Quantification of hexokinase mRNA in mouse blastocysts by ...

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Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic ...
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Molecular Human Reproduction vol.3 no.4 pp. 359–365, 1997

Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction*

M.D.Johnson1, D.W.Batey and B.Behr Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305–5317, USA 1To whom correspondence should be addressed at: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Harbor–UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90509, USA

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription–nested polymerase chain reaction (RT–nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT–nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95310–18 g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation. Key words: embryonic gene expression/embryonic metabolism/glucose phosphate isomerase/hexokinase/ preimplantation embryo

Introduction In mouse and human preimplantation development, pyruvate is consumed preferentially during embryonic cleavage; however, later in embryogenesis during morula compaction and blastocyst formation, glucose is the preferred energy substrate (Brinster, 1965; Biggers et al., 1967; Whitten and Biggers, 1968; Leese and Barton, 1984; Gardner and Leese, 1986; Conaghan et al., 1993; Bavister, 1995). Studies have shown that the enzyme that catalyses the first reaction in glycolysis, hexokinase (HX), increases during these later stages of human and mouse embryonic development to peak activities in the blastocyst (Brinster, 1968; Hooper and Leese, 1989; Martin et al., 1993; Ayabe et al., 1994; Houghton et al., 1996). In a previous study investigating the genetic activities of HX during embryonic glucose metabolism, we analysed the transcription patterns of HX in individual mouse morulae and blastocysts incubated in culture conditions with and without glucose and phosphate by qualitative RNA assays using reverse transcriptase–nested polymerase chain reaction (RT–nPCR) amplification (Johnson et al., 1997). We observed an increased *Presented in part as abstracts at the 43rd Annual Meeting of the Society of Gynecologic Investigation, Philadelphia, PA, USA, March 20–23, 1996 and the 44th Annual Meeting of the Pacific Coast Fertility Society, Indian Wells, CA, USA, April 17–21, 1996. © European Society for Human Reproduction and Embryology

incidence of HX gene transcripts in blastocysts in comparison with morulae, and differences in the incidence of HX gene transcripts between individual embryos at the same stage of development. These observations of the presence of variable HX gene transcripts prompted us to quantify the amount of HX mRNA in late-stage mouse embryos as a preliminary step towards analysing whether the amount of HX mRNA transcript in individual embryos correlates with embryo viability and competence for further development and implantation. In order to quantify the minute amounts of HX mRNA transcribed in individual embryos, we used competitive RT– PCR in which a known quantity of internal standard DNA is coamplified with the sample DNA within the same PCR reaction. Following PCR amplification, the relative amount of each product is determined. The original amount of cDNA that was reverse transcribed from the RNA sample can be calculated by comparison with the known amount of internal standard that underwent competitive amplification within the same reaction. We designed a RT–nPCR assay for amplification of HX mRNA in single embryos that reliably generates an expected 533 bp product. In order to quantify HX mRNA amounts by competitive RT–nPCR, a 340 bp internal standard was designed as a competitive template for co-amplification with the 533 bp product. The recombinant 340 bp competitor template is flanked by the nested HX primer sequences in their respective 359

M.D.Johnson, D.W.Batey and B.Behr

Figure 1. The extended primer pairs used to incorporate the nested hexokinase (HX) primer sequences into the flanking ends of the HexTemp-340 competitor template. The HXTEMP-FI primer encompasses the 18 bp 59 upstream inner HX primer and a nested downstream 15 bp HX cDNA sequence with an intervening 66 bp deletion between the native cDNA sequences. The HXTEMP-RI incorporates the 39 downstream inner primer and an upstream nested 15 bp sequence with an internal 40 bp deletion between the native cDNA sequences. The HXTEMP-FO and HXTEMP-RO primer pair contain the full-length 59 upstream outer and inner primers and the full-length 39 downstream outer and inner primers in tandem.

positions on the DNA template. In the competitive PCR reactions, the 340 bp internal standard competes with the HX cDNA target for primer binding and amplification. Known quantities of the 340 bp internal standard are added to the reverse transcription products to compete with the 533 bp target in the sequential nested PCR reactions. Following nested PCR, the quantitative amounts of the 340 bp internal standard and the 533 bp target products are compared. The amount of competitor internal standard that yields the equivalent amount of product when compared with the 533 bp target product provides an estimate of the original amount of reverse transcribed HX mRNA.

