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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 953-959
Copyright © 1998 by The Endocrine Society

Original Studies

Single Blastomeres within Human Preimplantation Embryos Express Different Amounts of Messenger Ribonucleic Acid for ß-Actin and Interleukin-1 Receptor Type I1

Jan S. Krüssel2, Hong-Yuan Huang3, Carlos Simón, Barry Behr, Andrea R. Pape, Yan Wen, Peter Bielfeld and Mary Lake Polan

Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University School of Medicine (J.S.K., H.-Y.H., B.B., A.R.P., Y.W., M.L.P.), Palo Alto, California 94305; Instituto Valenciano de Infertilidad (C.S.), Valencia, Spain; the Department of Obstetrics and Gynecology, Heinrich Heine University (P.B.), Dusseldorf, Germany; and the Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital (H.-Y.H.), Taipei, Taiwan

Address all correspondence and requests for reprints to: Dr. Jan S. Krüssel, Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University School of Medicine, 300 Pasteur Drive, Palo Alto, California 94305. E-mail: krussel{at}leland.stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gaining knowledge about the physiological timetable of gene expression during preimplantation embryo development is crucial, and a better understanding of cytokine and growth factor expression in early embryonic development could lead to improved in vitro culture conditions and enhance in vitro fertilization implantation rates. Our aim was to detect the patterns and levels of two messenger ribonucleic acids [mRNAs; ß-actin and interleukin-1 receptor type I (IL-1R tI)] in single human blastomeres by RT-nested PCR and to compare possible variations in the gene expression both between different embryos and in multiple blastomeres within the same embryo. Single blastomeres from nine human tripronucleic preimplantation embryos were examined by one round of RT and two rounds of nested competitive PCR. ß-Actin mRNA was detected in each blastomere, and IL-1R tI mRNA was found in 72% of the blastomeres examined. ß-Actin was expressed at a level of 511–12185 molecules of complementary DNA/blastomere, and IL-1R tI was expressed at a level of 2–290 molecules of complementary DNA/blastomere. Our results suggest that the mRNA pattern of an embryo cannot be reliably quantitated from the mRNA pattern of a single blastomere and therefore imply limitations for the use of this method for preimplantation diagnosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DETECTING messenger ribonucleic acids (mRNAs) in single blastomeres for preimplantation diagnosis presents a dual challenge: first, to overcome the methodological problems of DNA amplification by PCR during preimplantation genetic diagnosis, and second, to learn more about the physiological course of gene expression during preimplantation embryonic development. Single cell PCR is a powerful tool in preimplantation diagnosis, using blastomere biopsy of human embryos at the 8–12 cell stage. The ability to rapidly genotype a single cell using PCR and the fact that biopsy of early cleavage stage human embryos does not decrease their viability allow identification of affected embryos from patients with various inherited diseases (1, 2, 3, 4, 5), so that only unaffected embryos are transferred back to the uterus. Reports of misdiagnoses and complete reaction failures using this technique, however, have diminished enthusiasm for its widespread clinical use (6, 7, 8). These misdiagnoses originate primarily from a single allele amplification, i.e. in a single cell, only two alleles or templates of DNA are present, and consumption of reagents by the allele that first encounters both primers or complex DNA structure considerations may result in a homogeneous population of amplification products derived from either the wild-type or the mutant allele, although the genotype of the investigated cell is heterozygous. Although techniques such as fluorescence PCR have greatly increased the method’s sensitivity and accuracy (9), it remains questionable whether the allelic dropout and/or preferential amplification can be totally avoided. To overcome these methodological problems, the use of RT-PCR of mRNA to detect genetic diseases caused by heterozygous mutations has been reported (10) for preimplantation genetic diagnosis.

