Developmental consequences of alternative Bcl-x splicing during preimplantation embryo development Alagammal Perumalsamy1, Roxanne Fernandes1,2,3, Ingrid Lai1,2,3, Jacqui Detmar1,2, Sue Varmuza4, Robert F Casper1,2,3 and Andrea Jurisicova1,2,3 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, University of Toronto, Canada Department of Physiology, University of Toronto, Canada Department of Cell and System Biology, University of Toronto, Canada Keywords apoptosis; Bcl-x; blastocyst; embryo; in vitro culture Correspondence A Jurisicova, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 6-1001-AJ, Toronto, ON, Canada Fax: +1 416 586 8588 Tel: +1 416 586 4800 ext 2052 E-mail: jurisicova@lunenfeld.ca (Received 23 June 2009, revised 10 November 2009, accepted 18 December 2009) doi:10.1111/j.1742-4658.2010.07554.x Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and ⁄ or dead cells Altering the Bcl-xL ⁄ Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development Introduction For one in 10 couples, failure to achieve a pregnancy remains a major problem Clinical results of assisted reproductive technology procedures in Canada suggest that clinical pregnancy is achieved in only 34% of patients under the age of 39 years [1] The cellular and molecular reasons behind such a high rate of failure are unclear; however, low developmental competence of conceived embryos is the most significant factor affecting pregnancy outcome Successful development of mammalian preimplantation embryos requires the integration of a series of molecular and cellular events, culminating in the formation of two distinct cell types – the trophectoderm (TE) and the inner cell mass (ICM) These two cellular Abbreviations AS, antisense; CDI, cell death index; DAPI, 4¢,6-diamidino-2-phenylindole; DCHFDA, 2¢,7¢-dichlorodihydrofluorescein diacetate; E, embryonic day; ICM, inner cell mass; IGF-1, insulin-like growth factor-1; MM, mismatched; PCD, programmed cell death; qPCR, quantitative PCR; ROS, reactive oxygen species; SC, scrambled; SEM, standard error of the mean; TE, trophectoderm; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DWm, mitochondrial membrane potential FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1219 Human blastocyst formation and Bcl-x expression A Perumalsamy et al lineages are committed to differentiation by activation of a set of distinctive transcription factors, which engage diverse downstream molecular pathways [2] In addition, the TE and ICM not only differ in their contribution to separate cellular compartments (e.g placenta and epiblast respectively), but also exhibit different susceptibilities to cell death The presence of dead and dying cells in blastocysts has been confirmed in a number of mammalian species by the use of several techniques, implicating apoptosis as a mode of cellular demise during blastocyst formation [3,4] In vitro culture of mammalian embryos from the zygote to blastocyst increases the rate of cell death, particularly in the ICM lineage [4,5] This becomes even more evident in embryos that have been conceived, manipulated and cultured in vitro [6–8] However, under these conditions, death occurs in both ICM and TE cells [4] Several growth factors, cytokines and nutrients have been implicated in the regulation of cell death of mammalian blastocysts [9,10] In particular, imbalances in glucose metabolism have been linked to increased activation of several death pathways in rodent preimplantation embryos Cell death induced by exposure to high glucose levels in vitro downregulates glucose transporters, resulting in a drop in intraembryonic free glucose [11], triggering massive apoptosis in the ICM lineage [12], causing malformations, congenital abnormalities, and cranial and neural tube defects [13,14] The molecular pathways mediating cell death in these animal models have been partially dissected, and involve p53, Bax and caspase-3 activation [14–17] At the present time, even with improved formulations for culture medium, only approximately 50% of human zygotes develop to the blastocyst stage, and only a subset of these blastocysts ( 40%) are able to implant after transfer [18,19] As excessive cell death is one of the logical candidates for early developmental failure, we explored cell death activation in human blastocysts under suboptimal culture conditions, and correlated this with cell death expression of several cell death regulatory genes The data presented here indicate that alternative processing of Bcl-x, which may be mediated in part by culture conditions, is a key determinant regulating the developmental fate of human and murine embryos in vitro Results Cell death rates in human blastocysts cultured in vitro We have previously shown that the development of human preimplantation embryos in vitro is influenced 1220 by the culture medium used [20] In order to understand the effect of culture medium on embryo quality, we have reanalyzed this cohort of normally fertilized human embryos (n = 96) for the extent and cellular location of cell death A low level of cell death was detected in almost all blastocysts, and this rate increased with prolonged culture in vitro, as the cell death index (CDI) increased from embryonic day (E)6 to E7 In addition, we confirmed that cells showing condensed chromatin also contained fragmented DNA, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays (Fig 1A–D) Occasionally, we noticed a few cells that displayed features of necrosis (0.8 ± 0.6 per blastocyst, data not shown) These cells were most likely arrested blastomeres from earlier cleavage divisions [21] In the majority of embryos, cell death activation appeared to be random, affecting both the ICM and TE; however, in  13.5% of embryos (13 ⁄ 96), the majority of cells in the ICM exhibited extensive chromatin condensation and ⁄ or fragmented DNA (Fig 1D), indicating elimination of the whole ICM Furthermore, some blastocysts (15 ⁄ 96) had an increased overall CDI (up to 56%), which, on average, did not exceed 14% on E6 and 21% on E7 (Fig 1E) Finally, there was a trend towards a higher rate of