Hum. Reprod. Advance Access originally published online on July 10, 2007
Human Reproduction 2007 22(8):2214-2224; doi:10.1093/humrep/dem147
Human embryos developing in vitro are susceptible to impaired epithelial junction biogenesis correlating with abnormal metabolic activity
1 School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK 2 Department of Biology, University of York, PO Box 373, York YO10 5YW, UK 3 Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Leeds LS1 9NS, UK 4 Reproduction and Early Development Research Group, Leeds Institute of Genetics, Health and Therapeutics, D Floor, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK 5 Developmental Origins of Health and Disease Division, School of Medicine, University of Southampton, Princess Anne Hospital, Southampton SO16 5YA, UK 6 Present address: Developmental Origins of Health and Disease Division, School of Medicine, University of Southampton, Princess Anne Hospital, Southampton SO16 5YA, UK 7 Present address: Centre for Human Development, Stem Cells and Regeneration, Division of Human Genetics, University of Southampton, Duthie Building (MP808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
8 Correspondence address. Developmental Origins of Health and Disease Division, Institute of Developmental Sciences, D08/MP887 Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK. Tel./Fax: +44 2380594401; E-mail: jje{at}soton.ac.uk
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Abstract |
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BACKGROUND: Blastocyst biogenesis occurs over several cell cycles during the preimplantation period comprising the gradual expression and membrane assembly of junctional protein complexes which distinguish the outer epithelial trophectoderm (TE) cells from the inner cell mass (ICM). In the human, TE integrity and the formation of a junctional seal can often be impaired. Embryos likely to result in a successful pregnancy after transfer are mostly selected according to morphological criteria. Recent data suggest that non-invasive measurement of amino acid turnover may be useful to complement such morphological scores. Whether morphological and metabolic criteria can be linked to poor TE differentiation thereby underpinning developmental predictions mechanistically remains unknown.
METHODS: We examined TE intercellular junction formation in human embryos by immunofluorescence and confocal microscopy and correlated this process with morphological criteria and amino acid turnover during late cleavage.
RESULTS: Our results show that TE differentiation may be compromised by failure of membrane assembly of specific junction constituents. This abnormality relates more closely to metabolic profiles than morphological criteria.
CONCLUSION: Our data identify that amino acid turnover can predict TE differentiation. These findings are the first to link two mechanisms, metabolism and junction membrane assembly, which contribute to early embryo development.
Key words: human embryo/trophectoderm/blastocyst/tight junction/amino acid metabolism
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Introduction |
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In vitro fertilization and embryo transfer is a well-established procedure to overcome human infertility, but success rates are low, with an average live birth rate for women under 35 at only 28% (HFEA Guide to infertility, 2006/7). High embryonic losses occur during cleavage with, at best, only 50% in vitro fertilized oocytes competent to reach the blastocyst stage (reviewed in Hardy et al., 2002
). Since high order births are the major side effect of assisted reproductive technologies, the number of embryos transferred to the uterus is generally limited to reduce the risk of multiple pregnancies. It is therefore essential to identify and select the most viable embryos for transfer to maintain success rates. The common practice is to transfer embryos during early cleavage (day 2–3) after selection based upon morphology and timing of cleavage, although the use of blastocyst culture has increased in recent years (Gardner and Lane, 1997; Blake et al., 2005). Blastocyst culture allows continued morphological evaluation of embryos and, where relevant, screening for aneuploidy or genetic disease after biopsy (Gardner and Lane, 1997; Gardner et al., 2002; Hardy et al., 2002; Racowsky, 2002). However, the ability of blastocyst transfer to increase pregnancy rates is subject to debate (Gardner et al., 1998; Milki et al., 2002; Summers and Biggers, 2003; Blake et al., 2005), and extended culture may lead to adverse programming of later fetal or post-natal growth (reviewed in Fleming et al., 2004; Gardner and Lane, 2005). Hence, there is a need for reliable prediction of embryo viability during early cleavage based upon non-invasive selection criteria (reviewed in Hardy et al., 2002; Sakkas and Gardner, 2005).