Materials and methods cDNA target sequence and PCR primer design cDNA sequences from the 4198 bp mouse cDNA of hexokinase I (Arora et al., 1990) were selected for RT–PCR as described previously (Johnson et al., 1997). Nested sets of primer pairs were designed to amplify products spanning exon–intron boundaries to differentiate the desired 533 bp HX cDNA sequence from the larger genomic DNA products containing introns. The HX-specific nested primer sequences are shown in combination as extended primer pairs in Figure 1. The extended HX-specific oligonucleotide primers were synthesized in the DNA and Protein Synthesis Laboratory of the Beckman Center, Stanford University School of Medicine, CA, USA. Following our initial RT–nPCR assays performed on mouse embryonic stem cell RNA, the identity of the cloned 533 bp HX RT–nPCR product was confirmed by nucleotide sequencing to be the specific mouse cDNA sequence targeted for amplification. Construction of the hexokinase competitor cDNA template for quantitative PCR We constructed the competitor internal standard template by a twostep PCR amplification strategy using two sets of overlapping extended primer pairs. As shown in Figure 1, the extended primer pairs are

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composed of the nested HX primer sequences in tandem with additional downstream nested sequences derived from the human hexokinase cDNA (Arora et al., 1990; Stoffel et al., 1992). The extended primers were designed with internal deletions between the original primers and the downstream native sequences to generate a competitor template that is 106 bp shorter than the native target cDNA sequence. The difference in size between the shortened competitor template and the native target HX cDNA sequence allows for differentiation between the two products by gel electrophoresis. Hexokinase-specific cDNA templates were reverse transcribed from 1 µg of mouse embryonic stem cell total RNA with 0.5 µM of 39 downstream outer primer in 20 µl of RT buffer (5 mM MgCl2; 1 mM each dGTP, dTTP, dATP, dCTP; 50 mM KCl; 10 mM Tris–HCl, pH 8.3) with 20 IU of RNase inhibitor and 2.5 IU of Malony murine leukaemia virus (M-MLV) RT enzyme at 42°C for 20 min followed by 99°C for 5 min as described in the GeneAmp RNA PCR Kit (Perkin Elmer Cetus, Inc., Norwalk, CT, USA). The RT product underwent two rounds of PCR amplification in a DNA Thermal Cycler (Perkin Elmer-Cetus). The initial series of amplifications used 0.15 µM each of the HXTEMP-FI and HXTEMP-RI primers in PCR buffer (2.5 IU of Taq polymerase, 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 1 mM each of dATP, dCTP, dGTP, and dTTP) with a ‘hot start’ at 99°C for 2 min, followed by 35 cycles of amplification (94°C for 1 min, 60°C for 1 min, 72°C for 90 s, and a final extension at 72°C for 6 min). The second series of 35 amplification cycles was performed with 0.15 µM each of the HXTEMP-FO and HXTEMP-RO primers under the same conditions except for an increased annealing temperature of 62°C for 1 min. Electrophoresis of the final PCR products revealed two DNA fragments, an expected size of 427 bp and a smaller 340 bp product. The 340 bp (HexTemp340) and 427 bp (HexTemp-427) PCR products were isolated from agarose gels followed by cloning, plasmid amplification, repurification, and nucleotide sequencing. Each recombinant fragment was successfully tested as a competitive template by amplification with each primer pair. The 340 bp fragment (HexTemp-340) was selected for use since it was more easily isolated from the 533 bp HX gene target by electrophoresis, thereby allowing for accurate excision of

Quantification of blastocyst hexokinase mRNA

Figure 2. The nucleotide sequence of the HexTemp-340 competitor template. The HXTEMP-FO and HXTEMP-RO primers containing the outer and nested hexokinase (HX) primer pairs in tandem are delineated by underlined type. The HXTEMP-FI and HXTEMP-RI primers containing the nested primers and the native HX cDNA sequences are delineated by bold type. only the HexTemp-340 product. As shown in Figure 2, the HexTemp340 competitor internal standard fragment contains the required fulllength 59 upstream outer and inner primers and the full-length 39 downstream outer and inner primers in tandem at their 59 and 39 ends respectively. Although our initial design was to generate a competitor fragment with native HX sequence and inserted deletions flanked by the nested HX primers at the 59 and 39 ends, our final competitor fragment is heterologous with the targeted HX sequence except for the critical flanking primer sequences in tandem at the ends of each fragment.

