Abstract. Introduction. RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 23 January 2004

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 23 January 2004 Article Real-time reverse transcription-polymerase chain reaction analysis of translation initiation factor 1A (eif-1a) in human and mouse preimplantation embryos Maria Lindeberg is a doctoral student at the Department of Clinical Science at the Karolinska Institutet in Stockholm. Her research is aimed at gaining further comprehension of the expression pattern of genes involved in preimplantation embryo development and oocyte maturation. The objective of the research team is to gain more knowledge regarding the molecular mechanisms involved in the reproductive system and thereby try to improve human assisted reproduction techniques. Ms Maria Lindeberg M Lindeberg 1,3, O Hovatta 2, L Ährlund-Richter 1 1 Clinical Research Centre, 2 Department of Clinical Sciences, Karolinska Institute Novum, Karolinska University Hospital, Stockholm, Sweden 3 Correspondence: Tel: ; Fax: ; maria.lindeberg@crc.ki.se Abstract Fluorescence-monitored real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to study steady state concentrations of translation initiation factor eif-1a mrna in mouse and human preimplantation embryos. Its expression in human embryos has not been described previously. oocytes, and 2-cell and 4-cell embryos all showed comparable total concentrations of eif-1a RNA, indicating a gradual decrease in the average concentration per blastomere during these developmental stages. A 4-fold increase was observed in the 8-cell embryos. This concentration remained at the morula stage, followed by a 7- to 8-fold further increase at the blastocyst stage. preimplantation embryos already showed increased concentrations of eif-1a RNA at the 2-cell stage. Thus, transcription levels of the eif-1a gene are associated with embryonic gene activation (EGA) in both species. The method used, real time RT-PCR, proved to be sensitive enough to detect quantitative expression in single mouse blastomeres, the observed values for steady-state concentrations of mrna in single blastomeres correlating well with the values for whole embryos. The possibility to study gene expression quantitatively in single blastomeres may be useful in preimplantation genetic diagnosis. Keywords: eif-1a/human, preimplantation, real-time RT-PCR, RNA concentrations 338 Introduction During oocyte maturation and preimplantation development, important events concerning cell growth and differentiation occur and altered transcription levels of specific genes often reflect these events. The switch from maternal to embryonic control of the genome is commonly referred to as embryonic gene activation (EGA) (Schultz, 1993). Early minor transcription activation has been observed in several species as early as at the G2 phase of the first cleavage after fertilization (Thompson, 1996). The major transition occurs at different time points in different species, in mice at the 2-cell stage (Telford et al., 1990; Schultz, 1993) and in humans between the 4- to 8-cell stages (Tesarik et al., 1986). Successful occurrence of EGA is obviously one of the crucial events for further survival of the mammalian embryo. The fluorescence-monitored real-time reverse transcription polymerase chain reaction (RT-PCR) has previously been shown to be a suitable method for analysis of gene expression in single embryos (Steuerwald et al., 1999, 2000). Compared with conventional RT-PCR, several steps involved in the quantification procedure can be omitted. The increased sensitivity excludes the need for nested PCR, which reduces the risk of contamination artefacts. Furthermore, there is no need for gel electrophoresis to visualize the PCR products, or phosphoimaging or densitometry to quantify bands on the gel.