RT-PCR has also been used to gain knowledge about the stage-specific expression of various genes during preimplantation development (11, 12). Cytokine and growth factor mRNAs have been detected in blastomeres and in preimplantation embryos from different species (13, 14, 15, 16) as well as in the human endometrium throughout the menstrual cycle, and a better understanding of these factors during early embryonic development could lead to improved in vitro culture conditions and enhance the outcome of human in vitro fertilization. The interleukin-1 (IL-1) system is believed to play an intimate role in implantation by acting as a communication signal between embryonic and maternal surfaces. In humans, the IL-1 receptor type I (IL-1R tI) has been detected in total human endometrium (17) and, more specifically, in endometrial epithelial cells, with maximal protein and mRNA expression during the luteal phase (18), i.e. the time of embryonic attachment and implantation. IL-1ß mRNA was detected in secretory human endometrium beginning on day 23 of the menstrual cycle (19). Recently, all major components of the IL-1 system, namely IL-1ß, IL-1 receptor antagonist (IL-1ra), and IL-1R tI, were detected immunohistochemically in single preimplantation embryos (20), perhaps suggesting an autocrine role of the IL-1 system. In vitro fertilized, cultured human embryos have been shown to produce both IL-1{alpha} and IL-1ß, and high concentrations (>60 and >80 pg/ml) of these cytokines in culture medium have been correlated with successful implantation after intrauterine transfer of these embryos (21), although other researchers could not detect IL-1{alpha} or IL-1ß in culture fluids of human embryos (22). We have also demonstrated the presence of IL-1ß and IL-1R tI mRNA and proteins in the human fallopian tube in all phases of the menstrual cycle in epithelial and stromal cells of the human tubal mucosa (23). This allows the preimplantation embryo to communicate with maternal surfaces through its IL-1 production throughout tubal transport during the first 5 days of preimplantation development.

In mice, both IL-1{alpha} and IL-1ß mRNA and protein have been detected and localized in endometrial endothelial cells (24) in increasing levels from day 3 of pregnancy, peaking between days 4 and 5 (25), with blastocyst implantation known to occur late on day 4. Furthermore, systemically administered recombinant human IL-1ra given ip from days 3–6 of pregnancy inhibited embryonic implantation in mice (26). We have previously demonstrated expression of the components of the IL-1 system (IL-1ß, IL-1ra, and IL-1R tI) at the mRNA level in murine preimplantation embryos as early as in the eight-cell stage (16, 27).

Another hypothetical benefit of RT-PCR could be the ability to examine the mRNA expression of several genes in a single blastomere and to thereby gain information about the viability and quality, in terms of successful implantation, of the particular preimplantation embryo in order to select the most suitable embryos for transfer. The basic assumption for this, however, is that the mRNA expression of a single blastomere is representative of the mRNA expression of the remaining embryo. The aim of our study was to detect the patterns and levels of different mRNAs in multiple single human blastomeres from the same embryo by RT-nested competitive PCR and to compare levels of transcripts in different blastomeres. In the present study, we have chosen to examine the mRNA expressions of IL-1R tI, whose protein is expressed at this stage of preimplantation development in humans (20), and a housekeeping gene (ß-actin) that is known to be expressed at all stages of development in mice (13, 16, 27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
For both ethical and practical reasons, this investigation was performed on pathologically fertilized embryos (three pronuclei). All patients who participated in this study by donating their polyspermic embryos signed an informed consent; the protocol and the consent form had been approved by the human subjects in medical research committee at Stanford University. Polyspermic human preimplantation embryos obtained from the in vitro fertilization program at the Stanford University Department of Gynecology and Obstetrics were cultured in vitro until the six- to eight-cell stage in P1 medium with gentamicin (Irvine Scientific, Santa Ana, CA) and then separated into single blastomeres, using the procedure commonly described for blastomere biopsy. Briefly, embryos were placed in a single drop of medium (25 µL) under a micromanipulator, and the zona pellucida was partially dissolved by the punctual application of acidified Tyrode’s solution adjusted to pH 2.0 (Sigma Chemical Co., St. Louis, MO). Embryos were mechanically separated into single blastomeres by use of a biopsy pipette with an inner diameter of approximately 40 µm (Fig. 1Go). Single blastomeres (four to eight from each embryo) were examined by one round of RT followed by two rounds of nested PCR using a modification of methods described previously (28, 29) for ß-actin and IL-1R tI mRNAs. Sequences of complementary DNA (cDNA) clones for the mRNAs that should be detected in single blastomeres [ß-actin (30) and IL-1R tI (31)] were obtained from the GenBank database of the National Center for Biotechnology Information of the NIH (internet address: http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank). One set of corresponding outer primer sequences and one set of corresponding inner primer sequences were constructed with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN) and synthesized at the Beckman Center, Stanford University Medical Center (Palo Alto, CA). The ß-actin outer primers were obtained from Clontech Laboratories (Palo Alto, CA). To ensure that the product detected resulted from amplification of cDNA rather than contaminating genomic DNA, primers were designed to cross intron/exon boundaries. The primer cDNA sequences and the sizes of the amplified fragments are listed in Table 1Go.