Embryo collection The study was approved by the Administrative Panel on Laboratory Animal Care, Stanford University and was carried out in accordance with the principles and procedures described in the Resource Manual for the Care and Use of Animals in Research and Teaching at Stanford University and the NIH Guide for the Care and Use of Laboratory Animals. Females of the hybrid mouse strain B6C3F1/Sim (C57BL/63C3H/ He; Simonsen Laboratories Inc, Gilroy, CA, USA) aged 6–8 weeks old underwent ovulation stimulation and mating followed by zygote retrieval. Each female received pregnant mare’s serum gonadotrophin (PMSG) 10 IU (0.1 ml) injected i.p. in the morning, followed by human chorionic gonadotrophin (HCG) 10 IU (0.1 ml) 54 h later. Each female was then mated with a B6C3F1/Sim (C57BL/63C3H/ He) stud male. The following morning, zygotes with adherent cumulus cells were removed from the female mice and incubated in hyaluronidase (~300 µg/ml) at room temperature for several min until complete digestion of the cumulus cells. The zygotes were then rinsed twice in P1 medium, and placed in 200 µl microdroplets of preequilibrated glucose/phosphate-containing human tubal fluid (HTF) medium (Irvine Scientific Inc, Irvine, CA, USA) under mineral oil. Cultures were maintained in a 37°C incubator, in 90% N, 5% O2, 5% CO2 atmosphere until the embryos had reached the fully expanded blastocyst stage. Competitive RT–nPCR HX assays of multiple blastocysts Embryo thermolysis and reverse transcription reactions Gene-specific cDNA templates were reverse transcribed from RNAs using a primer designed from the 39 end of the HX targeted sequence, rather than with random hexamer or oligo-dT primers. A single RT master mix (5 mM MgCl2; 1 mM each dGTP, dTTP, dATP, dCTP; 50 mM KCl; 10 mM Tris–HCl, pH 8.3) with 0.5 µM of HX 39 downstream outer primer was prepared for each group of embryos as described for the GeneAmp RNA PCR Kit (Perkin Elmer Cetus). The RT master mix was divided into 18 µl aliquots in individual PCR reaction tubes to minimize pipetting error and provide uniformity between individual reactions. Two drops of sterile Nujol oil were added to cover the reaction and prevent evaporation. Approximately 2 ng of embryonic stem cell RNA was added to a single RT master mix which had been divided into aliquots and analysed with each batch of embryo assays as a positive control. The negative controls

contained only RT mix without RNA template. Control reactions were handled exactly as the sample reactions throughout the entire sequence of nested RT–PCR analyses. Ten groups of fully expanded blastocysts, composed of eight groups of five, one of 10, and one of 13, underwent HX mRNA quantification by competitive RT–nPCR. Blastocysts were removed from the culture media by sterile micropipette and washed twice in drops of sterile water for 30–60 s to remove residual media. The embryos were then transferred into a PCR reaction tube containing the aliquoted RT mix under oil as described above. Embryos underwent thermolysis to release nucleic acids by heating the reaction mix under PCR oil for 1 min at 100°C (Kumazaki et al., 1994). Reaction tubes were then removed and placed on ice for 30 s before adding 20 IU of RNase inhibitor and 2.5 IU of MMLV RT enzyme. Each final reaction volume was ~20 µl. The reaction was allowed to proceed for 1 h at 42°C; the samples were then stored at –20°C until required for nPCR amplification. Competitive PCR amplification Quantification of cDNA templates synthesized in the blastocyst RT reactions was performed by nested PCR using the HexTemp-340 competitive template as an internal standard and the nested primer pairs specific for HX. Each 20 µl RT reaction performed on a group of blastocysts was split into four 5 µl aliquots to facilitate four competitive PCR reactions per sample using concentrations of the HexTemp-340 competitor (10–7 pg, 2.5310–7 pg, 5310–7 pg, and 10–6 pg) previously determined to be optimal for the reaction. The initial round of PCR amplifications was performed with 0.1 µM of the HX 59 upstream outer primer in 50 µl of buffer (2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 1 mM each of dATP, dCTP, dGTP, and dTTP and 2.5 IU of Taq polymerase) as described in the GeneAmp RNA PCR Kit (Perkin Elmer Cetus) for 35 cycles (94°C for 45 s, 55°C for 45s, 72°C for 1 min) in a DNA Thermal Cycler (Perkin Elmer-Cetus). An aliquot (5 µl) of first round reaction product was used as template for a second round of 35 nested PCR amplifications performed with 0.2 µM concentrations of the HX upstream and downstream nested pairs of primers using reaction buffers and amplification conditions as described previously in the ‘first round’ of PCR analysis. The PCR products were radiolabelled by the addition of 5 µCi of [32P]-dCTP to each reaction prior to beginning the nested (second) amplification. The final nested RT–PCR products were electrophoresed in 13 TBE on 2.5% agarose gels containing ethidium bromide (~0.5 µg/ml) and photographed. Bands corresponding to the 533 bp fragment of the endogenous hexokinase transcript and the 322 bp competitive template were excised from the gel and counted by liquid scintillation in Opti-fluor (Packard, Meriden, CT, USA).