2 The expression of eif-1a increases at the time of EGA in murine and bovine preimplantation embryos (De Sousa et al., 1998). The expression of this factor in human embryos has not been reported. This factor may be of importance as regards the developmental potential of the embryo. Hence, it was necessary to study its expression in human embryos using a sensitive method, fluorescence-monitored real-time RT-PCR, which was successfully applied both in embryos and in single blastomeres. Materials and methods Oocyte and embryo preparations Female (CBA B6)F1 mice underwent ovulation by hormonal induction according to standard procedures (Hogan et al., 1994). Fertilized oocytes were collected and incubated in M2 medium with 0.1% hyaluronidase to remove the adherent cumulus cells. The zygotes were then washed five times in M2 media before they were placed in microdrops of KSOM medium covered with mineral oil and cultured at 37 C in 5% CO 2. After culture to the indicated stages, the zona pellucida was removed using acidified Tyrode s solution (Vitrolife AB, Göteborg, Sweden), and zygotes, and 2-cell, 4-cell and 8-cell embryos were transferred to 2 µl ddh 2 O with RNasin 1 IU/µl (Promega, Madison, WI, USA), snap-frozen in liquid nitrogen and stored at 70 C. Single blastomeres were obtained by first removing the zona pellucida from 2-cell to 8-cell embryos (using acidified Tyrode s solution), which were then mechanically separated by pipetting up and down, using a thin glass pipette. Single blastomeres were placed in 2 µl ddh 2 O with RNasin (1 IU/µl) (Promega). Preimplantation embryos were donated by couples undergoing fertility treatment at the IVF unit at Huddinge University Hospital. Written consent concerning the fact that the embryos could be used for research purposes was obtained from the couples and the Research Ethics Committee of the Karolinska Institute approved this procedure. The embryos were supernumerary from IVF treatment, either fresh non-optimal quality, or frozen embryos. The couples underwent treatment with a long protocol according to Fridström et al. (1999). Decisions on IVF versus intracytoplasmic sperm injection (ICSI) were based on sperm quality according to WHO criteria, and strict morphological criteria (Krüger et al., 1987; WHO, 1992). The oocytes were collected into IVF medium (Vitrolife AB), and then IVF or ICSI was performed. Fertilization was checked h after insemination, with embryos containing two pronuclei being judged as normally fertilized. After fertilization control, fertilized oocytes were transferred to fresh droplets containing IVF medium. On day 2 of embryo culture, good quality embryos were transferred to the subject or cryopreserved for later use. Surplus embryos were cultured further in 20 µl droplets of CCM medium (Vitrolife AB) and transferred to fresh medium on day 4 of embryo development. The morphology of the embryos was evaluated according to a system derived from Mohr and Trounson (1985) and described in detail by Fridström et al. (1999). Oocytes that failed to be fertilized, embryos that were of too poor a quality to be transferred and surplus embryos following transfer and freezing were used in this study. No distinction was applied between embryos derived via ICSI or IVF in the present study. Oocytes and embryos from the indicated preimplantation stages were treated with acidified Tyrode s solution to remove the zona pellucida and washed in Dulbecco s phosphate-buffered saline (PBS) (Gibco BRL, Grand Island, USA) before being lysed in ddh 2 O with RNasin (Promega) at 1 IU/ µl, snap-frozen in liquid nitrogen and stored at 70 C until reverse transcription was performed. Reverse transcription Synthesis of cdna was performed using the Superscript First Strand Synthesis System for RT-PCR (Gibco BRL). Random hexameres (50 ng), 1 µl dntp-mix (10 mmol/l) and DEPCtreated water up to 10 µl were added to the cell lysate. The mixture was incubated at 65 C for 5 min and then immediately placed on ice. Next, a mixture of 2 µl RT-buffer (20 mmol/l Tris-HCl, 50 mmol/l KCl), 4 µl MgCl (5 mmol/l), 2 µl DTT (10 mmol/l) and 40 IU RNaseOUT recombinant ribonuclease inhibitor was added and the samples were incubated at room temperature for 2 min. Finally, 50 IU of Superscript II reverse transcriptase was added and cdna synthesis was carried out at room temperature for 10 min and at 42 C for 50 min. The reaction was stopped by incubating at 70 C for 15 min. The samples were then stored at 20 C until real-time PCR was performed. Primers and probes For primer and probe design Primer Express Software (Applied Biosystems, Foster City, CA, USA) was used. The design was based on cdna sequences obtained from GeneBank: mouse (AF026481) (Davis and Schultz, 1998) and human (L18960) (Dever et al., 1994). Primers were designed to generate short amplicons of between 50 and 150 bp, which is reported to be optimal for real-time PCR. The ABI 7700 sequence detector requires the annealing temperatures for the primers to be between 58 and 60 C and for the probe between 65 and 70 C. Primer sequences and expected size for each product are shown in Table 1. The same probe was used to detect transcripts of both human and mouse eif-1a; in an area where homology between the murine and human sequences is 100%. Pre-developed primers and probes from Applied Biosystems were used for the controls, 18S rrna and GAPDH. Real-time PCR Real-time PCR was performed using an ABI PRISM 7700 sequence detector (Applied Biosystems), in which the thermal cycler, fluorescence detector and analysis software is integrated in one instrument. The reaction mixture consisted of 300 nmol/l of each primer, 100 nmol/l of the TaqMan probe 1x TaqMan universal mastermix and an aliquot of the RT reaction mixture, corresponding to one-quarter of an embryo (human) and one embryo (mouse) in a total reaction volume of 339

