1 Institute and Department of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, 2 Institutes of Biomedical Sciences and Molecular Biology, College of Life Sciences, National Chung-Hsing University, Taichung, 3 Department of Internal Medicine, National Taiwan University Hospital, Taipei, 4 Department of Physiology, College of Medicine, Taipei Medical University, Taipei and 5 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Taipei Medical University Hospital, Taipei, Taiwan
6 To whom correspondence should be addressed at: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Taipei Medical University Hospital, No. 252, Wu-Shing Street, Taipei, Taiwan. Email: tzengcr{at}tmu.edu.tw
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Abstract |
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Key words: blastocyst/cDNA microarray/gene expression/hatching/implantation
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Introduction |
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In the past few decades, only a few factors could be studied in each experiment. Since the cDNA microarray technique has been developed in the mid-1990 s, this high-throughput tool has been the most powerful technique in answering the physiological or pathophysiological questions (Chen et al., 1998; Schneider et al., 2004
). We have reported the differential gene expression profiles between early gestational decidua and chorionic villi using cDNA microarray technology (Chen et al., 2002
). Using this technique, the endometriosis-related genes have also been identified in our previous study (Yang et al., 2004
). The global gene profiles of human and mice endometrium during the window of implantation have also been investigated (Reese et al., 2001
; Kao et al., 2002
). However, a successful implantation not only depends on the maternal endometrium, but also the embryo. To the best of our knowledge, the gene expression pattern in the embryo during the blastocyst hatching stage for implantation remains undiscovered.
Recently, failure of the embryonic zona pellucida to rupture following blastocyst expansion has been put forward as a possible contributing factor in implantation failure. In order to help embryos escape from their zona during blastocyst expansion, different types of assisted hatching have been developed (Cohen, 1991; De Vos and Van Steirteghem, 2000
). Early loss of pregnancy after hatching and implantation is also very high, estimated at 2540% (Wilcox et al., 1988
). What is wrong with this process? Although many losses involve genetic abnormalities, there is often no known cause. Several factors, including trypsin-like proteinases, hormonal factors, leukaemia inhibitory factor and prostanoid pathways, might play important parts in successful hatching and implantation (Simpson, 1980
; O'Sullivan et al., 2001
). But, given the complexities of early development, it is likely that many other genetic and/or epigenetic (DNA methylation) regulatory mechanisms are also involved (Carlone and Skalnik, 2001
; Norwitz et al., 2001
). In order to study these, the gene expression profile of normal embryo development during the hatching process has become more important.
Preimplantation embryo development, especially the formation of blastocyst, has been studied extensively over the past decade. These studies were focused on specific molecules or a few members of a given family such as nitric oxide (NO)-related factors (Chen et al., 2001; Sengoku et al., 2001
), cytokines/hormones (Harvey et al., 1995
; Diaz-Cueto and Gerton, 2001
; Das et al., 2002
), proteinases (O'Sullivan et al., 2001
; Whiteside et al., 2001
), and signal transductions (Armant et al., 2000
) using a one-by-one approach. To overcome the limitation of trace amounts of RNA from a certain number of embryos, the T7 RNA polymerase-based in vitro linear RNA amplification was used to amplify the mRNA from blastocyst (Schneider et al., 2004
). Herein, the global transcriptomic analyses of blastocyst before or after hatching were investigated by cDNA microarray.
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Materials and methods |
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In vitro transcription of amplified antisense RNA (aRNA)
Total RNA was extracted from each set of blastocysts using RNAzolTM B reagents (Life Tech, Gaithersburg, MD, USA) and linearly amplified (independently) an estimated 106-fold using T7 RNA polymerase as previously and according to the manufacturer's specifications from MessageAmpTM aRNA Kit (Ambion Inc., Austin, TX, USA) (Polacek et al., 2003; Wang et al., 2003
; Schneider et al., 2004
).