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  		Figure 1. Separation of a human eight-cell embryo into single blastomeres. a, Punctual digestion of the zona pellucida with acidified Tyrode’s solution adjusted to pH 2.0. b and c, Single blastomeres are successively removed from the embryo. d, Empty zona. e, Morphologically intact single human blastomeres. The horizontal bar in a represents 100 µm (scale for a–d); the horizontal bar in e represents 50 µm.

 

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  		Table 1. Primers used for RT/(nested) PCR

 
RT

For each blastomere, 17.5 µL RT-MasterMix were prepared [4 µL 25 mmol/L MgCl2 solution, 2 µL 10 x PCR buffer, 2 µL diethylpyrocarbonate (DEPC)-treated H2O (distilled), 2 µL deoxy (d)-ATP, 2 µL dCTP, 2 µL dGTP, 2 µL dTTP (all from Perkin-Elmer, Foster City, CA), and 1.5 µL outer 3' primer mix] and filled into a 0.5-mL thin wall PCR tube (Applied Scientific, South San Francisco, CA). RT-MasterMix in PCR tubes was covered with 50 µL light white mineral oil (Sigma) and kept on ice until the RNA extraction. One single blastomere was added to the RT mix, allowing a culture medium carry-over of 1 µL. Samples were immediately heated up to 99 C for 1 min in a DNA Thermal Cycler 480 (Perkin-Elmer) to release the total RNA and denature the proteins. Samples were cooled to 4 C, and 1 µL ribonuclease inhibitor (Perkin-Elmer) was added, followed by 0.5 µL Moloney murine leukemia virus RT (Life Technologies, Grand Island, NY). The RT was carried out in the DNA Thermal Cycler 480 using a program with the following parameters: 42 C for 30 min, 99 C for 5 min, and 4 C for 5 min. After the reaction was complete, samples were stored at -20 C until the first PCR.

Construction of the competitive and target cDNA fragments for IL-1R tI

A 284-bp fragment of native IL-1R tI-cDNA (i.e. target) was obtained by PCR amplification of reverse transcribed total RNA from a luteal phase endometrial biopsy (Fig. 2aGo) with the regular 3'- and 5'-outer primers (Table 1Go). The PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide (ETB), and the cDNA was extracted from the gel, purified with an agarose gel extraction kit (Boehringer Mannheim, Mannheim, Germany), and quantitated by spectrophotometry (GeneQuant, Pharmacia, Cambridge, UK). Sequence analysis was performed to confirm the identity of the expected sequence and the amplified cDNA.



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  		Figure 2. a, Sizes of target PCR products and locations of the primer binding sites for the 3'- and 5'-outer primers (black), the 3'- and 5'-inner primers (gray), and the primer to construct the competitive cDNA (white) on the native IL-1R tI cDNA. b, Construction of the competitive cDNA. Three artificial deletions are created to synthesize a shorter cDNA fragment with the same primer binding characteristics as the target cDNA. c, Size of competitor PCR products and location of the primer binding sites for the 3'- and 5'-outer primers (black), and the 3'- and 5'-inner primers (gray).