ICM depletion in blastocysts cultured in HTF medium To determine whether caspase-3 ⁄ activity is associated with induction of cell death, we employed cleavage assays of rhodamine-conjugated DEVD peptide This approach revealed a high level of caspase3 ⁄ activity in cells exhibiting chromatin condensation in human blastocysts that were cultured in HTF medium (Fig 1F,G), whereas limited caspase activity was detected in blastocysts with low death rates Taken together, our results suggest that cell death rate and CDI are elevated upon extended in vitro culture of human embryos Furthermore, the increased cell death might be correlated with high caspase-3 ⁄ activity Expression of cell death regulatory genes in human blastocysts In order to correlate cell death rates with the gene expression profile in individual preimplantation embryos, we exposed live embryos to a nuclear fluorochrome [4¢,6-diamidino-2-phenylindole (DAPI)], and determined the total number of cells with normal or condensed chromatin Embryos were immediately lysed and subjected to quantitative RT-PCR dot blot analysis We first performed preliminary experiments on FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al E B Cell death index (%) A Human blastocyst formation and Bcl-x expression n = 42 n = 48 Day D Human blastocysts (%) C 20 18 16 14 12 10 25 Day Day 20 15 10 Dead ICM F Day Elevated CDI G Fig Cell death in human blastocysts Representative photomicrographs of expanded human blastocysts analyzed for cell death by assessing chromatin status and fragmentation of DNA (A) Embryo stained with DAPI Very few cells show signs of apoptosis (B) Blastocyst with extensive activation of cell death in many cells, as shown by condensed chromatin with DAPI staining (C) Blastocyst with preferential elimination of ICM (arrow), as assessed by DNA condensation using DAPI staining (D) The same embryo depicted in (C) analyzed by TUNEL using biotinylated nucleotides and streptavidin ⁄ Texas Red conjugate The same cells that show intense DAPI fluorescence are also TUNELpositive, as indicated by the bright red fluorescence (E) CDI ± SEM (proportion of cells exhibiting hallmarks of apoptosis); incidence of either ICM depletion or elevated rates of cell death in human blastocysts at day or day of in vitro culture (F, G) Differential interference contrast (DIC) light microscopy (F) and cleavage of the DEVD substrate (red) merged with nuclear DAPI staining (blue) (G) in a day fully expanded human blastocyst (containing over 100 cells and an ICM of normal appearance), with many cells (arrow) of the ICM showing higher activity for caspase-3 mouse blastocysts to determine whether this approach would result in altered gene expression None of the assessed genes (Bax, Mcl-1, Bag-1, Bcl-x, or b-actin) showed significant changes induced by exposure to DAPI (data not shown) We next analyzed the expression levels of genes implicated in regulating cell survival (Mcl-1, Bcl-x, Bcl-w, and Bag-1), cell death inducers (Bax and Hrk), and cell death executioners (Casp-2 and Casp-3) After hybridization of cDNA amplified from 37 single blastocysts against a probe for EF-1a (used as a housekeeping gene), only those embryos giving a robust signal (n = 32) were used for further analysis Levels of both cell death suppressors and activators were variable among the embryos, often ranging from undetectable to highly expressed Using multiple regression analysis, we determined that only Bcl-x transcript level correlated significantly with CDI FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1221 Human blastocyst formation and Bcl-x expression A A Perumalsamy et al DAPI C Bcl-xS Bcl-x Cpm bound 1500 1000 500 0 10 15 20 25 Number of dead cells per blastocyst B Bcl-xL Bcl-xS Actin Fig Bcl-xL ⁄ Bcl-xS expression in human blastocysts (A) Correlation between Bcl-x expression and the number of dead cells in human blastocysts The levels of Bcl-x mRNA are expressed as units of specifically bound c.p.m Two populations of blastocysts with either high levels (squares, n = 5) or low levels (circles, n = 27) of Bcl-x expression are present (B) Analysis of Bcl-x splice variants in human blastocysts RT-PCR southern blot gel hybridized with a radiolabeled human Bcl-x cDNA probe that recognizes both splice variants (see arrows) Differences in the long ⁄ short ratio were observed among individual embryos Shown below is ethidium bromide staining of gel-separated products following RT-PCR amplification of b-actin Each lane represents a single human expanded blastocyst (C) Expression of Bcl-xS protein in three E7 fully expanded human blastocysts All embryos were obtained from different patients, had good morphology, with distinct ICM cells, contained > 80 cells, and had low (4–8%) CDI Magnification · 400 Left panles: nuclear DAPI staining; right panels: Bcl-xS Whereas some embryos did not express detectable levels of Bcl-xS, others exhibited faint diffuse nuclear Bcl-xS immunostaining in a subset of healthy cells but strong punctuate immunostaining in some, but not all, cells showing evidence of DNA condensation (arrowheads) Inset: control blastocyst exposed to the primary antibody preabsorbed with Bcl-xS peptide (P = 0.005) In addition, after plotting Bcl-x transcript levels versus the number of dead cells per blastocyst, it became clear that two distinct populations of embryos could be detected (Fig 2A), those with high (n = 5) and those with low (n = 27) Bcl-x expression 1222 Alternative splicing of the Bcl-x transcript and subcellular distribution of Bcl-x in human blastocyst It was previously shown that Bcl-x is alternatively spliced into at least two different isoforms: Bcl-xL FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al (long or death-suppressing) and Bcl-xS (short or death-inducing) Thus, we next determined whether this alternative splicing of the Bcl-x transcript could explain the cell death profile observed in human blastocysts Using RT-PCR and Southern blot analysis, we simultaneously detected variable levels of Bcl-xL and Bcl-xS in individual human blastocysts (representative results are shown