3 25 µl. The cdna was then heated to 50 C for 2 min and denatured at 95 C for 10 min. The template was then amplified over 50 cycles of 15 s melting at 95 C and 1 min at 62 C for annealing and extension. During the PCR, an argon ion laser excites fluorescent dyes in each reaction. Fluorescence data were acquired by measurements taken approximately every 7 s and presented as a plot of fluorescence intensity versus cycle number. Synthesis of cdna was run on pools of embryos of each developmental stage studied. An aliquot of the RT reaction mixture corresponding to one embryo was then added to each PCR. Four PCR were run for each stage, duplicates of eif-1a and the endogenous reference gene GAPDH. This experiment was carried out three times, which means that a total of six embryos of each stage were studied per gene. When analysing single blastomeres, 10% of the RT reaction mixture was added to each of two PCR, duplicates of eif-1a. Samples of single whole embryos of each stage were treated similarly. Blastomeres from four 2-cell and two 4- and 8-cell embryos were studied; in total 8, 8 and 10 blastomeres from the respective stages. Of the 16 blastomeres from 8-cell embryos, one was excluded because of unacceptably low values from both the control and test gene, and five blastomeres were damaged during separation. The experiment was carried out twice for eif-1a and once for the control 18S rrna. Synthesis of cdna was run on a pool of 10 human oocytes. For PCR a volume corresponding to 1 oocyte was taken from the RT reaction mixture and split into four (duplicates of eif-1a and the control 18S rrna). For 2-cell stages and later stages, reverse transcription was run on single human embryos. A volume corresponding to one-quarter of an embryo was then added to four PCR (duplicates of eif-1a and the control 18S rrna). As each embryo was used in four PCR (two controls and two test gene) the experiment could not be repeated with the same embryo. However, the experiment was carried out three times with different embryos and the total numbers of embryos studied at each stage were: oocyte n = 3, 2-cell n = 3, 4-cell n = 5, 8-cell n = 4, morula n = 2 and blastocysts n = 3. Standard curve construction Standard curves were constructed using duplicates of six serial dilutions of cdna prepared from RNA of known concentration. Total liver RNA (mouse) covering a concentration range of ng and total placenta RNA (human; range ng) were used. One standard curve was generated and run in parallel with the unknown embryo samples in each experiment. Fluorescence was measured and the threshold cycle (C t ) values at each point in the standard curve were plotted against the log (ng) of the initial concentration (Higuchi et al., 1993). The relative concentration of unknown samples was determined from the standard curve and calibrated against the zygote (mouse) or the oocyte (human). The inter-assay coefficients of variation were 3.6% for the mouse standards and 2.7% for the human standards. The standard curves displayed linear correlation for both species. The efficiency of the reactions was close to 100% (m-values around 3.3). Results Successful DNA amplification signals were observed from all oocyte/embryo samples subjected to reverse transcription. Samples without reverse transcription, and control samples without template were all negative. Using the indicated mouse primers (Table 1), a product of the expected size (143 bp) from mouse liver cdna was demonstrated (Figure 1). The experiments were carried out three times and the results presented are the averaged relative values for each developmental stage. Duplicate data within the same run were consistent, and individual experiments showed similar trends. Figure 1. Ethidium bromide-stained 2% agarose gel. M = 50 bp ladder, A = PCR product human eif-1a (148 bp) and B = PCR product mouse eif-1a (143 bp). Table 1. Polymerase chain reaction primers and probe sequences (eif-1a). PCR primers/ Sequence Amplicon probe length (bp) 340 Forward 5 -AAG AAT AAA GGC AAA GGA GGC A-3 Reverse 5 -ATT GCT TCC AAC CGT CCA TT Forward 5 -ATG CCC AAG AAT AAA GGT AAA GGA-3 Reverse 5 -TTG CTT CTA GCC GTC CAT TTC Probe (mouse 5 -ACC TGA GCA TAC TCC TGC CCA TCC TCT TT-3 and human)

4 In the experiments with mouse embryos, both GAPDH and 18S rrna were tested as endogenous controls. Their expression profile was different to that of eif-1a at the developmental stages studied. Using GAPDH or 18S rrna as a common denominator would therefore result in a misleading pattern. Nevertheless, a control was run in parallel with the test gene in each experiment to make sure the assay worked properly and also as a control of embryo quality. All the embryos included in the analysis showed an acceptable expression level of the control. A large increase in the steady state concentration of eif-1a mrna was observed as early as after the first cell division (20-fold in the whole embryo and 10-fold per blastomere; Figure 2A). Concentrations from 4-cell embryos were similar to those found in 2-cell embryos, i.e. indicating a decrease in the average blastomere concentration. Results from 8-cell embryos indicated a subsequent increase of eif-1a RNA (approximately two-fold increase per blastomere). This observation is in line with that in an earlier report by De Sousa et al. (1998), who also observed an initial increase at the 2-cell