 
After sequence confirmation, 1 ng target cDNA was amplified using a modification of a method previously described (29) to construct a competitive cDNA fragment; a "floating" primer was designed from a sequence complementary to the cDNA between the inner primer binding sites followed by the reverse complementary 3'-binding site of the inner primer (Fig. 2Go, a and b). After 30 cycles of PCR (45 s at 95 C, 45 s at 55 C, and 60 s at 72 C) with the regular inner 5'-primer and the 3'-floating primer, the PCR product was visualized by agarose gel electrophoresis and stained with ETB, and the cDNA was extracted from the gel and purified with an agarose gel extraction kit (Boehringer Mannheim). This first step resulted in a cDNA fragment with an approximately 150-bp deletion compared to the cDNA to be detected (target cDNA) and with the 3'- and 5'-end inner primer binding sites. In the second step, special primers were designed with the 5'-end inner and outer primers and the 3'-end inner and outer primers attached to each other. One nanogram of the cDNA obtained after step 1 was amplified with these special primers (PCR program described above), resulting in a cDNA sequence with both inner and outer primer binding sites attached to the deleted target sequence (Fig. 2cGo). This PCR product was extracted from an agarose gel as described above and quantitated by spectrophotometry (GeneQuant, Pharmacia, Cambridge, UK). The identities of all PCR products were confirmed by independent sequence analysis (data not shown).

Construction of the competitive and target cDNA fragments for ß-actin

Target cDNA (838 bp) and competitive cDNA (437 bp) for ß-actin were obtained in basically the same way as described above for IL-1R tI with one difference: because only one round of PCR was necessary to amplify a sufficient amount of ß-actin target cDNA from the single blastomeres, the second step of adding the complementary sequences of the outer primer pair to the cDNA sequence synthesized in step 1 could be omitted.

Standard curve and competitive PCR

The standard curves for ß-actin and IL-1R tI were constructed by coamplification of a constant amount of competitive cDNA (5 x 10-21 mol for ß-actin and 3.5 x 10-23 mol for IL-1R tI) with declining amounts of target cDNA obtained by serial dilution. The amounts of target cDNA that were added to each PCR are shown in Figs. 3Go and 4Go. For ß-actin one round of PCR, and for IL-1R tI two rounds of PCR were conducted. PCR specifications are listed in Table 2Go. Two microliters of the cDNA mix were added to 48 µL PCR-MasterMix containing 3.4 µL 25 mmol/L MgCl2 solution, 4.7 µL 10 x PCR buffer, 1 µL dATP, 1 µL dCTP, 1 µL dGTP, 1 µL dTTP, 0.5 µL Polymerase-Gold (all from Perkin-Elmer), 2.4 µL 3' plus 5' primer mix (5 µmol/L of each) for either ß-actin or IL-1R tI and 33 µL DEPC-treated H2O. The reaction mix was covered with 50 µL light white mineral oil, placed in the DNA Thermal Cycler 480, and heated to 99 C for 9 min to denature all proteins and to activate the Polymerase-Gold. After completion of the PCR, ß-actin products were stored at -20 C until 2% agarose gel electrophoresis was carried out in the presence of ETB. For IL-1R tI, a second round of PCR was carried out. Five microliters of the first round PCR product were added to 95 µL PCR-MasterMix containing 7.2 µL 25 mmol/L MgCl2 solution, 9.5 µL 10 x PCR buffer, 2.2 µL dATP, 2.2 µL dCTP, 2.2 µL dGTP, 2.2 µL dTTP, 0.5 µL Polymerase-Gold (all from Perkin-Elmer), 4 µL 3' plus 5' inner primer mix (5 µmol/L of each) for IL-1R tI, and 65 µL DEPC-treated H2O. The reaction mix was covered with 50 µL light white mineral oil, placed in the DNA Thermal Cycler 480, and heated to 99 C for 9 min to denature all proteins and to activate the Polymerase-Gold. After completion of the PCR, IL-1R tI products were stored at -20 C until 2% agarose-gel electrophoresis was carried out in the presence of ETB. After completion of electrophoresis, the agarose gels were analyzed on the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, CA). DNA size calculation and UV densitometry were carried out using the Molecular Analyst Software (Bio-Rad Laboratories).



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  		Figure 3. Standard curve for ß-actin. Upper panel, Two percent agarose gel stained with ETB. Decreasing amounts of target cDNA (838 bp) are coamplified with a constant amount (5 x 10-21 mol/PCR) of competitive cDNA (437 bp). Lower panel, Composite of two standard curves obtained from two independent experiments. The log ratio of target to competitor band intensity was plotted against the log amount of target initially added to each PCR.