in Fig 2B) Whereas some embryos (Fig 2B, lanes 1, 3, and 6) predominantly expressed the long isoform, others (lane 4) expressed either low levels of the Bcl-xL isoform, or approximately equal levels of the long and short Bcl-x splice variants (lane 2) Minimal differences were observed in the levels of b-actin expression We further investigated the presence and subcellular localization of Bcl-xS Confirming our data obtained by RT-PCR, we observed variable levels of Bcl-xS immunostaining, ranging from undetectable to high (Fig 2C) A moderate level of immunostaining was observed in both the cytoplasmic and nuclear compartments of healthy cells, whereas elevated levels of nuclear staining were observed in some cells undergoing apoptosis In many cases, these distinct staining patterns of Bcl-xS were observed in the same blastocyst Moreover, in some embryos, staining in the mural TE lineage was largely restricted to the nucleus, whereas in the ICM and overlaying polar TE, staining was mostly cytoplasmic (Fig 2C) As Bcl-xS is the logical candidate for the cell death inducer, our results suggest that elevated levels of Bcl-xS may be responsible for activation of cell death in human embryos cultured in vitro under suboptimal conditions To confirm our hypothesis that Bcl-xS expression is associated with induction of cell death, we designed antisense (AS) experiments targeting the splice junction boundaries of Bcl-x [22] This approach is based on the premise that a complementary oligodeoxynucleotide, binding to the 5¢-splice site of the Bcl-xL template strand in exon II, will downregulate expression of this isoform via suppression of Bcl-xL splicing Moreover, this approach would simultaneously induce Bcl-xS transcription, owing to redirection of the splicing machinery to the 5¢ Bcl-xS site First, we tested a wide range of oligodeoxynucleotide concentrations from to 50 lm on mouse morulae Doses of 1, 2.5 and lm had no effect on Bcl-x expression, or on embryo development, whereas a dose of 50 lm decreased the rate of blastocyst formation to below 50%, even in the control [scrambled (SC)] group, and thus was deemed unsuitable for further studies An oligodeoxynucleotide concentration of 25 lm both downregulated Bcl-xL expression and induced Bcl-xS expression in mouse blastocysts in the AS treatment group only Correspondingly, Human blastocyst formation and Bcl-x expression mismatched (MM) oligodeoxynucleotide completely downregulated Bcl-xL, but failed to induce Bcl-xS, at this dose, whereas SC oligodeoxynucleotide had no effect (Fig 3A,B) It has been shown previously that MM oligodeoxynucleotide with a bp mismatch as compared with AS decreased the ability to induce Bcl-xS transcript, implying that the induction is sequence-dependent [22] Thus, having established the optimal concentration for experiments as 25 lm, we performed further experiments to assess the impact of altered isoform expression Embryonic development was not affected by simultaneous downregulation of Bcl-xL and Bcl-xS (MM group), as 81% of embryos (n = 113) formed fully expanded or hatching blastocysts 48 h after initiation of treatment However, developmental competence was dramatically affected by upregulation of Bcl-xS, as only 54% of embryos (n = 118) reached blastocyst stage in the AS group (P < 0.0001) Control (untreated; n = 111) and SC (n = 93) groups were not significantly different from the MM group (86% and 79%, respectively) Furthermore, upon evaluation of the cell number and death rates, it was evident that Bcl-xS induction was accompanied by decreased cell numbers and elevated cell death rates (P < 0.0001; Fig 3C) Embryos in the AS group appeared to be developmentally delayed, and frequently degenerated in culture (Fig 3D) To determine whether caspase-3 ⁄ activity is associated with the induction of cell death mediated by altered Bcl-x splicing, we again employed the caspase cleavage assay As was observed in human blastocysts, caspase-3 ⁄ activity was detected in cells undergoing programmed cell death (PCD) in murine blastocysts However, DEVD cleavage was also noted in cells prior to evidence of nuclear collapse (DNA condensation) As expected, treatment of murine embryos with AS oligodeoxynucleotides resulted in dramatically upregulated caspase-3 ⁄ activity, which was not observed in the control or MM groups (Fig 3E) The results suggest that, as seen with human blastocysts, induction of Bcl-xS expression is positively correlated with cell death and caspase-3 ⁄ activity in mouse embryos Altered Bcl-x splicing affects reactive oxygen species (ROS) production and mtDNA copy number Bcl-xL has been previously implicated in inhibiting ROS production [23] To determine whether decreasing Bcl-xL and increasing Bcl-xS expression increases ROS production in murine blastocysts, individual murine embryos were treated with AS, MM and SC oligodeoxynucleotides and subsequently exposed to FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1223 Human blastocyst formation and Bcl-x expression A A Perumalsamy et al D Bcl-xL Bcl-xS B Total cell number C SC Control 80 * 60 40 MM AS 20 E Dead cell number Con SC MM AS * SC Con SC MM MM AS AS Fig Altered Bcl-xS expression causes induction of cell death in murine embryos (A) Representative results of analysis of Bcl-x splice variants and b-actin levels in murine blastocysts upon treatment with antisense oligodeoxynucleotides Each lane represents five embryos subjected to RT-PCR analysis Con, untreated; SC; 25 lM scrambled oligodeoxynucleotides; MM, 10 or 25 lM mismatched oligodeoxynucleotides; AS, 10 lM AS or 25 lM antisense oligodeoxynucleotides Amplification of alternative splicing products (long versus short isoforms) of the Bcl-x gene hybridized with a radiolabeled murine Bcl-x cDNA probe that recognizes both splice variants Note that increased accumulation of Bcl-xS in the SC group represents variability in the abundance of this isoform in murine blastocysts, as similar results were observed in untreated embryos in other experiments (B) Ethidium bromide staining of gel-separated products following RTPCR amplification