To date, embryos are routinely selected for transfer based on their morphology and time to first cleavage (Racowsky, 2002; Scott, 2003; Sakkas and Gardner, 2005). An alternative method to select embryo viability is to monitor embryo metabolism non-invasively (Houghton et al., 2002; Brison et al., 2004; Houghton and Leese, 2004; Stokes et al., 2007). It was found that embryos capable of developing to the blastocyst stage were metabolically more quiescent during early cleavage compared with embryos that arrested prior to blastocyst formation (Houghton et al., 2002). Subsequently, it was shown that amino acid turnover of embryos measured from day 1 to day 2 of development was also capable of predicting pregnancy after transfer (Brison et al., 2004). More recently, amino acid turnover has been shown to predict development to the blastocyst stage of cryopreserved embryos as well as being able to differentiate between the developmental capacity of embryos of the best morphological grade (Stokes et al., 2007). We now investigate the relationship between amino acid turnover and expression of junctional proteins.
The two major morphological changes occuring in the mammalian embryo during preimplantation development are ‘compaction', when blastomeres polarise and cell–cell adhesion is initiated, and ‘cavitation', when the blastocoel cavity forms and the two distinct cell lineages of the blastocyst (trophectoderm epithelium, TE; inner cell mass, ICM) become apparent (reviewed in Fleming et al., 2001). These morphological events represent the initiation (compaction) and completion (cavitation) of TE epithelial differentiation and are dependent upon an underlying programme of gene/protein expression and cellular re-organization. Mechanisms coordinating TE differentiation have largely been studied in the mouse; in particular, associated with apico-basal transport processes involving the Na+/K+-ATPase and intercellular adhesion and tight junction formation (Fleming et al., 2001; Watson and Barcroft, 2001; Violette et al., 2006). Compaction and cell polarity initiate TE differentiation in the human embryo (Nikas et al., 1996) and a similar pattern of epithelial gene expression associated with intercellular junction formation occurs during human cleavage as in the mouse (Bloor et al., 2002; Ghassemifar et al., 2003). However, human embryos cultured in vitro commonly exhibit poor or absent expression of epithelial junction genes and show deficiency in membrane assembly of expressed proteins at junctional contact sites, indicative of loss of embryo viability (Hardy et al., 1996; Bloor et al., 2002; Ghassemifar et al., 2003).
In the present study, we have examined in more detail the process of TE intercellular junction formation in human embryos and sought to correlate this process with both morphological and metabolic markers of embryo viability during late cleavage.
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Human embryos
Spare donated human embryos were obtained with full patients' consent from the Assisted conception unit (ACU), Leeds General Infirmary. Ethical approval for the work was granted by the Human Fertilisation and Embryology Authority (HFEA) and the local Ethics Committees of the collaborating institutions. Ovarian stimulation, oocyte collection and culture in Medi-cult IVF medium (Medicult UK Ltd, UK) and in vitro fertilization overnight in the same medium were carried out at the Leeds ACU before transport of embryos at day 2 post-insemination to the University of York (HJL laboratory) using methods previously described (Balen, 2001
; Houghton et al., 2002; Ghassemifar et al., 2003). Culture of human embryos in York was in individual 4 µl drops of Earle's balanced salt solution (EBSS) supplemented with 1 mmol/l glucose, 5 mmol/l lactate, 0.47 mmol/l pyruvate, 0.5% human serum albumin (Zenalb 20; Bioproducts Lab, UK) and a near physiological mixture of amino acids (0.02–0.2 nmol/l depending on the amino acid; Tay et al., 1997) under oil at 37°C in 5% CO2 in air from day 2 to the morula or blastoycst stage. Developmental morphological grade and stage were recorded daily from embryos as described previously (Table 1; Houghton et al., 2002). Only embryos of grades 1–3 were used in our experiments. On average, morulae were collected and fixed for staining after 5.2 ± 0.27 days, and blastocysts after 5.9 ± 0.06 days with mean morphological grades of 1.75 ± 0.16 and 1.94 ± 0.15, respectively (Table 2). Culture media were renewed every 24 h and spent media were stored frozen at – 80°C for future amino acid analysis.