 


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  		Figure 4. Standard curve for IL-1R tI. Upper panel, Two percent agarose gel stained with ETB. Decreasing amounts of target cDNA (284 bp) are coamplified with a constant amount (3.5 x 10-23 mol/PCR) of competitive cDNA (191 bp). Lower panel, Log ratio of target to competitor band intensity was plotted against the log amount of target initially added to each PCR.

 

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  		Table 2. PCR cycles and temperatures for amplification of the different cDNAs

 
The logarithmically transformed ratio of the ETB densities of target cDNA to competitive cDNA were plotted against the log amount of initially added target cDNA in each PCR to obtain the standard curves shown in Fig. 3Go (ß-actin) and Fig. 4Go (IL-1R tI). These standard curves were highly reproducible and linear over 2 orders of magnitude. The values obtained from the regression line of the standard curves (y = b + mx) allowed us to calculate the amount of cDNA transcripts in an unknown sample; 5 x 10-21 mol competitive cDNA for ß-actin and 3.5 x 10-23 mol competitive cDNA for IL-1R tI were added to each unknown sample before PCR. The ratio of the densities of the competitive cDNA band (437 bp for ß-actin and 191 bp for IL-1R tI, respectively) and the cDNA band obtained from the sample (838 bp for ß-actin and 284 bp for IL-1R tI, respectively) were logarithmically transformed and compared to the values obtained from the standard curve (x = (y - m)/b). Quantitative competitive PCRs were carried out on at least two aliquots from the RTs of each blastomere.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have successfully established a quantitative competitive PCR system to detect ß-actin and IL-1R tI mRNA in single blastomeres. To validate the accuracy of the quantification, several series of PCR were carried out on the same randomly chosen blastomeres, with a new standard curve for each series (Fig. 5Go); mRNA levels differed by ±5% in the same blastomeres for ß-actin and by ±11% for IL-1R tI.



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  		Figure 5. Assessment of the method’s accuracy. Randomly chosen blastomeres underwent competitive PCR for ß-actin and IL-1R tI (data not shown) in two independent experiments. Upper panel, Two percent agarose gel of PCR for ß-actin stained with ETB. Coamplifications of various samples with a constant amount (5 x 10-21 mol/PCR) of competitive cDNA (437 bp). L, One hundred-base pair DNA ladder; standard curve as described in Fig. 3Go. nc, Negative control (P1 medium without blastomere). 1–10, Single blastomeres from human preimplantation embryos. Lower panel, Molecules of ß-actin cDNA as determined by calculation from the standard curves for series 1 (upper gel) and series 2 (lower gel). Values could be reproduced in a range of ±5% for ß-actin and ±11% for IL-1R tI.

 
A total of 53 single blastomeres from 9 preimplantation embryos (4 at the 8-cell stage, 2 at the 7-cell stage, and 3 at the 6-cell stage) were examined. The amounts of ß-actin and IL-1R tI mRNA expression differed widely, not only between the different preimplantation embryos, but also between the individual blastomeres from the same embryo. We detected ß-actin mRNA in each blastomere of each embryo. Within different embryos, ß-actin mRNA was expressed at a maximal number of 14,300 copies and in a minimal amount of 5 copies cDNA/blastomere (Fig. 6Go, a and c). All embryos expressed IL-1R tI mRNA. However, IL-1R tI mRNA was detected in only 78% of the blastomeres examined. IL-1R tI mRNA was expressed in a maximum of 354 copies and in a minimum of 2 molecules cDNA/blastomere (Fig. 6Go, b and c). The overall expression of mRNA per blastomere was much higher for ß-actin than it was for IL-1R tI (mean ± SD, 4,803 ± 4,867 vs. 47 ± 76 molecules cDNA/blastomere). There was no correlation between the amounts of ß-actin and IL-1R tI mRNAs within the different embryos (Fig. 6cGo).