of b-actin (C) Effect of oligodeoxynucleotide treatment on embryo quality, as evidenced by total cell number and cell death Data shown are means ± SEMs Statistical significance was observed in the AS group (significantly different from all three other groups, as denoted with asterisk) in all categories assessed, with P < 0.0001 (D) Nuclear staining (DAPI in blue) of murine embryos exposed to 25 lM phosphothiolated oligodeoxynucleotides (C, SC, MM, and AS), as described above Magnification: · 250 Only AS treatment, associated with induction of Bcl-xS, resulted in embryos with poor developmental competence, caused by reduced cell numbers and induction of cell death, as observed by DNA condensation (see arrows) (E) Caspase-3-like activity in mouse blastocysts Images were obtained by serial sectioning on a deconvolution microscope (· 200) SC, MM or AS embryos (dose: 25 lM) Only AS treatment associated with induction of the Bcl-xS splicing isoform was accompanied by upregulation of caspase-3-like activity 2¢,7¢-dichlorodihydrofluorescein diacetate (DCHFDA), a fluorescent ROS indicator that reflects accumulation of H2O2 (Fig 4A–D) In the AS group, decreasing Bcl-xL expression and increasing Bcl-xS expression significantly increased the level of H2O2 as compared with control or SC embryos (P < 0.05; Fig 4E) In addition, significant increases in ROS levels were also seen in the MM group (P < 0.05) (Fig 4E), indicating that depletion of Bcl-xL is sufficient to cause elevated oxidative stress 1224 As there is a close relationship between ROS generation and mitochondrial activity, we also measured changes in mitochondrial membrane potential (DWm); however, no significant differences were observed due to either induction of Bcl-xS or downregulation of Bcl-xL (data not shown) In order to determine whether the difference in ROS production was due to the difference in mtDNA content, quantitative PCR (qPCR) was performed to analyze the amount of mtDNA present in embryos of the SC, MM and AS FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al B E Relative fluorescence units (millions) A Human blastocyst formation and Bcl-x expression Control b b a a SC Con SC C 180 160 140 120 100 80 60 40 20 MM AS D MM AS Relative fluorescence ratio F b 1.3 1.28 1.26 1.24 a a 1.22 1.2 1.18 1.16 1.14 SC MM AS Fig Altered Bcl-xL ⁄ Bcl-xS expression results in increased ROS production in murine embryos (A–D) Representative photomicrographs of mouse blastocysts exposed to 25 lM phosphothiolated oligodeoxynucleotides [control (Con) (n = 15), SC (n = 18), MM (n = 22), and AS (n = 23)], treated with 10 lM DCHFDA, and analyzed for ROS (E) Analysis of ROS in oligodeoxynucleotide-treated mouse blastocysts Values represent relative fluorescence units ± SEM Increased ROS production was observed in MM and AS oligodeoxynucleotide-treated murine blastocysts Different letters indicate that the treated groups are significantly different (P < 0.05) (F) Quantitation of mitochondrial DNA copy number in treated murine blastocysts Elevated mitochondrial copy number was observed with MM oligodeoxynucleotide-treated murine blastocysts Different letters indicate that they are significantly different (P < 0.05) groups As we did not observe any difference in ROS levels between the control and SC groups, a control group was not included in the mtDNA content assay Surprisingly, only Bcl-xL downregulation resulted in increases in mtDNA in the MM group (Fig 4F) In summary, the data presented here show that induction of Bcl-xS with a concomitant decrease in Bcl-xL transcript level increases ROS levels without any change in DWm and mtDNA content Culture in HTF medium and exposure to high glucose levels alters Bcl-x splicing in mouse embryos As we observed variable ratios of Bcl-xL ⁄ Bcl-xS splicing in human embryos, we investigated whether HTF medium could be responsible for induction of Bcl-xS expression We first determined that HTF medium significantly decreased the rate of blastocyst formation (P = 0.0303) (Fig 5A) and caused an increased rate of cell death by E4.5 (Fig 5B) This was accompanied by a change in Bcl-x splicing, favoring formation of Bcl-xS (Fig 5C) and accumulation of ROS (Fig 5D), thus recapitulating many features of the AS experiments KSOM and HTF medium differ in the presence or absence of several compounds; one of the most obvious differences is a 10-fold higher glucose level in HTF medium Previously, in vitro exposure to high glucose levels has been linked to poor embryo development, causing increased rates of cell death and embryonic malformations [12] Therefore, we explored whether exposure to glucose could alter Bcl-x splicing In order to confirm the biological effect, we assessed the developmental progression of embryos exposed to in vitro hyperglycemia The rate of embryonic progression was reduced, particularly at E4.5, when a significantly lower proportion of embryos in high-glucose (KSOM + 50 mm d-glucose) conditions developed to the appropriate stage (P = 0.0265) (Fig 6A) We next determined whether an alteration in Bcl-x splicing could be associated with the lower rates of progression in high-glucose conditions In vitro-cultured embryos reaching the morula stage or later at E4.5 in KSOM + 50 mm d-glucose were assessed for alteration in Bcl-x splicing Indeed, embryos cultured in FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1225 Human blastocyst formation and Bcl-x expression A A Perumalsamy et al C 4.50 Bcl-xL:Bcl-xS splicing ratio Embros reaching successive developmental stage (%) 120 100 * 80 60 HTF KSOM 40 3.00 2.50 2.00 1.50 1.00 0.50 0.00 HTF E2.5 E3.5 D E4.5 a CDI MI a a b KSOM 450 Relative fluorescence units (millions) E1.5 Cell death and mitotic index (%) 3.50 20 B * 4.00 400 350 300 250 * 200 150 100 50 HTF KSOM HTF KSOM Fig Developmental progression, cell death and altered Bcl-xL ⁄ Bcl-xS expression in murine embryos (A) Zygotes cultured either in HTF medium (n = 102) or in KSOM (n = 101) were assessed daily for developmental progression from E1.5 to E4.5; the proportion of embryos reaching the appropriate developmental