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Antibodies
All antibodies used in this study were tested and optimized using the human CaCo2 cell line as described previously (Ghassemifar et al., 2003
). All antibodies showed strong, reliable staining of the membrane-assembled junctional proteins analysed. Rat polyclonal antibody for mouse E-cadherin (1:500; Sigma), rabbit polyclonal antibodies for human occludin (1:1000, Zymed), bovine desmocollin 2 (1:500; Collins et al., 1995), mouse pan ZO-1 (1:100; Sheth et al., 1997) and human ZO-1
+ isoform (1:200; Balda and Anderson; 1993), and guinea-pig polyclonal antibodies for human ZO-1- isoform (1:100; Balda and Anderson; 1993) and mouse ZO-1 + (1:250; Sheth et al., 1997) were used as previously described.
Immunocytochemistry and confocal microscopy
Human embryos were fixed in PBS supplemented with 1% formaldehyde (Analar or Sigma) for 10 min and processed for immunofluorescence as described previously (Fleming et al. 2002; Eckert et al., 2004a,b, 2005) with minor modifications. Briefly, individual fixed zona pellucida-intact embryos were permeabilized in 0.25% Triton x 100 in PBS, washed with PBS, and blocked in 2.6 mg/ml ammonium chloride in PBS, respectively. Embryos were placed and sealed within specially designed chambers for immunolabelling (Fleming et al., 2002) and transported overnight from York to Southampton (TPF laboratory) in the appropriate dilution of primary antibody. On arrival, the embryos were washed three times over 30 min in 0.1% Tween 20 in PBS and incubated in appropriate cross-purified ALEXA-488, ALEXA-546 or ALEXA-568 labelled anti-rat, anti-guinea pig or anti-rabbit secondary antibodies (Molecular Probes) diluted 1:500 either alone or in combinations for double labelling experiments for 1 h at room temperature. After final washes in PBS-Tween, individual embryos were attached to coverslips coated with Cell-TAK (BD Bioscience) and sealed. Specimens were visualised with a x 40 or x 63 oil-immersion Nikon inverted microscope linked to a Bio-Rad MRC-600 or – 1024 series confocal imaging system using a krypton-argon laser. Images were analysed and processed using the Bio-Rad Software system (Confocal Assistant version 4.01). The staining pattern of all embryos was scored according to the degree of membrane assembly on at least three separate occasions for all embryos after 3D z-series projection of 1 µm sections through almost the entire embryo (Fig. 1) as follows: score 0 = no specific staining; 1 = perinuclear staining, no membrane assembly; 2 = membrane assembly in some areas of the embryo with or without simultaneous perinuclear staining; 3 = membrane assembly within most areas of the embryo without perinuclear staining.
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Amino acid analysis
Amino acid concentrations within the spent culture medium drops were analysed by reverse-phase HPLC as previously described (Houghton et al., 2002
). Diluted embryo-containing and embryo-free control samples were examined in parallel (Houghton et al., 2002). Spent medium obtained during embryo compaction, cavitation or blastocyst expansion was analysed. Amino acid depletion/appearance was calculated in pmol/embryo/h ± SEM. Total amino acid turnover was defined as the net sum of amino acids depleted from, or appearing in, the medium.
Statistical analysis
Depletion from or appearance in the culture medium (i.e. significantly different from zero) during the pre-compact to compacting period (compaction), morula-to-blastocyst transition (cavitation) or during blastocyst expansion (expansion) of individual amino acids were analysed by one-sample Student's t-test or Mann–Whitney Rank Sum test where appropriate after testing for normal distribution and equal variance (SigmaStat software package, version 2.0, Jandel Scientific). Overall, amino acid depletion, production and turnover during the different time periods were compared by ANOVA or ANOVA on Ranks followed by a Dunn's test where appropriate after testing for normal distribution and equal variance (SigmaStat). Significant correlations between morphological grade, staining pattern and amino acid turnover during compaction and cavitation were analysed by a Spearman's rank order correlation (SigmaStat).