Results Preliminary competitive RT–nPCR analyses In order to confirm the functional capacity of the HexTemp340 competitor DNA template to compete with the targeted HX 361

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Figure 3. The results of preliminary competitive reverse transcription–nested polymerase chain reaction (RT–nPCR) hexokinase (HX) assays using 100 ng of embryonic stem cell total RNA as template. Each lane is labelled with the amount (pg) of HXTEMP-340 competitor template that was added to that reaction; – 5 negative and 1 5 positive controls. M 5 molecular weight standard. HXTEMP-340 internal standard is observed to compete with the 533 bp target template within each reaction. In order to improve the precision of our quantification, the polymerase chain reaction (PCR) products were radiolabelled by the addition of 5 µCi of [32P]-dCTP to each reaction prior to beginning the nested (second) amplification. Electrophoresis of the products on 2.5% agarose gels allowed for excision of the 533 and 322 bp products, radioactivity was quantified by scintillation counting, and plotted in Figure 4.

cDNA product in PCR amplifications reactions, preliminary competitive nested PCR HX assays were performed. Initial competitive RT–nested PCR assays revealed that the HexTemp340 internal standard template reliably generated a single 322 bp fragment that was easily differentiated from the targeted 533 bp HX fragment. A series of competitive RT–nPCR HX reactions were performed using 100 ng of embryonic stem (ES) cell total RNA as template for the RT reaction followed by the addition of decreasing concentrations of 10–10–4 pg of competitor template to each individual PCR reaction. Figure 3 shows the results of the second nested round of PCR amplification. Each lane contains a specific amount of HexTemp-340 competitor template that was added to each reaction (i.e. 0, 10–4, 10–3, 10–2, 10–1, 1, and 5 pg). The HexTemp-340 internal standard is observed to compete with the 533 bp target template within each reaction. Excision of the radiolabelled 533 bp and 322 bp products from the 2.5% agarose gel in Figure 3 allowed for quantification of radioactivity by scintillation counting. Net values for the samples and the internal standards were calculated, linearly plotted, and the points at which the samples and standards reached equivalent masses were determined. As shown in Figure 4, the amount of HexTemp-340 competitor that yields the equivalent amount of product when compared to the 533 bp target product is 0.00339 pg.

Competitive RT–nPCR HX assays of multiple blastocysts The results of the 10 RT–nPCR assays performed on groups of blastocysts were analysed following agarose excision of the DNA products and scintillation counting. Background activity 362

Figure 4. Net values for the samples and the internal standards were calculated, linearly plotted, and the points at which the samples and standards reached equivalent masses were determined. Black diamonds denote the concentration of HexTemp-340 template added to each reverse transcription–nested polymerase chain reaction (RT–nPCR) reaction (horizontal axis) and the net value of radioactivity in its final amplified product (vertical axis). White squares denote the net value of radioactivity measured in each hexokinase (HX) gene product (vertical axis) that was generated in each RT–nPCR reaction co-amplified with the amount of competing HexTemp-340 template added to each reaction (shown on the horizontal axis). The amount of HXTEMP-340 competitor that yields the equivalent amount of product when compared to the 533 bp target product is 0.00339 pg.