stage, a decrease at the 4-cell stage and a subsequent increase at later stages. Similar results were obtained using single blastomeres. Compared with the zygote, a 10-fold increase was observed in single blastomeres at the 2-cell stage, followed by a slight decrease at the 4-cell stage and a 1.5-fold increase at the 8-cell stage. Adding the values from single blastomeres gave a total value similar to that from a whole single embryo of the respective stage (Figure 2B). Complementary DNA synthesized from single human embryos was readily amplified. Strong fluorescence signals were obtained from all developmental stages tested and threshold values from duplicate samples were consistent. Different embryo samples from the same stages showed some variation but a common trend was observed. Negative controls were not amplified, and product identity (148 bp) was confirmed by gel electrophoresis (Figure 1). B A Figure 2. (A) Relative eif-1a mrna concentrations at the indicated stages of mouse preimplantation embryos, as determined by real-time RT-PCR. Values for later developmental stages are calibrated against values for the zygote. (B) Steady state concentrations of mouse eif-1a mrna in single blastomeres and in single whole embryos, as determined by real-time RT-PCR. The values on the y-axis correspond to the amounts (ng) of eif-1a RNA found in mouse liver RNA (used to construct the standard curve). Figure 3. Relative eif-1a mrna concentrations at the indicated stages of human preimplantation embryos, as determined by real-time RT-PCR. Values for later developmental stages are calibrated against values for unfertilized oocytes. * indicates significant difference from 4- cells result (P = 0.05, Student s t-test). 341

5 342 In the experiments with human embryos, 18S rrna was run as an endogenous control. Its expression profile was different from that of eif-1a at the developmental stages studied. Using 18S rrna as a common denominator would therefore result in a misleading pattern. Similarly to the mouse experiments, the control was run in parallel with the test gene in each experiment and all the embryos included in the analysis showed an acceptable expression level of the control. Oocytes, 2-cell and 4-cell embryos all showed a similar degree of amplification (Figure 3), indicating a gradual decrease of the average blastomere steady-state level. A four-fold total increase was observed in 8-cell embryos (i.e. a two-fold increase at the blastomere level). The difference in expression level between the 4- and 8-cell stage reached statistical significance (P = 0.05; Student s t-test). No further increase was observed at the morula stage (i.e. a decrease at the blastomere level), followed by a seven- to eight-fold increase in blastocysts (Figure 3). Discussion It has been shown that human embryos express eif-1a, and an increase in the steady state concentrations of its mrna was observed in early embryogenesis. This increase is related to the time of embryonic gene activation (EGA). A 4-fold increase occurred at the 8-cell stage, and a 7- to 8-fold increase at the blastocyst stage. Morphologically, some of the embryos studied were classified as bad quality and not suitable for transfer. It has been suggested that failed EGA is associated with bad embryo quality in cultured human embryos (Tesarik, 1989). Nevertheless, significant up-regulation between the 4- and 8-cell stage has been observed, so even if some of the 8-cell embryos studied here were suboptimal, they tended to have entered the EGA phase. This encourages further studies of this characteristic to add more information on genetic markers that possibly could be added to the morphological criteria when selecting embryos. In the mouse, the change in expression level was earlier, corresponding to earlier EGA. A 20-fold increase was seen at the 2-cell stage, followed by a decrease at the 4-cell stage and an increase again at the 8-cell stage. It has been suggested that the onset of EGA in the mouse requires the first round of DNA replication, which provides an opportunity for the inherited transcription machinery of the oocyte to gain access to promoters and enhancers. In contrast, the second round of DNA replication may repress transcriptional activity. This repression is caused by displacement of the previously assembled transcription complexes and it provides an opportunity for histones to out-compete the transcription factors This chromatin remodelling is suggested to be the reason for the downregulation of eif-1a at the 4-cell stage (Davis et al., 1996). The large amounts of mrna produced at the 2-cell stage may be enough to produce proteins sufficient for the needs of the 4-cell embryo. The increase again at the 8-cell stage probably reflects a general increase of transcriptional activity necessary for further developmental stages. Real-time RT-PCR proved to be a very useful method to detect gene expression even in single blastomeres. The expression levels of eif-1a were studied in blastomeres from the same embryo. There were small differences, illustrated in Figure 2B, each point representing the mean value of duplicate measurement of one blastomere. As this gene is a translation factor, the expression is likely to be evenly distributed in the different blastomeres irrespective of their developmental destiny. It is not probable that its expression would reflect any early differentiation of cleavage stage embryos. However, studies by Hansis et al. (2002) have shown that differences between blastomeres in cleaving embryos can be revealed by the expression pattern of genes involved in blastocyst