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Impaired membrane assembly of junctional proteins in human morulae or blastocysts
A total of 8 morulae and 28 blastocysts of different morphological quality were stained for the different junctional components, in some cases for two proteins in one embryo (see Table 3 for details). When stained at the morula stage, E-cadherin (n = 3) and pan ZO-1 or ZO-1
– (n = 3) could be detected at the membrane in all embryos examined. However, perinuclear (n = 2) as well as membrane staining was found for occludin whereas ZO-1 + was only diffusely present within the cytoplasm (n = 2) (Fig. 1a–d). In blastocysts, pan ZO-1/ZO-1– (n = 7) and E-cadherin (n = 7) were assembled at the junctions in all embryos except one of very poor morphology (grade 2.5) (Fig. 1e–h). In contrast, only two out of seven or two out of nine blastocysts showed membrane assembly of occludin and ZO-1 +, respectively, whereas the remaining blastocysts had predominantly perinuclear localization of these two junctional proteins (Fig. 1i–m). Similarly, DSC2 membrane assembly was often (four out of six blastocysts) incomplete (Fig. 1n and o). The degree of membrane assembly of both occludin and ZO-1 + together in resultant blastocysts correlated positively and significantly with morphological grade during compaction (n = 8 observations; R = 0.72, P = 0.04; Table 4) but not with morphology of the resultant blastocyst (n = 15 observations; R = 0.23, P = 0.41; Table 4). In contrast, membrane assembly of the other junctional proteins individually as well as overall was independent of morphological grade during either transition phase (Table 4; compaction: n = 21 observations, R = 0.01, P = 0.97; cavitation: n = 35 observations, R = 0.05, P = 0.77).
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Double labelling of the same blastocysts with both ZO-1
– and - +-specific antibodies revealed that membrane assembly of ZO-1 – was never impaired, whereas ZO-1 + was mainly found in the cytoplasm within the same embryo (n = 4, two morulae and two blastocysts; Fig. 2a). Both our antibodies directed against ZO-1 + co-localized perfectly when used in reverse double labelling experiments in blastocysts (n = 2; data not shown). Occludin and ZO-1 + co-localized in all embryos examined by double labelling (n = 3; Fig. 2b) and the frequent perinuclear staining as well as membrane staining (two out of three embryos) suggested deficient membrane incorporation of both junctional proteins at the same time (Fig. 2c and d, tangential section).
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In order to minimize embryo numbers required for this study, a cocktail of all secondary antibodies employed (anti-guinea pig, -rat, -rabbit ALEXA 488 and anti-guinea pig ALEXA 546 and anti-rabbit ALEXA 568) was used on two compacting day 6 embryos in the absence of a primary antibody to act as a negative control. No specific staining pattern was observed (Fig. 2e). This demonstrates the specificity of the junctional staining patterns seen with the respective primary antibodies.
Turnover of specific amino acids correlates with the degree of junctional membrane assembly
Only developmentally competent embryos that reached the blastocyst stage were considered for amino acid turnover in our study. During compaction, a total of 19 embryos were assayed for amino acid turnover. Fifteen developed further to blastocysts the next day and were included in the statistical analysis for amino acid turnover, whereas the remainder were used for immunolabelling only. During cavitation, a total of 17 and during blastocoel expansion, a total of 5 embryos were analysed for amino acid turnover (see Table 3 for details). Similar to previous reports (Houghton et al., 2002), the total amino acid turnover increased significantly (P < 0.05) during cavitation (8.049 ± 1.016 pmol/embryo/h during compaction versus 10.932 ± 0.802 pmol/embryo/h during cavitation; Fig. 3A). During blastocoel expansion, total amino acid turnover remained at a similar level as during cavitation (11.228 ± 2.362 pmol/embryo/h; Fig. 3A). Similarly, total depletion and production significantly increased between compaction and cavitation but remained relatively unchanged during expansion (Fig. 3A). In principle, our amino acid profiling largely confirmed previous reports of amino acid turnover in developing embryos (Fig. 3B; Houghton et al., 2002; Stokes et al., 2007). Mainly essential amino acids (E) were significantly (P < 0.05, difference from zero) depleted from the medium (during compaction: SerNE, LeuE, IsoE; during cavitation: ArgNE, LeuE, IsoE), and mainly non-essential amino acids (NE) were produced by the embryos (during compaction: AlaNE, TyrNE, PheE; during cavitation: AspNE, GluNE, GlnNE, GlyNE, ThrE, AlaNE).