Microarray system
Preparation of cDNA targets and microarray hybridization
Five micrograms of the aRNA derived from blastocysts before or after hatching were labelled with biotin during reverse transcription. All hybridization experiments were performed in triplicate. The details of target preparation, hybridization and colour development have been described previously (Chen et al., 1998; Hong et al., 2000
; Chen et al., 2002
, 2004a
). The 6144 mouse expressed sequence tag (EST) clones with a putative gene name for the mouse cDNA microarray were obtained from the IMAGE consortium libraries through its distributor (Research Genetics, Huntsville, AL, USA). These mouse IMAGE clones were derived from various tissues and in different library constructs, including unfertilized oocytes, whole embryos (from 2-cell to blastocyst), inner cell mass, embryonic stem cells and germ cells, and several other organs (brain, heart, liver, and so on) in different development stages (http://www.image.llnl.gov/image/html/muslib_info.shtml#NIH_MGC_256). Most of the clones have been partially sequenced and verified, and the sequence information is available as EST from dbEST of GenBank. It has been used in several other mice studies and has been published in our recent works (Chen et al., 2004b
; Yu et al., 2004
).
Image processing and digitization
After colour development, the microarray images were scanned and digitized using a flat-bed scanner (PowerLook 3000; UMAX, Taipei, Taiwan) (Chen et al., 2004a,b). The scanner provided a high resolution and was suitable for larger arrays such as arrays of 6144 elements. The microarray was processed by commercial image processing programs to convert the true-colour images into gray-scale images, and then the image analysis and spot quantification were done by the GenePix 3.0 (Axon, Union City, CA, USA) or by the MuCDA program, which was written in-house and is available online (http://www.w3.mc.ntu.edu.tw/department/genechip/supplement.htm).
Real-time quantitative RTPCR
To confirm the expression patterns of up-regulated or down-regulated genes in the blastocyst hatching process, several re-sequenced and known genes were selected for further analysis using real-time quantitative RTPCR in a 96-well format as previous described (Chen et al., 2004a). Total RNA from pre-hatched or hatched blastocysts without amplification was used for real-time quantitative RTPCR. Primers were designed using the Primer Express v2.0 Software (Applied Biosystems Inc., Foster City, CA, USA). All of the primers used in this study have been listed in the Table I. All reactions were carried out in 50 µl volumes containing 25 µl of SYBR Green PCR Master Mix (Applied Biosystems Inc.). The amount of tested gene cDNA relative to the amount of TBP cDNA was measured as
. The ratio of tested gene mRNA copies relative to TBP (TATA box binding protein, used as a housekeeping control) mRNA copies was defined as 2
CTxK (K: constant).
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Statistical analysis
Gene expression data obtained from the microarray experiments were processed and normalized using the protocol and programme that has been previously described (Chen et al., 2002). For each gene, the mean and SD of expression as well as the ratios of the mean pre-hatched blastocyst expression versus mean hatched blastocyst expression were calculated and used for comparison. The differentially expressed genes were chosen beyond the 95% predicted regression line. Next, the conventional criteria of 3.0-fold differences were used to sub-classify the significantly different genes. The differentially expressed genes were considered to be significantly down- or up-regulated by a factor of
3.0-fold between pre-hatched and hatched blastocysts and were sequence-verified. Expression differences of <2.0- or <3.0-fold have usually been considered at the limit of detection in previous analyses (Popovici et al., 2000
; Tanaka et al., 2000
; Cavallaro et al., 2002
). The genes whose expression corrected with the blastocyst development were grouped into categories by their putative functions on the basis of literature reports. A repeated measure analysis of variance (ANOVA test) was performed to determine any significant difference between the development stages of blastocyst in the real-time quantitative RTPCR analysis. Where appropriate, the data are expressed as mean ± SEM.
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Results |
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After analysis, it was determined that the expression of 85 detected genes was up-regulated by a factor of 3-fold in blastocysts at the hatching stage and only 13 genes were
2-fold in blastocysts before hatching. Table II lists the categories of 47 up- and down-regulated known and novel genes which have been verified via re-sequencing, which were significantly altered in hatched blastocysts (Table II). The ratios showed the difference between two stages in mRNA gene expression level. The complete gene expression profiles in both pre-hatching blastocyst and hatched embryo and other supplement data are posted on our website: http://www.w3.mc.ntu.edu.tw/department/genechip/supplement.htm.