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  		Figure 6. Expression of ß-actin mRNA (a) and IL-1R tI mRNA (b) as determined by cDNA detection via competitive PCR. Dots represent the mRNA amount of a single blastomere (n, number of blastomeres examined from that embryo), columns of dots represent one individual embryo, vertical lines represent the mean value of mRNA molecules per embryo. c, Comparison between the mRNA levels of ß-actin (black) and IL-1R tI (gray) within the different embryos. Bars represent the mean value; vertical lines indicate +1 SD. Stages of preimplantation development for the examined embryos were: 1) 8-cell (8c); 2) 6c; 3) 8c; 4) 7c; 5) 8c; 6) 8c; 7) 7c; 8) 6c; and 9) 6c.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first report of a quantitative determination of differences in gene expression within the individual blastomeres of a human preimplantation embryo by RT-PCR. The coamplification of target cDNA and competitive cDNA for ß-actin and IL-1R tI not only obviated tube to tube variations (a frequent problem when dealing with high numbers of PCR cycles), but also allowed us to calculate the absolute amount of cDNA transcripts for the genes examined.

The great variation from blastomere to blastomere in the mRNA levels observed for both ß-actin and IL-1R tI allows speculation about the cause. Possible explanations include the mitotic cell cycle, different onset of gene expression, or simply the fact that we examined only tripronucleic embryos.

It is a well known fact that early preimplantation embryos cleave in an asynchronous way. It is also clear that cells produce mRNA mainly in the interphase of their mitotic cell cycles, which is of variable length. Given the relatively high cleavage rate and the short time between these cleavages during the first days of preimplantation development, the interphase is considered to be relatively short. Although it remains uncertain how long the blastomeres stay in the interphase and how long the half-life time of the mRNA actually is, it is possible that the different amounts of mRNA in single blastomeres of the same embryos mirror the different segments of their mitotic cell cycle.

The onset of human embryonic gene expression is known to occur past the four-cell stage, as determined by changes in the pattern of polypeptides synthesized during the preimplantation stages of human development (32) and by incorporation of [3H]uridine into total embryonic RNA (33). Any mRNA detected before the four-cell stage is believed to be of maternal origin. It is therefore possible that the wide variety of mRNA levels observed in the blastomeres within a single preimplantation embryo reflect variations in the onset of expression of the embryonic genome. Blastomeres expressing high amounts of mRNA may have started the process of transcription, whereas blastomeres with a low amount may still use the maternal RNA. If this speculation is correct, then the amounts of mRNA within the blastomeres of a preimplantation embryo should converge during further development, and we are planning to examine preimplantation embryos beyond the 12-cell stage to test this hypothesis.

Human blastomeres within preimplantation embryos of the 8- to 12-cell stage are believed to be developmentally omnipotent, i.e. they have not started to differentiate into either embryoblast or trophoblast. This allows removal of one or two blastomeres for the purpose of preimplantation genetic diagnosis without affecting the further development of the embryo, and the data collected worldwide from preimplantation genetic diagnosis programs show that this is indeed the case. Another possible explanation for our results, however, may be that they reflect the beginning differentiation of the blastomeres. The earliest marker of trophoblast differentiation is hCGß, which is detected as early as the 8-cell human preimplantation embryo (34). The next step of our investigation, therefore, will be the detection of hCGß mRNA in the single blastomeres and to examine whether there are differences in expression levels in hCGß between the single blastomeres.

For both ethical and practical reasons, we conducted our experiments on triploid embryos by assessment of the pronuclear number 16–18 h after in vitro fertilization. These embryos were unsuitable for embryo transfer. The most crucial question regarding the biological significance of the findings described here is whether they are influenced by the triploidy of the blastomeres. Some polyploid embryos are capable of an extensive, morphologically normal development far beyond the eight-cell stage (35, 36), and occasional triploid births have been reported (37). It therefore seems unlikely that the pattern of transcriptional activity in these cells is totally abnormal compared to that of regular diploid embryos. It is also uncertain whether a tripronucleic egg results in triploid blastomeres; about 40% of tripronucleic eggs may revert to diploidy after the first mitotic cleavage (38). Although we assume that tripronucleic embryos reflect transcriptional processes that occur during normal diploid development, we have no proof, and this experiment should be repeated on diploid human preimplantation embryos.