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During compaction, morphological grade was significantly correlated with the depletion or appearance of seven specific amino acids (AspNE, GluNE, AlaNE, TyrNE, TrpE, ValE and IsoE; n = 15, R = – 0.58 to 0.67; P = 0.005–0.04; shown as net sum in Fig. 4A). During cavitation, morphology and depletion or appearance of three specific amino acids (HisNE, TrpE and PheE) had a significant positive correlation (n = 14–17; R = 0.54–0.66, P = 0.003–0.03; shown individually and as sum in Fig. 4B). During both transition periods, embryos with higher morphological grades mostly depleted amino acids from the medium, whereas those of lower morphological grade largely released amino acids into the medium. Overall, the net total amino acid turnover was lower in embryos of better morphology during compaction (n = 15; R = 0.73, P = 0.001; Fig. 4A), but this relationship was lost during cavitation (n = 14; R = 0.45, P = 0.10, Fig. 4B).
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In contrast to morphology, the degree of overall membrane assembly of all junctional proteins in blastocysts improved with higher total amino acid turnover, but this was only seen during cavitation (n = 14; R = 0.61, P = 0.02). Amongst the individual amino acids, only two, HisNE (n = 14) and TyrNE (n = 17), showed a significant relationship to membrane assembly (R = 0.56–0.66, P = 0.01–0.02; Fig. 5A). In total, blastocysts with better membrane assembly of junctional proteins mainly released amino acids into the medium whereas compromised membrane assembly paralleled amino acid depletion from the medium. During compaction, no significant relationships were found between the degree of membrane assembly and either total amino acid turnover (n = 15; R = – 0.06, P = 0.87, not shown) or any individual amino acids (R = – 0.46 to + 0.39, P = 0.08–0.99, not shown). Membrane assembly of most individual junctional proteins in blastocysts showed no significant relationship to amino acid turnover at any time examined (DSC2: n = 3 and 5, E-cadherin: n = 6 and 4, Pan ZO-1/ZO-1
–: n = 4 and 4 during compaction and cavitation, respectively, with R = – 0.87 to + 0.87, P = 0.18–1 during compaction and R = – 0.86 to + 0.78, P = 0.33–1 during cavitation). This was mainly attributable to the high degree of membrane assembly in all embryos examined at least in the case of E-cadherin and Pan ZO-1/ZO-1–. In contrast, the degree of membrane assembly of occludin and ZO-1 + together was significantly related to amino acid depletion or appearance. During both transitions, membrane assembly of occludin and ZO-1 + in blastocysts was more complete the higher the total net amino acid turnover (compaction: R = 0.72, P = 0.04, n = 8; cavitation: R = 0.82, P = 0.005, n = 8; Fig. 5b). Depletion or appearance of six (AspNE, GluNE, HisNE, TrpE, ValE and IsoE) or three (AlaNE, IsoE and LeuE) amino acids during compaction or cavitation, respectively (compaction: n = 8, R = ± 0.72 to 0.82, P = 0.004–0.05; cavitation: n = 8, R = ± 0.73, P = 0.03–0.04) correlated with the degree of membrane assembly of occludin and ZO-1 + (shown as net sum in Fig. 5B). Two different patterns in this relationship were apparent: (i) better membrane assembly was associated with either greater release (e.g. AlaNE, HisNE, TrpE) or greater depletion of certain amino acids (e.g. IsoE, LeuE), (ii) a low degree of membrane assembly coincided with amino acid release, higher degree of membrane assembly paralleled amino acid depletion (e.g. AspNE, GluNE, ValE). In addition, it is predominantly the essential amino acids that are depleted from the medium when a higher degree of junctional membrane assembly is achieved within the TE.