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The gene expression profiles of three hatching-related proteinases [pitrilysin metalloprotease 1 (Pitrm1), proteinase 3 (Prtn3), and RIKENCDNA6820428-006, similar to human serine proteinase 23 (prtse23)] and other novel hatching-stage expressed factors [HO-1, protein inhibitor of nitric oxide synthase (PIN), and proteasome 26S subunit], which have higher expression level in hatched blastocyst, have also been identified from 2-cell stage, 8-cell stage, morula, pre-hatching blastocyst to hatched blastocyst via real-time quantitative RTPCR (Figure 2B). The prtn3 and pitrm1 are two novel proteinases, which were up-regulated during the embryo development and attained a higher expression level at hatched blastocyst as compared with other stages of preimplantation embryo. The novel serine proteinase, 12 days mice embryo female Müllerian duct includes surrounding region cDNA, similar to human serine proteinase 23 (prtse23), was almost undetectable from 2-cell stage to pre-hatching blastocyst, but showed >10-fold increase at hatched blastocyst stage (12.45±0.81-fold). Other hatching-related factors (HO-1, PIN and proteasome 26S subunit) also show the related higher expression level at hatched blastocyst (Figure 2B).
Immunohistochemistry
Figure 3 showed that the protein level and location of NCAM, E-cadherin and IL-4R were highly expressed in hatched blastocyst, and of these, the NCAM was more highly expressed in the inner cell mass side of the hatched blastocyst, which might be the implantation site to the decidual endometrium. The E-cadherin was distributed almost in all blastocyst membrane, but it showed stronger staining in inner cell mass. IL-4R has been found to be expressed in both the pre-hatching and hatched embryo, but the hatched embryo also showed more IL-4R staining.
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Discussion |
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Previously, using suppression subtractive hybridization (SSH) and heterologus cDNA array, the gene expression profiles in mice and bovine oocyte and/or preimplantation embryos have been screened (Dalbies-Tran and Mermillod, 2003; Zeng and Schultz, 2003
). Using human cDNA array,
300 genes have also been identified in bovine oocyte, and of this amount, 70 transcripts were expressed differently during in vitro maturation (IVM) (Dalbies-Tran and Mermillod, 2003
). The mRNA expression patterns of 10 genes [including Na/K-ATPase alpha1, E-cadherin, zonula occludens protein-1 (ZO-1), glucose transporter-1 (Glut-1), Glut-2, Glut3, and others] have also been studied during blastocyst expansion by RTPCR (Wrenzycki et al., 2003
).
Recently, the global gene expression profiles of mice and human pre-implantation embryos from germinal vesicle (GV) oocyte to blastocyst have already been published via in vitro transcription and microarray techniques (Dobson et al., 2004; Hamatani et al., 2004a
; Wang et al., 2004
; Zeng et al., 2004
). The differential expressions of genes between inner cell mass and trophoblast in blastocyst were also reported (Dreesen et al., 2002
). For the first time, the blastocyst hatching-specific gene expression profile was identified in this study. Here, we compared the previous reports and ours to focus on discussing the hatching-related genes, including cell adhesion molecules, epigenetic regulators, immunoresponse modulators and hatching-related proteinases, which were and/or might be related to some critical events during embryo development, hatching, cell differentiation and implantation.
Cell adhesion molecules (CAM) are important during embryo development not only in cellcell adhesion to maintain the structure of blastocyst, but also in cellcell interaction and communication in embryo implantation (Aplin, 1997; Kimber and Spamswick, 2000
). There were at least four adhesion molecules (NCAM, E-cadherin, galatin 7 and vanin 3) up-regulated after blastocyst hatching in this study, and the immunohistochemical staining also showed that NCAM and E-cadherin were localized in the inner cell mass of the embryo, which might also be the site for implantation (Duc-Goiran et al., 1999
). NCAM not only plays an important role in neural migration, differentiation and nervous system development, but also as a survival factor against teratogen pyrimethamine (Klementiev et al., 2002
). E-Cadherin deletion might lead to development defects in several development stages and has been suggested as playing the critical role in embryo implantation (Larue et al., 1994
). Vanin 3 and galatins (1, 3 and 4) have been reported to be involved in preimplantation embryo development and blastocyst activation (Hamatani et al., 2004a
,b
). Galatin3 has been reported to be expressed at higher levels in blastocyst compared with morula (Ponsuksili et al., 2002
). Herein, according to the microarray data, the vanin 3 and galatin7 were both up-regulated at hatched blastocyst, which might suggest that these two adhesion molecules could play a role in blastocyst adhesiveness for embryo implantation. Further studies are underway to identify the detailed mechanisms of these CAM in regulating cellcell connection within the embryo or between embryo and maternal endometrium.