stage is expressed as a percentage A significant decrease in blastocyst formation in HTF mediumcultured embryos (black line) as compared with KSOM-cultured embryos (gray line) was observed by E4.5 (B) Cell death and mitotic index (MI) were calculated by staining the embryos with DAPI, and are expressed as percentages based on total cell number A significantly increased CDI (black bar) was seen in the embryos cultured in HTF medium (n = 20) as compared with those cultured in KSOM (n = 19) (P = 0.006) Different letters indicate that values are statistically significant (C) Pools of five embryos at the morula ⁄ blastocyst stage cultured in HTF medium (n = 8) and KSOM (n = 9) were assessed for changes in Bcl-xL ⁄ Bcl-xS ratio Embryos cultured in HTF medium had a significantly decreased ratio as compared with those cultured in KSOM (P = 0.027), owing to the decreased expression of Bcl-xS (D) Analysis of fluorescence generated by DCHFDA probe, reflecting ROS generation, was performed in embryos cultured in either HTF medium (n = 13) or KSOM (n = 10) at E3.5 Values represent relative fluorescence units ± SEM Significantly increased ROS generation was observed in the HTF medium group (P = 0.0009), as indicated by the asterisk Representative images for ROS are shown glucose-enriched KSOM medium had a significantly lowered Bcl-xL ⁄ Bcl-xS ratio as compared with the KSOM-only group (P = 0.031) (Fig 6B) Furthermore, culture of embryos in the presence of high glucose concentrations caused elevated production of ROS (Fig 6C) In summary, our results indicate that developmental rate is compromised by culture in HTF medium or in 1226 the presence of high glucose levels, and that this can be attributed to an increase in Bcl-xS transcript levels Discussion Our results on cell death rates in the human embryos indicate that almost 15% of human blastocysts cultured in vitro completely eliminate the embryonic FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al Relative fluorescence units (millions) C 100 90 80 70 60 50 40 30 20 10 Embryos reaching successive developmental stage (%) A Human blastocyst formation and Bcl-x expression KSOM KSOM + 50 mM glucose * 80 70 60 50 40 30 20 10 KSOM E2.5 E3.5 E4.5 KSOM + 50 mM Glucose Bcl-xL:Bcl-xS splicing ratio B 4.50 4.00 3.50 3.00 * 2.50 2.00 1.50 1.00 0.50 0.00 KSOM KSOM + 50 mM Glucose Fig Exposure to glucose is associated with decreased embryo development and induction of Bcl-xS (A) Embryos were assessed daily from E1.5 to E4.5, and the proportion of cleaved embryos reaching the correct developmental stage at the correct time was calculated Embryos in high-glucose conditions (KSOM + 50 mM D-glucose; gray line, n = 170) had compromised developmental competence, as a significantly lower proportion of these embryos than of those cultured in KSOM only (black line, n = 60) reached the blastocyst stage at E4.5 (P = 0.0265) (B) At E4.5, pools of five embryos at the morula stage or later were assessed for changes to Bcl-x splicing KSOM-cultured embryos had an average Bcl-xL ⁄ Bcl-xS ratio of 3.49 ± 0.74 (same as in Fig 5C), which was significantly greater than the average Bcl-xL ⁄ Bcl-xS ratio of 2.03 ± 0.27 in embryos cultured in KSOM + 50 mM D-glucose (P = 0.031) (C) Representative images for analysis of ROS are shown ROS analysis was performed in embryos cultured in KSOM and KSOM + 50 mM glucose at E3.5 A significant increase in ROS was seen in embryos cultured with 50 mM glucose (P < 0.0001) lineage destined to form the fetus (i.e the ICM), leaving only precursor cells for the placenta Such blastocysts may initiate implantation, but are capable of creating only extra embryonic tissues with an empty embryonic sac, a condition clinically recognized as a ‘blighted ovum’ [24] The absence of ICM cells has been previously described in human embryos cultured in vitro [25,26], but this was attributed to an abnormal allocation of cells into the ICM and TE lineages [26] Our results suggest that, in some embryos, cells of the ICM are established, but are subsequently eliminated An elevated rate of cell death in human ICM cells has been previously described [27]; however, the molecular pathways responsible for this phenomena are unclear Of the genes examined in this study, Bcl-x was the only one whose expression showed a statistically significant correlation with CDI in human blastocysts Importantly, alternative splicing within the coding region of Bcl-x results in the production of several distinct protein products with opposing functions [28] Bcl-xL has been reported to function as a prominent death-suppressing molecule during postimplantation embryonic development [29], as targeted mutation of the murine Bcl-x gene that disrupts both isoforms triggers midgestational embryonic lethality [30] By comparison, very little is known about the mode of action and physiological importance of Bcl-xS, which lacks the BH1 and BH2 domains Bcl-xS renders cells more susceptible to apoptotic signals [21], even when these cells simultaneously express Bcl-xL [31,32] In addition, it has been observed that Bcl-xS is able to form homodimers as well as heterodimers with Bcl-xL [33], but this ability may depend on cell type Moreover, cell death induction is associated with Bcl-xS FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1227 Human blastocyst formation and Bcl-x expression A Perumalsamy et al localization to mitochondria, as deletion of the transmembrane region prevented mitochondrial localization and diminished the death-inducing properties of this molecule [33] Our experiments on native Bcl-xS showed either diffused cytoplasmic or nuclear accumulation of Bcl-xS in early embryos, with colocalization to condensed DNA in pyknotic cell corpses Although a role of Bcl-2 family members in regulating survival has been extensively established, new evidence exists suggesting a role of these proteins in regulation of cell cycle progression Both Bcl-x and Bcl-2 delay cell cycle entry and enhance the differentiation potential of immature cells [34,35] Furthermore, Bcl-xS not only increases susceptibility to apoptosis [22], but