synovial cell carcinoma1LAD, left anterior descending artery; RCA, right coronary arteryFig. 3A 63-year-old female patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (RCX) allow excluding significant coronary stenosis in this patient. Mean heart rate during DSCTA was 71 bpm. (b) Thin-slab transverse maximum intensity projection shows no evidence of pulmonary embolism. (c) Transverse image at the level of the pulmonary trunk demonstrates acute aortic dissection type B (arrow) with mild left-sided pleural effusionFig. 4A 58-year-old female patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (LCX) demonstrate normal coronary arteries and no evidence of stenosis. Mean heart rate during DSCTA was 63 bpm. (b) Thin-slab transverse maximum intensity projection show bilateral pulmonary embolism (arrows). (c) Oblique-sagittal thin-slab maximum intensity projection demonstrates the thoracic aorta without evidence of diseaseFig. 5A 71-year-old male patient admitted to the emergency department with acute chest pain. (a) Curved multiplanar reformations along the centerline of the right coronary (RCA), left anterior descending (LAD), and the left circumflex artery (LCX) show occlusion of the proximal RCA (long arrow) and vessel wall calcifications without significant stenosis in the proximal and middle segment of the LAD and LCX (short arrows). Mean heart rate during DSCTA was 73 bpm. (b) Thin-slab transverse maximum intensity projection demonstrates normal opacification of pulmonary arteries with no evidence of embolism. (c) Oblique-sagittal thin-slab maximum intensity projection demonstrates the thoracic aorta with minimal atherosclerotic wall changes, but with no evidence of potential causes for acute chest pain
Discussion
Our study demonstrates that DSCT allows performing an ECG-gated chest examination for visualizing the different thoracic vascular territories at the same time-by employing an adjusted contrast media protocol-within a reasonable breath-hold period, and thus provides a diagnostic image quality in almost all patients. One of the most important findings of this study is that diagnostic data of coronary arteries could be obtained without foregoing heart rate control.
Contrast media protocol and scan time
With increasing gantry rotation times and faster volume coverage of newer CT scanners, scan times successively shorten and higher injection rates are required to achieve sufficient contrast in the vascular territory of interest. Similar to a previous 64-slice CT study [15], we have set the ROI for bolus tracking in the ascending aorta. With a relatively low threshold of 80 HU above which the scan was initiated, a homogenous attenuation in both aortic/coronary and pulmonary arteries could be achieved. By doing so, we aimed at the time interval where the contrast enhancement curve of the pulmonary circulation overlaps with enhancement of the aorta [25]. This has also resulted in comparable attenuation in left and right cardiac cavities. Recommended mean attenuation values for diagnostic image quality are approximately 300 HU for the pulmonary arteries [26] and 250–350 HU in the ascending aorta and coronary arteries [27, 28]. We obtained a mean attenuation of 291 HU in the ascending aorta, 334 HU in the pulmonary trunk, 285 HU in the RCA, and 286 HU in the LMA with only small intra- and inter-individual variations. These attenuation values were considered diagnostic in 98% of our patients and are comparable to previously published data [15, 29]. We encountered only one patient with non-diagnostic attenuation of segmental and subsegmental pulmonary arteries. However, this may have been also caused by other reasons negatively affecting contrast attenuation in pulmonary arteries such as Valsalva maneuver [30].
The other major issue for CT in acute chest pain represents the issue of scan duration. As ECG-gated CT requires low pitch [31], scan time is increased as compared to non-gated chest CT examinations. This, however, negatively affects image quality by making examinations more prone to breathing artifacts. This holds particularly true for patients with acute chest pain who often suffer from dyspnea. In our study, mean scan time was approximately 12 s, which represents a further improvement as compared to a mean scan time of 21 s with 64-slice CT [15].
Role of CT in the diagnostic pathway of acute chest pain
Figure 6 represents a simplified flow-chart of a generally accepted clinical pathway for patients with cardiac and non-cardiac causes of acute chest pain [9, 32, 33]. While CT is the accepted reference modality for the diagnosis and exclusion of pulmonary embolism and aortic dissection [10, 11], the role of CT in patients with a suspected cardiac cause for chest pain is currently under investigation. Gallagher and colleagues [6] have shown that 64-slice CT has accuracy that is comparable to that of stress nuclear imaging for the detection of acute coronary syndrome in low-risk patients with negative serial ECG and biomarker results. Hoffmann and coworkers [8] have demonstrated that 64-slice CT has good performance characteristics for ruling out acute coronary syndromes in patients presenting to the emergency department with acute chest pain. The authors demonstrated in patients in whom initial triage was inconclusive that the absence of coronary artery plaque or significant stenosis on CT angiography had an excellent negative predictive value for the subsequent diagnosis of acute coronary syndrome. Furthermore, in those patients with CAD on CT, the extent of coronary atherosclerotic plaque provided incremental information to standard baseline patient variables and clinical risk assessment. Goldstein and colleagues [7] have shown that 64-slice CT coronary angiography is able to definitely establish or exclude CAD as the cause of acute chest pain. The high negative predictive value of the studies suggests that CT coronary angiography may be useful for facilitating and optimizing triage of patients with acute chest pain and/or inconclusive initial emergency department evaluation [7, 8].
Fig. 6Flow chart of a generally accepted clinical pathway for patients with cardiac and non-cardiac causes of acute chest pain. CT represents the standard of reference in patients with suspicion of acute aortic syndromes or pulmonary embolism (light gray boxes). The added value of CT in the evaluation of acute coronary syndrome has been already demonstrated [8] (moderate gray box). The potential future role of CT in patients with acute chest pain might be at an even earlier point of diagnostic work-up (dark gray boxes) to rule out life-threatening coronary, pulmonary, and aortic disease and to guide adequate therapeutic interventions
Study limitations
We included only a relatively small number of patients and thus a small number of pathologies. Second, our study lacks a standard end-point to assess the final diagnosis and misses comparison with the reference standard invasive coronary angiography for investigating CAD for which diagnostic accuracy could be calculated. However, only a minority of our patients underwent invasive coronary angiography because in our emergency department standard management in patients with suspected acute coronary syndrome directly undergoing invasive coronary angiography without prior CT. That is in line with most studies involving patients referred to the emergency department [34]. Third, we did not investigate the optimal contrast media technique for obtaining homogenous contrast attenuation of different thoracic vascular territories. It remains to be determined if the use of the test bolus technique would yield better results with regard to vessel opacification. Finally, it needs to be assessed whether caudo-cranial or caudo-cranial scan direction will result in better image quality.
Conclusion
First experience indicates that DSCTA is feasible in patients with acute chest pain and provides diagnostic image quality of the aorta, pulmonary arteries, and coronary artery system as well as of the lung parenchyma and mediastinum in a patient population without foregoing heart rate control. A dedicated contrast media protocol allows for homogenous attenuation of the different thoracic vascular territories.

acute chest pain
coronary angiography
dual-source ct