(d.p.m.) was defined as the activity (d.p.m.) present in the gel slice from a reagent blank PCR sample. This was subtracted from the activity detected in each excised 533 or 322 bp fragment to determine the net amount of activity in each product. The net amount and activity of the 533 and 322 bp product for each reaction were plotted, and regression lines were developed from each sample’s data set, followed by calculation of the coefficients of determination (r2). The data from each of the 10 reactions had to be plotted individually, because each reaction was independently radiolabelled with [32P]-dCTP of a different age and specific activity, resulting in reaction products with different radioactivity levels. Seven of the reactions had widespread data distributions that resulted in the derivation of regression lines that were unsatisfactory (r2 ,0.8) for predictive analysis. Three of the reactions, shown in Figures 5, 6, and 7, had regression lines that were satisfactory for accurate quantitative estimation (r2 .0.8). The point at which the samples and standards reached equivalent masses was determined for each reaction. The amount of HX mRNA was calculated by extrapolating from the point of intersection to the horizontal axis, and corrections were made for the number of embryos per reaction (n), the four template concentrations, and the different dCTP contents of the 533 bp HX targeted product (120 dCTP) and the HexTemp-340 product (65 dCTP). Corrections were performed by multiplying the quantity of HexTemp-340 at the intersecting points by the correction factor (65/120 3 4/n). This corrected amount of

Quantification of blastocyst hexokinase mRNA

Figure 5. Data from reverse transcription–nested polymerase chain reaction (RT–nPCR) reactions performed on 13 expanded blastocysts. The black diamonds denote the concentration of HexTemp-340 template added to each RT–nPCR reaction (horizontal axis) and the net value of radioactivity in its final amplified product (vertical axis). The white squares denote the net value of radioactivity measured in each hexokinase (HX) gene product (vertical axis) that was generated in each RT–nPCR reaction co-amplified with the amount of competing HexTemp-340 template added to each reaction (shown on the horizontal axis). The amount of HXTEMP-340 competitor that yields the equivalent amount of product when compared with the 533 bp target product is 11.2310–18 g.

Figure 6. Data from reverse transcription–nested polymerase chain reaction (RT–nPCR) reactions performed on five expanded blastocysts. The black diamonds and white squares are as defined in Figure 5. The amount of HXTEMP-340 competitor that yields the equivalent amount of product when compared with the 533 bp target product is 3.4310–18 g.

HexTemp-340 was equivalent to the mean quantity of HX mRNA transcribed from a single blastocyst in the reaction. As shown in Table I, the data derived from these three competitive RT–nPCR HX assay reactions calculated an average of 1.95310–18 g of HX mRNA present in a single expanded blastocyst from this hybrid mouse strain cultured in HTF medium.

Discussion Glucose is the preferred energy substrate during morula compaction and blastocyst formation (Brinster, 1965; Biggers et al., 1967; Whitten and Biggers, 1968; Leese and Barton, 1984; Gardner and Leese, 1986; Conaghan et al., 1993; Bavister, 1995). In parallel with the consumption of glucose during these later stages of human and mouse embryonic development, HX enzyme activity increases dramatically to peak values in the blastocyst (Brinster, 1968; Hooper and Leese, 1989; Martin et al., 1993; Ayabe et al., 1994; Houghton et al., 1996). In our previous investigations of the genetic activities of HX in mouse embryos developing in vitro with and without exposure to glucose and phosphate, we observed an increased incidence of HX gene transcripts in the populations of blastocysts compared to the morulae, and differences in the incidence of HX mRNA transcripts between individual embryos at the same

Figure 7. Data from reverse transcription–nested polymerase chain reaction (RT–nPCR) reactions performed on five expanded blastocysts. The black diamonds and white squares are as defined in Figure 5. The amount of HXTEMP-340 competitor that yields the equivalent amount of product when compared with the 533 bp target product is 5.84310–18 g.

stage of development (Johnson et al., 1997). These observations of variability of HX gene transcript content between embryos of the same developmental stage suggested to us that individual 363

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Table I. Competitive polymerase chain reaction of mouse blastocyst reverse transcription products Sample no.

Blastocyst no.

Value of ‘X’ at intersection (g 3 10–18)

Hexokonase mRNA/blastocyst (g 3 10–18)

A B C

13 5 5

11.2 3.4 5.84

1.86 1.47 2.53

Mean 5 1.95; SD 5 0.54; SEM 5 0.44.