also alters the kinetics of the cell cycle progression [36] Thus, nuclear subcellular localization in healthy cells in some embryos may reflect the ability of Bcl-xS to interfere with cell cycle progression, as we also observed decreased cell numbers with upregulated Bcl-xS transcript Alternative splicing of the Bcl-x gene appears to play an important role in human blastocyst development Human blastocysts have variable implantation potential and exhibit variable Bcl-x long ⁄ short form ratios Alternative splicing favoring increased levels of Bcl-xS may be one mechanism involved in upsetting the balance of PCD regulatory molecules in the developing embryo Indeed, AS experiments performed on murine embryos confirmed this hypothesis Abnormal embryonic development, associated with delayed cleavage, and activation of cell death accompanied by activation of caspase-3 ⁄ 7, causing embryonic degeneration, was only observed in the group of embryos with upregulation of Bcl-xS and was not affected by downregulation of Bcl-xL These data, which fully agree with previous reports on the action of Bcl-xS in some somatic cell types [22,36], implicate alternative splicing of the Bcl-x gene as a major determinant of successful embryonic development In conjunction with its antiapoptotic activity, Bcl-xL has been implicated in the regulation of ROS production [37] Both of these cellular roles have been linked to the BH4 domain [38] The results of our study indicate that altering the Bcl-x isoform ratio (e.g decreasing the amount of Bcl-xL and increasing the amount of Bcl-xS) results in an elevated ROS level, without any obvious effect on DWm Concomitantly, altered mtDNA content was detected only in the Bcl-xL knockdown, suggesting the initiation of premature mtDNA replication, which otherwise occurs only after the blastocyst stage [39] It is thus possible that the increase in ROS production in the Bcl-xL knockdown group is due to increased mitochondrial copy 1228 number, as previously observed in yeast [40] As mtDNA is in close proximity to the site of H2O2 generation, the effect on mtDNA could be immediate, and this might result in premature replication of mitochondria [40] However, mtDNA number remained unchanged in the AS group, implying that the increased level of ROS caused by downregulation of Bcl-xL and induction of Bcl-xS was not due to the increased mitochondrial number It has been previously reported that increased ROS levels in mitochondria signals the nucleus and, as a result, telomere shortening occurs, leading to the loss of proliferative capacity and replication senescence [41–43] In addition, elevated ROS levels have been linked to permanent early embryo arrest, and this may be due to mitochondrial dysfunction and activation of p66Shc [44] Thus, we propose that altering the Bcl-xL ⁄ Bcl-xS ratio in blastocysts results in mitochondrial stress, and increased ROS production, causing diminished cell numbers and cell senescence, resulting in compromised embryo development In addition, Bcl-xL has been postulated to have a role in cellular metabolism, although this role is not well defined Bcl-xL overexpression affects several metabolic parameters, most notably causing a shift from oxidative phosphorylation to aerobic glycolysis and promoting glucose consumption [45] In conditions of nutrient deprivation, Bcl-xL facilitates the metabolism of endogenous substrates such as phosphocreatine [45] The ability to maintain metabolic function in conditions of limited nutrient availability is thought to contribute to the antiapoptotic function of Bcl-xL [46] However, transgenic mice with Bcl-xL overexpression showed significant disturbances of mitochondrial metabolism associated with defective insulin secretion and induction of diabetes [47] Thus, these data suggest that fine regulation of Bcl-xL expression, perhaps via alternative splicing, regulates metabolism Hyperglycemic conditions have been previously linked to embryopathies [48] that result in changes in mitochondrial morphology along with elevated ROS production [49,50] Furthermore, in mice, hyperglycemia-induced cell death and embryo death were correlated with the induction of the proapoptotic members bax, p53 and caspase-3 [14,17,51] As hyperglycemia has been previously established as a potent inducer of cell death in murine embryos, we assessed whether glucose can alter the Bcl-xL ⁄ Bcl-xS ratio Recent findings suggest that insulin and insulinlike growth factor-1 (IGF-1) are capable of altering cell death via downregulation of Bcl-xS without affecting Bclx-L in a caspase-dependent manner [52] This is very exciting, as IGF-1 has been shown to improve rates of blastocyst formation and decrease rates of PCD in FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al human embryos [10] However, whether IGF-1 acts through regulation of Bcl-xS splicing in preimplantation embryos remains to be established Experimental procedures Human embryos and in vitro cultures The major cohort of human embryos described here consists of a subset of embryos previously used for the analysis of developmental competence in different culture conditions and the gene expression of trophoectodermal markers [20] Additional blastocysts used for analysis of Bcl-x splicing, immunocytochemistry and caspase activity assay were obtained under identical conditions from the IVF Program, Division of Reproductive Endocrinology and Infertility, at the Mount Sinai Hospital, Toronto Some of these embryos were of poor quality and are thus unsuitable for cryopreservation Also, the good quality embryos obtained were not used for long-term storage as decided by the patients In all cases, the project was approved by the human ethics committee at Toronto General Hospital, Mount Sinai Hospital and the University of Toronto Embryos were incubated in HTF medium (supplemented with 10% synthetic serum substitute; Irvine Scientific, Irvine, CA, USA) at 37 °C in a humidified chamber gassed with 5% CO2 ⁄ 95% air Murine blastocyst cultures Female mice of the ICR strain were stimulated with IU of pregnant mare serum gonadotropin (Sigma-Aldrich, St Louis, MO, USA) followed, 48 h later, by IU of human chorionic gonadotropin (Sigma), and mated overnight with ICR males of proven fertility Embryos were recovered by flushing the oviducts at day 0.5 or day 1.5 postmating These embryos were subsequently cultured in microdrops of HTF medium (Irvine Scientific), KSOM with amino acids (Speciality Media, now Millipore, Billerica, MA, USA) or KSOM with amino acids supplemented with 50 mm d-glucose (Sigma) under mineral oil (Speciality Media), and were followed in culture up to the expanded blastocyst stage (E4) Gene expression studies were performed at E3.5 or E4.5 with all embryos that reached at least the morula stage All animal experiments were conducted in accordance with the Canadian Council of Animal Care guidelines, and protocols were approved by the Animal Care Committee of Mount Sinai Hospital Analysis of cell number and cell death To analyze chromatin status, we used a combined technique for simultaneous nuclear staining with the fluorochrome DAPI (Sigma), and, in a subset of embryos, TUNEL Briefly, blastocysts were fixed in 4% paraformaldehyde Human blastocyst formation and Bcl-x expression diluted in NaCl ⁄ Pi on microscope slides and air-dried, and slides were stored at )70 °C until further use After thawing, slides were washed in NaCl ⁄ Pi, and TUNEL was performed as previously described [53] After TUNEL, the slides were stained with DAPI for 10 The slides were then examined and photographed using a Zeiss fluorescence microscope with appropriate filters at · 400 magnification The CDI was calculated as the percentage of total cells that exhibited intense DAPI staining due to condensation of chromatin Cells were considered to be necrotic when they contained cytoplasmic staining with DAPI and or TUNEL, as previously described [21,53] Analysis of gene expression A subset of 37 human blastocysts was stained unfixed by adding DAPI to the culture medium After a 30 incubation, the zona pellucida was removed, and embryos were briefly viewed in depression slides under the fluorescence microscope, as above The numbers of normal cells and cells with condensed DNA (pyknotic nuclei) were recorded, and single embryos were then used for analysis of gene expression The levels of Mcl-1, Bcl-x, Bcl-w, Bag-1, Hrk, Bax, caspase-2 and caspase-3 mRNA were determined by quantitative RT-PCR-based assay, described previously [54,55] The amplified material was then analyzed by hybridization of dot blots with radiolabeled cDNA probes, followed by quantification on a phosphorimager as previously described [8] The cDNA probes used for analysis recognized the 3¢-UTR, and were either donated for this study or were cloned in our laboratory Reverse-transcribed products of single embryos were prescreened for the quality of cDNA by hybridization with a cDNA probe against the gene for elongation factor-1a (EF-1a) Using this control, we determined that five of the 37 samples failed to produce good-quality hybridization signals, and these cDNA samples were excluded from further analysis Alternative splicing of Bcl-x To determine the form of Bcl-x expression, we performed RT-PCR with specific primers spanning the alternative splice site of the Bcl-x gene [28] Single blastocysts (n = 10) were analyzed as previously described [8] The amplified products were separated through a 1.5% agarose gel, and analyzed by Southern blot analysis using a radiolabeled probe recognizing both Bcl-xL and Bcl-xS [8,56] RT-PCR was performed to amplify b-actin cDNA as an internal control (forward primer, 5¢-ATCATGTTTGAGACCTTC AA-3¢; reverse primer, 5¢-CATCTCTTGCTCGAAGTCCA -3¢) Murine embryos were subjected to identical analysis of Bcl-x expression as described above, as primer sequences for Bcl-x splicing are conserved b-Actin expression was assessed with the following primers: 5Â-GGCCCAGAG FEBS Journal 277 (2010) 12191233 ê 2010 The Authors Journal compilation ª 2010 FEBS 1229 Human blastocyst formation and Bcl-x expression A Perumalsamy et al CAAGAGAGGTATCC-3¢ and 5¢-ACGCACGATTTCCC TCTCAGC-3¢ Immunocytochemistry To characterize Bcl-x protein expression, we performed indirect immunocytochemistry using antibody against Bcl-xS (PC-89; Oncogene, Cambridge, MA, USA) Twenty embryos that reached the blastocyst stage were fixed in formalin, stored and thawed as described for the TUNEL procedure (see above) Following antigen retrieval in citrate buffer, samples were washed and preblocked in 10% normal porcine serum with 0.1% Triton X-100 in NaCl ⁄ Pi The rabbit polyclonal antibody against human Bcl-xS was diluted : 100 in blocking solution, and incubation was performed overnight A biotinylated Multilink secondary antibody (Dako, Mississauga, ON, Canada) was applied in a : 200 dilution, and the final localization of bound antibody was performed with streptavidin ⁄ Texas Red Embryos were counterstained with DAPI Samples not exposed to primary antibody (n = 2) and those exposed to primary antibody preabsorbed with Bcl-xS peptide (n = 3) (PP54; Oncogene) served as negative controls Antibody specificity was also confirmed by antibody preabsorption experiments followed by western blotting performed on MCF7 cells, according to the manufacturer’s instructions Single-cell caspase activity assay Caspase activity was assessed in blastocysts as previously described [57] Briefly, embryos were cultured in the presence of rhodamine-conjugated DEVD substrate (PhiPhiLux; OncoImmunin Inc., College Park, MD, USA) for h Samples were washed three times, fixed in 4% paraformaldehyde, transferred onto slides, and stained with DAPI Embryos were viewed by fluorescence microscopy using appropriate filters, and representative photomicrographs were taken For AS experiments, caspase-3-like activity was assessed under deconvolution microscope (Olympus IX70) Embryos were serially scanned, and optical sections (1 lm) were analyzed using deltavision software (Applied Precision, Issaquah, WA, USA) AS experiments Phosphothiolated oligodeoxynucleotides, previously designed and tested by Taylor et al [22], were synthesized by The Center