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Impaired membrane assembly of junctional proteins in human blastocysts
Our current study confirms that mouse and human preimplantation embryos display similar protein expression and membrane assembly profiles of key junctional components (Bloor et al., 2002
; Ghassemifar et al., 2003). However, in contrast to the mouse, human blastocysts often show impaired expression (Ghassemifar et al., 2003) and membrane assembly of junctional proteins during cleavage or when embryo morphology appears abnormal (Alikani, 2005). This could have detrimental consequences for embryo viability and TE integrity. The correct assembly of TJ proteins is required to form a permeability seal in the TE but also appears to participate in signalling pathways (Tsukita et al., 2001; Aijaz et al., 2006). Most recently, a functional role for tight junctions, in particular ZO-1, has been established in the regulation of transcription and RNA processing and stability by interacting with the transcription factor ZONAB and symplekin, a member of the RNA processing machinery which affects functions like proliferation and cell cycle progression (Kavanagh et al., 2006; Sourisseau et al., 2006). Deficiencies within such key functions could result in lethal or sublethal consequences becoming apparent later during development after activation of the embryonic genome and upon ongoing degradation of maternal mRNAs.
In our current dataset, key adherens junction and desmosome proteins appeared to be unaffected, whereas the tight junction was impaired. This fact seemed surprising but could indicate that embryos with deficient adhesion show cleavage abnormalities (Alikani, 2005) or severe morphological irregularities with extruded cells due to perturbed Wnt signalling (Na et al., 2007) and may, therefore, have been excluded from our analysis. Desmosome biogenesis may be an autonomous process initiated at cavitation and would, again, not be impaired in the embryo cohort examined. However, of the three types of junction, the tight junction is the most complex and might, therefore, show redundancies among its components. It seems likely that impaired assembly of the ZO-1 – isoform might also have resulted in earlier developmental arrest since, like the mouse, it appears to be the first tight junction component to be found at the membrane in the human (Sheth et al., 1997).
Occludin and ZO-1 +, two tight junction members shown to be critical for the formation of the final TE seal in the mouse (Sheth et al., 1997; 2000), were deficient in assembly in over 50% of human blastocysts, independent of morphology. This suggested that even high-grade human blastocysts had deficiencies in their differentiation capacity and may have suffered impaired epithelial transport and TE integrity. On the other hand, some blastocysts of poor morphology that may be discarded in current clinical IVF programmes could, due to a fully integrated and functional TE epithelial barrier, have a good potential to implant and give rise to offspring. Consequently, it seems plausible that not only expression profiling (Katz-Jaffe et al., 2006) but also localization of proteins is required for the assessment of embryo quality.
The complex mechanisms by which junctional proteins are transported to the membrane are only beginning to be understood and may be specific for certain tight junctional components, independent from adherens junction sorting (Macara and Spang, 2006). The underlying mechanisms causing impaired membrane assembly of occludin and ZO-1 +, as seen in our study, remain speculative but could involve: (i) insufficient transport to the membrane, indicative of insufficient activity of the membrane sorting machinery or of impaired tight junction protein modifications to facilitate such transport; (ii) deficiencies in the mechanisms of tight junction maintenance and, consequently, dislocation or increased protein turnover due to abnormalities of the proteins or the assembly and anchoring machinery; and (iii) insufficient protein production (see Macara and Spang, 2006, for review). Interestingly, in mouse embryos, occludin and ZO-1 + appear to connect with each other within the cytoplasm before being transported to the membrane together (Sheth et al., 2000), suggesting that a communal transport signal within both proteins may be impaired in the human embryo. It seems plausible that post-translational modifications such as phosphorylation could be compromised in both junctional components. This could lead to the inability of the transport machinery to bind efficiently and move both junctional components to their destination, resulting in increased turnover and protein degradation (Miyoshi and Takai, 2005; Macara and Spang, 2006). Alternatively, deficient junctional protein assembly may be due to abnormalities within regulatory signalling cascades; e.g. protein kinase C isoforms have a pivotal role in this context in the mouse embryo (Eckert et al., 2004a,b, 2005).