formation. If preimplantation differentiation is to be studied in single blastomeres the test genes should, of course, be very carefully selected, as there are limits to how much can be studied in single blastomeres if further amplification steps are undesirable. For studies of differentiation of blastomeres to the blastocyst stage, with marker genes that indicate development towards the inner cell mass or trophectoderm, as suggested by Hansis and coworkers (Hansis et al., 2002; Hansis and Edwards, 2003), this method could be very useful. To study polarization of the oocyte and blastomeres of early cleavage stages, it may be more illustrative to perform in-situ hybridization or protein-based assays such as immunostaining to investigate localization in the cell of the expressed gene or gene product of interest. In the present study, the expression pattern of eif-1a was similar to that reported earlier in mouse embryos (De Sousa et al., 1998). The increase is likely to reflect the transition to embryonic control of development. Several functions in the initiation of translation have been attributed to eif-1a. It stabilizes the binding of Met-tRNA to the 40S ribosomal subunit, promotes mrna binding and prevents premature association of the 40S and 60S ribosomal subunits (Thomas et al., 1980, 1981; Chaudhuri et al., 1997, 1999). For studies on gene expression at the single cell level, classical methods of RNA quantification, such as Northern blotting, insitu hybridization (Parker and Barnes, 1999) and the RNase protection assay (RPA) (Saccomanno et al., 1992), all have serious limitations, with inadequate sensitivity or quantification problems. RT-PCR has the potential for measurements of gene expression at single cell level (Rappolee et al., 1988). Improvements involving fluorescence techniques and instrumentation capable of combining amplification, detection and quantification, have increased the sensitivity and specificity of the RT-PCR method (Higuchi et al., 1993). When using real-time PCR, in addition to a relative standard curve to quantify the unknown samples, an endogenous control should be used to normalize the quantification of messenger RNA for differences in the amount of total RNA added to each reaction. This requires that the endogenous control should exhibit stable expression over the different developmental stages to be compared. Such a control is hard to find when studying preimplantation development, since each cell division involves large changes in the total RNA pool, including both degradation of maternal RNA and new synthesis from the embryonic genome. In the present study, 18S rrna (mouse and human) and GAPDH (mouse) were tested as endogenous controls. However, both showed a pattern different to that of the test gene and could not be used as common denominators. Hence it was decided to relate the values only to the standard curve and calibrate against the oocyte/zygote. Using this

6 approach, it is concluded that reliable data can be obtained. The addition of a sequence-specific probe to the PCR gave sufficient specificity and a single round of amplification was enough to reach the detection level. Access to human embryos donated to research after IVF treatment is limited and surplus embryos are obviously of varying quality. At the IVF clinic the embryos are routinely transferred or frozen at day 2, at the 4-cell stage. Donations of embryos with high viability scores before the transfer stage are rare. Thus, donated embryos from earlier stages (up to 4 cells) are commonly arrested or have a high degree of fragmentation (i.e. are not suitable for transfer). Embryos donated at the 8-cell stage or later have been selected for continued growth and are generally of higher quality than the earlier ones. Even if some embryos in this study were of poor grade the results indicate that EGA is likely to have occurred. This indicates that morphologically compromised embryos may also have passed this stage. It is therefore concluded that if the embryos have reached the 8-cell stage, the expression of such an important gene as eif-1a is unlikely to have been seriously affected by embryo quality. The present results showed that the sensitivity and accuracy of the method were satisfactory, and that reliable data concerning steady state mrna concentrations could be obtained. Thus, good possibilities are anticipated for further development of this application in studying human embryos. There may be applications in preimplantation genetic diagnosis. Acknowledgements The authors wish to thank Peter Sjöblom for useful discussions. We are very grateful to Michal Zmarzlak and José Inzunza at the Unit for Embryology and Genetics, for providing the mouse material. We also want to acknowledge the team at the IVF unit at Huddinge University Hospital, for their efforts and advice in this study and also for their help in obtaining the human embryos. Finally, we want to thank Hannele Laivuori for helpful discussions concerning methodology and Nicholas Bolton for revising the language. This study was supported by grants from The Karolinska Institute, The Swedish Research Council and The Swedish Board for Technical Development. References Chaudhuri J, Si K, Maitra U 1997 Function of eukaryotic translation initiation factor 1A (eif1a) (formerly called eif-4c) in initiation of protein synthesis. Journal of Biological Chemistry 272, Chaudhuri J, Chowdhury D, Maitra U 1999 Distinct functions of eukaryotic translation initiation factors eif1a and eif3 in the formation of the 40 S ribosomal preinitiation complex. 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