embryos may have different capacities for HX transcription which may influence their glucose metabolism. Although HX is generally very active in the morulae and blastocyst stages of development, lower amounts of HX mRNA may reflect an embryo’s condition and imply a reduced number of transcriptionally-active cells, transcript instability, or a possibly altered metabolic potential. These hypotheses prompted us to quantify the amount of HX mRNA in individual and cohorts of latestage mouse embryos as a preliminary step towards determining whether the observed variation in HX gene transcripts in individual embryos correlates with embryo viability and competence for further development and implantation. The aim of this study was to develop a method to accurately quantify HX RNA transcription in mouse blastocysts. On the basis of our current data, hexokinase is expressed in cultured mouse blastocysts (B6C3F1/Sim3B6C3F1/Sim) in minute amounts, averaging 1.95310–18 g of mRNA. Since this is the first published effort to quantitatively measure mRNA transcripts from a single gene in individual preimplantation embryos, we are unable to perform comparisons. The amount of HX mRNA measured may accurately reflect the gene’s embryonic expression, or it may represent a fraction of the gene’s activity, either as a result of instability of the HX RNA transcript or due to our method of measurement. This value was calculated from competitive RT–nPCR analyses of multiple rather than individual embryos. The majority of our competitive RT–nPCR HX expression analyses performed on single embryos exceeded the lower limits of our detection capabilities. We therefore considered these individual embryo assays to lack the level of precision required for confidence. As a result, we designed a strategy to measure HX in groups of embryos, which should enable HX gene expression to be compared between cohorts of embryos subjected to different experimental conditions. Whether investigations utilize samples containing single or multiple embryos, some variability of gene expression can be expected between embryos at the same developmental stage cultured in the same environment. This variation in gene expression between embryos may be the result of individual embryo variation concerning condition and cell number, as well as individual transcription rates. Our method for measuring HX gene transcripts in embryos has advantages and limitations characteristic of competitive RT–PCR techniques. Different types of internal standard have been designed for competitive PCR. In our strategy, we implemented a recombinant cDNA competitor rather than an RNA standard as a competitive template. Our strategy measures cDNAs generated from available RNA transcripts and does 364

not reflect the efficiency of the reverse transcription reaction. This potential limitation has been adequately addressed by Wang et al. (1989) and Becker-Andre and Hahlbrock (1989). In their initial investigations with competitive RT–PCR using competitor RNA fragments, these investigators observed highly efficient RT reactions with well-conserved synthesis of cDNAs. All competitive PCR methods require the target gene and competitive internal standard to amplify with equal efficiencies. In our strategy, the targeted HX gene cDNA and competitor HexTemp-340 fragments are heterologous; however, the HexTemp-340 competitor template has priming sequences flanking each end that are identical to the primer sequences of our targeted HX gene sequence. The published experience to date shows that amplification efficiencies between heterologous competitor and target cDNAs are primarily determined by the primer sequences, unless there are significant differences in denaturation or polymerase extension due to high G/C content or secondary structure (Siebert and Larrick, 1993). Several characteristics of our experimental design have the potential to influence the accuracy of our results. Precise measurement of the HexTemp-340 template in competitive PCR is critical for accurate results. We utilized spectrophotometric quantification methods with serial dilutions to accurately monitor the concentration of our competitor DNA to the 10–18 g range. In order to reduce the possibility of RNA loss or degradation, which can occur with standard RNA extraction methods, we have performed embryo thermolysis for release of RNA, followed by RT with initial PCR amplification in a single reaction tube. Embryonic thermolysis may be detrimental for RNA stability, since thermal effects have the potential to influence RNA structure. Furthermore, although protein denaturation is expected with thermolysis, protein-RNA interactions may impair the efficiency of the reverse transcription reaction, preventing complete RNA transcription into measureable cDNA molecules. In conclusion, the calculated average of 1.95310–18 g of mRNA per embryo is a very minute amount of product and further investigations are necessary to confirm this quantity of blastocyst HX gene transcript. In addition, we plan to utilize this strategy to monitor embryonic responses to various environments and ultimately determine whether lower or upper limits of hexokinase gene activity influence blastocyst viability and implantation. Competitive RT–PCR strategies are molecular instruments for measuring minute amounts of mRNAs, and are applicable for quantifying the genetic expression of other enzymes that are active during embryonic development. The ability to measure gene activity and correlate embryonic responses with embryo viability and metabolic state will eventually permit optimization of embryo culture methods, as well as improve the assessment of developmental competence and implantation potential in preimplantation embryos.

Acknowledgements We would like to thank Douglas Moore for his helpful assistance in mouse breeding and zygote retrieval. M.D.J. is a recipient of the Basil O’Connor Scholar Award of the March of Dimes Birth Defects Foundation through which this work was partially funded.

Quantification of blastocyst hexokinase mRNA

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