for Applied Genomics, Hospital for Sick Children (Toronto, Ontario, Canada) Three different oligodeoxynucleotides were used: AS targeting the splice site of Bcl-x (5¢-CTGGATCCAAGGCTCTAGGT-3¢), bp MM (5¢-CT GGTTACACGACTCCAGGT-3¢), and SC (5¢-TGACTG AGCCTATCTCGATA-3¢) These were prediluted in culture 1230 medium and added at different doses to culture microdrops containing embryos at the morula stage Embryos were cultured for 48 h, and five embryos from each experiment were used for RT-PCR for evaluation of Bcl-x transcript accumulation, as described above Remaining embryos were fixed and analyzed for cell number and cell death rates as described above Detection of ROS and DWm staining Morula-stage embryos were cultured in AS oligodeoxynucleotides (25 lm) for 48 h, as previously described, and subsequently analyzed for generation of ROS and DWm At E4.5, blastocyst-stage embryos from each treatment group were incubated with 10 lm DCHFDA or 10 lm JC-1 dye (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide) (Molecular Probes; Invitrogen, Carlsbad, CA, USA) for 30 at 37 °C in a humidified atmosphere of 5% CO2 JC1 allows simultaneous detection of mitochondria with low (green) and high (red) mitochondrial potential The red/green ratio was determined for all the experimental groups of individual embryos, and an average ratio of J-aggregate (red) to J-monomer (green) staining was determined as previously described [58] After incubation,they were washed once in mHTF medium with 0.5% BSA, and imaging was performed on live embryos with 10 lm optical interval scanning using deconvolution microscopy The fluorescence was detected using 528 nm and 572 nm (tetramethyl rhodamine iso-thiocyanate filter) intensity (fluorescein isothiocyanate filter), using the · 20 objective, and the intensity was measured and quantitated using delta vision image acquisition software (Applied Precision, Issaquah, WA, USA) Mitochondrial copy number To analyze mtDNA in the embryos, single blastocysts from all of the treatment groups were washed, transferred to lL of HTF medium, and stored at )80 °C until use Individual blastocysts were then lysed with lL of lysis buffer containing 200 mm KOH and 50 mm dithiothreitol at 65 °C for 15 After this period, samples were neutralized with lL of neutralization buffer containing 300 mm KCl, 900 mm Tris ⁄ HCl (pH 8.3), and 200 mm HCl qPCR was performed using the DNA engine Opticon (MJ Research Inc., Schaumbum, IL, USA), and opticon monitor software (MJ Research Inc.) was used to assess mitochondrial copy number using SYBR Green Reaction mix (ABI, Foster City, CA, USA), with the following cycle conditions: initial denaturation for 15 at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s Forty cycles apply to all three steps: denaturation, annealing and extension For comparison, a b-actin genomic locus was used to assess the FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS A Perumalsamy et al quantity of genomic DNA The relative fluorescence ratio was calculated Primer sequences used for qPCR for amplifying mtDNA were as follows: Mt11, 5¢-CATACCCCGAAA ACGTTGGTT-3¢; and Mt12, 5¢-AGGACCTAAGAAGA TTGTGAAGTAGATGA-3¢ The b-actin primer sequences used were as follows: Actin RT F1, 5¢-CCACAGCTGA GAGGGAAATC-3¢; and Actin TR R1, 5¢-AAGGAA GGCTGGAAAAGAGC-3¢ Statistical analyses Analysis of cell death and determination of the levels of expression of different genes were performed by multiple regression analysis and by Spearman rank correlation Analysis of the distribution of Bcl-x expression in human blastocysts was performed using the Kolmogorov–Smirnoff test The results of AS experiments were evaluated using v2-test or with Kruskal–Wallis one-way ANOVA followed by Dunn’s pairwise multiple comparison Analysis of Bcl-x splicing in mouse embryos was performed with the Mann– Whitney U-test, using spss software (SPSS Inc., Chicago, IL, USA) Values in the graph (mtDNA and ROS) represent the means ± standard errors of the mean (SEMs) Statistical analysis was conducted with the graphpad prism software package version (Graphpad Software Inc., San Diego, CA, USA) Comparisons between different groups were performed by one-way ANOVA combined with Newman–Keuls multiple comparison post hoc test Results were considered to be statistically significant if P < 0.05 Acknowledgements We would like to express our gratitude to all of the investigators who generously provided cDNA probes used in this study (Bcl-x, C B Thompson; Bcl-w, S Cory; Hrk, G Nunez; caspase 2, J Yuan; and ˜ Bag-1m, J C Reed) We would also like to thank G I Perez, K Moley and J Riley for advice on the glucose experiment and technical assistance, and N MacLusky for outstanding assistance with the statistical analysis Our special thanks go to members of RBU and TCART, who provided us with all human embryos required for this work This work was supported by grants from the Canadian Institutes of Health Research to A Jurisicova 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Casper RF (2003) Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and caspase-3 in blastomere fragmentation Mol Hum Reprod 9, 133–141 Perez GI, Tao XJ & Tilly JL (1999) Fragmentation and death (a.k.a apoptosis) of ovulated oocytes Mol Hum Reprod 5, 414–420 Acton BM, Jurisicova A, Jurisica I & Casper RF (2004) Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development Mol Hum Reprod 10, 23–32 FEBS Journal 277 (2010) 1219–1233 ª 2010 The Authors Journal compilation ª 2010 FEBS 1233 ... alters Bcl-x splicing in mouse embryos As we observed variable ratios of Bcl-xL ⁄ Bcl-xS splicing in human embryos, we investigated whether HTF medium could be responsible for induction of Bcl-xS... experiments to assess the impact of altered isoform expression Embryonic development was not affected by simultaneous downregulation of Bcl-xL and Bcl-xS (MM group), as 81% of embryos (n = 113) formed... Alternative splicing of the Bcl-x transcript and subcellular distribution of Bcl-x in human blastocyst It was previously shown that Bcl-x is alternatively spliced into at least two different isoforms: Bcl-xL