The amount of mRNA in preimplantation embryos is not known exactly. For murine embryos, values are reported to be between 50 pg (39) and less than 0.7 pg (40) per embryo at varying stages of preimplantation development. For this reason, most investigations have used pooled mRNA samples to obtain ~1 µg total RNA, which is necessary for one round of RT-PCR or ~5 µg for Northern blotting. To obtain a sufficient quantity of RNA for these methods, pools of 500-1000 embryos are lysed, and RNA is extracted (13). These methods therefore do not allow a statement about the mRNA levels of single preimplantation embryos or blastomeres, although methods to detect mRNA in single cells have been described (28). Using pooled samples, investigators have estimated that ß-actin is present at 18,700 copies of mRNA/mouse oocyte, 5,600 copies/2-cell embryo, 18,460 copies/8-cell embryo, and 41,480 copies/blastocyst, respectively (13). These results are comparable to the findings described in this article.

Methodologically, these results prove that the common practice of correlating the expression level of housekeeping genes such as ß-actin with the level of a functionally expressed mRNA may lead to incorrect conclusions. We have demonstrated that the level of ß-actin mRNA expression is not correlated with the level of IL-1R tI expression. This might be of minor importance when large amounts of cells or tissue are examined, as is the case when a Northern blot is performed, and the varying expression levels will be statistically equalized when the number of cells is large enough. However, the smaller the number of cells examined, the more important it may be to perform a quantitative mRNA analysis when a comparison of expression levels for various mRNAs is desired.

Our results suggest that the steady state mRNA level of an embryo cannot be reliably quantitated from the mRNA pattern of a single blastomere. This is of minor importance for preimplantation genetic diagnosis by RT-PCR, as it has been established for diseases caused by mutations resulting in mRNAs with different lengths or different sequences, such as Marfan syndrome (10); the diagnosis in these cases is qualitative, not quantitative. As long as the particular gene is expressed during early preimplantation development and the mutation does not reduce the levels and stability of mutant transcripts, it should be possible to determine whether the blastomere examined by RT-PCR is heterozygous or homozygous.

Several groups are examining gene expression in human and murine preimplantation embryos to gain knowledge about early embryonic physiology and to describe normal mRNA patterns that are predictive of normal embryonic development and, perhaps, successful implantation (11, 12, 13, 14, 15, 16, 27). Although our results may have been influenced by the aneuploidy of the embryos examined, they suggest that it will not be possible to predict the mRNA pattern of a preimplantation embryo by examination of one of its blastomeres, thereby limiting the use of RT-PCR for this particular type of preimplantation diagnosis. The method, however, is easily transferable to a larger number of cells, complete preimplantation embryos for example, where it would be suitable to compare the mRNA expressions of different genes at various stages of preimplantation development.


   Acknowledgments
 
The authors thank Prof. Uta Francke, M.D., Department of Genetics and Howard Hughes Medical Institute, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, and Prof. Roger A. Pedersen, M.D., from the Reproductive Endocrinology Center, University of California-San Francisco, for their helpful suggestions and discussions. We gratefully acknowledge the help of Amin Milki, M.D., and Douglas Moore, M.S., and the support of Prof. Linda Giudice, M.D., Ph.D., Francisco Raga, M.D., and Eva Maria Casañ, Ph.D.


   Footnotes
 
1 Presented in part at the 13th Annual Meeting of the European Society for Human Reproduction and Embryology (ESHRE), Edinburgh, UK, June 22–25, 1997. Back

2 Postdoctoral research fellow, currently on leave from the Department of Obstetrics and Gynecology (Chairman: H. G. Bender, M.D.), Heinrich Heine University (Dusseldorf, Germany). Back

3 Postdoctoral research fellow, currently on leave from the Department of Obstetrics and Gynecology (Chairman: Yung-Kuei Soong, M.D.) of the Chang Gung Memorial Hospital (Taipei, Taiwan). Back

Received August 18, 1997.

Revised November 6, 1997.

Accepted November 20, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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