Epigenetic modification of the genome could regulate several critical biological and pathological events, including development and carcinogensis (Li, 2002; Jones, 2002
). Epigenetic reprogramming was thought to be an important issue during mammalian development (Reik et al., 2001
). There are two major events to regulate this epigenetic modification, DNA methylation and histone acetylation. Several epigenetic regulator-related events (including DNA methyltransferase and histone deacetylase) have been identified in early embryo development. The DNA methyltransferase family (Dnmt1o, Dnmt1, Dnmt2, Dnmt3a, and 3b) has been shown to be involved in oogenesis, embryo development, and cell differentiation (Ding and Chaillet, 2002
; Ratnam et al., 2002
). Previous studies have shown that the DNA methylation was decreased after fertilization, increased after blastocyst, and maintained during fetal development (Reik et al., 2001
). Dnmt1o was thought to be critical for oogenesis and Dnmt3a and 3b for embryo differentiation (Bird, 1999
; Reik et al., 2001
). In the hatching process, the blastocyst transforms from low-methylated to higher-methylated status. In this study, Dnmt1, Dnmt3a and Dnmt3b were found to be up-regulated after blastocyst hatching, which might suggest that these Dnmt could be the epigenetic reprogramming regulator during this reverting process. The results are consistent with previous reports of the epigenetic regulation during the preimplantation embryo development (Bird, 1999
; Reik et al., 2001
). Other epigenetic regulators, SIN3a and SIN3b, were also increased after blastocyst hatching. The SIN3 complex shares four core proteins with NuRD (HD-1, 2, RbAP46 and 48) and SIN3 is proposed to act as a scaffold for the complex and this complex might be modulated by nuclear hormone receptor, which might play the role in histone deacetylation and lead to DNA methylation (Ahringer, 2000
). Recently, a role for SIN3 was demonstrated in cell survival, cell cycle regulation, as well as a regulatory role in mitochondrial respiration (Pile et al., 2003
). These results seem to indicate that Dnmt1, Dnmt3a, Dnmt3b, SIN3a and SIN3b might be the important epigenetic regulators in the blastocyst after the hatching process and might direct subsequent embryo development, implantation and differentiation.
Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cellcell communication in many organ systems, including the reproductive system. Although the presence of NO synthase (NOS) in development and its role in the regulation of embryo growth and apoptosis are well established (Shaul, 1995; Chen et al., 2001
), little is known about the expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during preimplantation embryo development. In this study, HO-1 has been found to be up-regulated during blastocyst hatching. This might suggest that the emzyme or its metabolites could be the regulator for embryo hatching or a survival factor for the hatched embryo. HO-1 has been reported to prevent CD95/FasL-mediated apoptosis, as an immunoregulator, which could significantly prolong allogeneic orthotopic liver transplantation survival via a downstream HO-1CO signalling pathway (Ke et al., 2002
).
Before implantation, the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, the proteinase enzyme, is necessary for the hatching process. Previously, a novel murine tryptase, implantation serine proteinase (ISP1) gene, has been reported (O'Sullivan et al., 2001). In this study, three novel proteinases were found during the blastocyst hatching process. The microarray data and real-time quantitative RTPCR show that the Pitrm1, Prtn3 and Prtse23 genes were expressed at the blastocyst stage and dominant at the hatched blastocyst stage. These early expressed proteinases might play a role in the embryo hatching and implantation processes.
In summary, blastocyst hatching is an important developmental process for embryo implantation, whereas assisted hatching in many cases is indicated in assisted reproduction and probably enhances clinical pregnancy in older women (Cohen, 1991; Edi-Osagie et al., 2003
). This study provides the last piece of the map to complete the profiles of the dynamic gene expression changes from GV oocyte to hatched blastocyst by using the T7 RNA polymerase-based in vitro linear RNA amplification and cDNA microarray, as compared with previous reports (Dobson et al., 2004
; Hamatani et al., 2004a
; Wang et al., 2004
; Zeng et al., 2004
). Our study has pointed out the usefulness of investigating the regulatory mechanisms and has selected some candidate genes in blastocyst hatching and further implantation. Not only cell adhesion molecules, but also epigenetic regulators, immunoresponse modulators, survival factors, and hatching-related proteinases might play important roles in this critical process of embryo development. This work also provides information for studying these hatching-specific genes which may become new drug targets for controlling fertility.
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Acknowledgements |
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Notes |
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Submitted on September 1, 2004; resubmitted on March 9, 2005; accepted on April 18, 2005.