Overall, impaired TE integrity due to deficient junction formation could result in the ability to implant into the uterine wall being compromised and failure to provide the ICM with a controlled and balanced microenvironment to support further development. Tight junction formation is increasingly recognized to be important in many processes, from spatial organization of cellular architecture including membrane domains, signalling networks up to regulation of downstream transcription (Kohler and Zahraoui, 2005; Matter et al., 2005; Kavanagh et al., 2006). Consequently, TE deficiency in the form of suboptimal junction assembly could not only be a consequence but a cause of disturbed lineage-specific transcription patterns as has been seen in the mouse (Kim et al., 2004).
Amino acid turnover is related to junction formation and may predict trophectoderm integrity
To our knowledge, this is the first report to relate amino acid turnover in preimplantation embryos to aspects of junctional protein assembly. Polypeptide synthesis profiles are affected by developmental stage as would be expected due to the activation of the embryonic genome and synthesis of new proteins essential for ongoing development (Capmany and Bolton, 1999). Moreover, it has been shown that arrested human oocytes or embryos, or embryos of poor morphology, have different polypeptide synthesis profiles when compared with healthy counterparts (Capmany and Bolton, 1999; Katz-Jaffe et al., 2006). Recently, it has been shown that amino acid turnover measured non-invasively during early development can predict blastocyst development and implantation capacity of human embryos with similar morphology (Houghton et al., 2002; Brison et al., 2004; Houghton and Leese, 2004; Stokes et al., 2007). In our current study, we found a significant relationship between the turnover of certain amino acids and morphology which changed during periods of developmental transition. Moreover, positive or negative correlations of uptake and/or release of several amino acids had opposite relationships with morphology. Taken together, these data raise serious doubts about the suitability of morphology alone to identify viable embryos.
The turnover of various amino acids during developmental processes was related in a timely manner to the degree of membrane assembly of key junctional components: the higher the total amino acid turnover, the better the membrane assembly of all junctional components and, hence, the better the functional integrity of the TE of the resultant blastocyst. At first glance, this may appear to contradict previous studies which suggest that embryos with a low amino acid turnover are developmentally more competent (Leese, 2002). However, these studies relate to the turnover of amino acids much earlier in development, from day 1–2 (Brison et al., 2004) and day 2–3 (Houghton et al., 2002; Stokes et al., 2007) in order to predict future developmental potential. In the present study, amino acid turnover was measured in a cohort of developing embryos much later in development; during compaction, cavitation and blastocoel expansion and the increased turnover in embryos displaying good membrane assembly is likely to reflect an increase in protein synthesis around the time of cavitation (Lamb and Leese, 1994; Leese, 1995). Embryos which are unable to make this metabolic transition may still progress to the blastocyst stage but might have developmental deficits, e.g. impaired TE differentiation. A relatively high amino acid turnover during compaction and particularly cavitation could also indicate a greater capacity to maintain homeostasis, a capability that increases during this developmental period, and, hence, might be related to embryos with better developmental potential (reviewed in Lane and Gardner, 2005). Moreover, morphology and amino acid turnover were inversely related during compaction further underpinning the idea of better developmental competence in quieter embryos (Leese, 2002). However, such inverse relationship was lost when measured during cavitation, similar to the time period when the positive relationship between amino acid turnover and junction membrane assembly was most significant. This coinciding change of both relationships may represent further confirmation that embryo metabolic behaviour and structural differentiation change dramatically over a very short time period when the blastocoel is formed. Intriguingly, a link between TJ membrane assembly and cellular energy status has been established recently showing that TJ membrane assembly requires AMPK activity which, in turn, is sensitive to the intracellular AMP/ATP ratio (Zheng and Cantley, 2007; Zhang et al., 2006). The Na+/K+-ATPase may be one of the main energy consumers in the human (Houghton et al., 2003). The deficits to establish and maintain TJ membrane assembly within the TE found in our current study could be a consequence of a limited ability to meet increasing energy demands during blastocoel formation and expansion due to a increasing Na+/K+-ATPase activity, hence involving AMPK and Na+/K+-ATPase-driven mechanisms (Violette et al., 2006; Zheng and Cantley, 2007).
Among the individual junctional components, only membrane assembly of occludin and ZO-1 + showed a significant relationship with amino acid turnover. Interestingly, although the identity of the specific amino acids showing this relationship differed during both developmental transitions, overall amino acid turnover remained highly, positively correlated with assembly degree for both proteins. It is unclear why certain amino acids showed net appearance and others net disappearance in a stage dependent manner, but we speculate that some amino acids may activate stage-specific signalling cascades or other intracellular metabolic processes for developmental progress. Interestingly, the essential amino acid Leu was taken-up by compacting embryos with less membrane assembly of occludin and ZO-1 + in the resultant blastocyst, whilst those embryos with well established assembly of these proteins in the resultant blastocyst tended to release Leu. Leucine, and in some cases also Isoleucine, are not only essential amino acids but also part of a signalling pathway that stimulates protein synthesis in a number of cell types, and can regulate transcription (Gingras et al., 2004; Martin and Hall, 2005; Kimball and Jefferson, 2006). A highly active protein synthesis machinery could have a beneficial impact on the amount of junctional proteins made and available for transport to the membrane just prior to TE maturation. In the case of an insufficient activity of the transport machinery for junctional proteins, an increased availability of transporters and the transport target might be able to overcome the otherwise limited transport capacity responsible for impaired membrane assembly of certain junctional proteins. Moreover, it has been shown that individual amino acids or small polypeptides (e.g. poly-L-arginine) can induce internalization of the tight junction proteins ZO-1 and occludin via PKC-dependent phosphorylation and dephosphorylation events, thereby inducing permeability changes in the epithelium (Ohtake et al., 2003). Essential amino acids like Leu can also increase the proportion of trophoblast outgrowth from mouse blastocysts (Van Winkle et al., 2006) and could be important in trophoblast differentiation generally.
It seems plausible that impaired TE integrity in human embryos, as shown by insufficient junctional membrane assembly, could result in dysregulation of amino acid transport systems. Regulation of the different amino acid transport systems in preimplantation embryos is poorly understood. At least 15 different transport systems have been identified within the TE, which interrelate with each other depending on amino acid availability in the environment (Van Winkle et al., 2006). In the placenta, a descendant of the TE, localization of such transporter networks appears highly complex, and responsive to the environment (Cariappa et al., 2003; Jones et al., 2006). Deficient membrane assembly may impair junction fence function and could allow for mislocation of directed transport systems or disturbances in balancing amino acid distribution within the blastocyst (Cariappa et al., 2003).
In conclusion, we have confirmed that morphology alone is not a good selection method to identify viable later-stage embryos from a developing cohort and that TE integrity can be impaired independently of morphological appearance. We have also shown that non-invasive measurement of the turnover of certain amino acids can predict TE integrity in blastocysts to a high degree, independently from morphology.
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Acknowledgements |
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This work was funded by an MRC Co-operative group grant, GO100558, to TPF and HJL. FDH is a recipient of a Wellcome Trust Research Career Development Fellowship.
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Footnotes |
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The first two authors contributed equally to this study. 
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References |
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Submitted on January 9, 2007; resubmitted on March 9, 2007; accepted on May 2, 2007.
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