Molecular Human Reproduction, Vol. 5, No. 9, 861-865,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of embryo development |
Expression of the insulin-like growth factor-1 gene and its receptor in preimplantation mouse embryos; is it a marker of embryo viability?
The Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, The New York Hospital–Cornell Medical Center, New York, NY 10021, USA
Abstract
Recent studies have demonstrated the importance of insulin-like growth factors (IGF) in mouse preimplantation development. We examined IGF-1 and IGF-1 receptor (IGF-1R) gene expression in a single blastomere of an early mouse embryo and compared it with subsequent embryo development in culture. Fertilized eggs and 2-cell embryos were obtained by tubal flushing in superovulated and mated female mice. Single cells were removed from embryos at cleavage stage between 3 and 8 cells using the standard embryo biopsy techniques. Individual blastomeres from each embryo were then assayed for the presence of IGF-1 and IGF-1R mRNA using reverse transcription–polymerase chain reaction. The biopsied embryos were washed in medium and placed in co-culture with murine endometrial cells. Embryonic development in culture was assessed and blastocyst grading was performed. IGF-1 gene expression was then examined for an association with in-vitro development. Eighty-seven embryos were biopsied. IGF-1R gene expression was detected in the majority of embryos tested and IGF-1 gene expression was detected in 34 of 81 (42%) embryos. A significant association between IGF-1 expression and blastocyst formation in vitro was found (P < 0.01). There was no association between IGF-1R expression and subsequent embryo development. We conclude that IGF-1 gene expression could potentially be used as a marker of embryo quality.
embryo biopsy/growth factors/IGF-I/RT–PCR
Introduction
Preimplantation embryos have been shown to synthesize a multitude of growth factors and growth factor receptors. The importance of growth factors and growth factor receptors has been studied indirectly by demonstrating that receptor activation can stimulate cell proliferation and embryo differentiation. Incubation of mouse embryos in insulin-like growth factor (IGF-1) increases the cell numbers in the blastocysts formed. (Harvey and Kaye, 1992
; Smith et al., 1993) Alternatively, reduction in the amount of growth factor available (IGF-2), by introducing antisense IGF-2 oligonucleotides decreases the rate of development to blastocyst stage as well as the cell numbers within the blastocyst (Rappolee et al., 1992).
The development of more sensitive reverse transcription–polymerase chain reaction (RT–PCR) techniques allows detection of gene activation and mRNA expression in the early embryonic stages. Utilization of this technique can increase our understanding of the importance of various genes and their products in the early embryonic growth and differentiation, which up to this point has been limited to study of pooled samples of same-stage embryos.
We report here on IGF-1 mRNA detection in single cells of individual cleavage embryos between 3-cell and 8-cell stage. We were able to demonstrate differential rates of expression by same-stage embryos. Furthermore, we show a correlation of IGF-1 with the in-vitro development of the source embryo.
Materials and methods
Collection of mouse embryos, embryo biopsy and culture
Female B6D2F1 mice underwent superovulation by intraperitoneal injection of 5 IU of pregnant mare serum gonadotrophin, which in 48 h was followed by 5 IU of human chorionic gonadotrophin (HCG) administered intraperitoneally. Female mice were subsequently mated and examined 12–18 h after HCG injection for the presence of copulation plugs. Fertilized oocytes and 2-cell embryos were subsequently obtained from these mice by flushing the oviducts 24 h after HCG injection. Collected embryos were then cultured in 1640 Roswell Park Memorial Institute medium (Gibco) supplemented with 10% fetal bovine serum until the time of embryo biopsy.
Embryo biopsy was performed using the standard biopsy techniques (Wilton and Trounson, 1989). Acidic Tyrode's solution was used to breach the zona pellucida and a single blastomere was isolated from biopsied embryos at various stages of cleavage using a pulled glass pipette and gentle suction.
The biopsied embryos were washed thoroughly in medium and were placed in co-culture with murine endometrial stromal cells as previously described (Liu et al., 1995). Embryonic development in culture was assessed and blastocyst grading was performed using described criteria (Dokras et al., 1993). The accuracy of the embryo grading system was confirmed by differential in-situ labelling of trophectoderm and inner cell mass nuclei utilizing polynucleotide specific fluorochromes (Handyside et al., 1984).
Extraction of RNA from single blastomeres obtained by embryo biopsy
Each blastomere was handled separately and was dissolved in 30 µl of buffer consisting of 0.01 mmol/l Tris–HCl, pH 7.8, 3 mmol/l ethylenediaminetetra-acetic acid, 0.5% sodium dodecyl sulphate, and 10 µg of glycogen. After repeat freezing and thawing, a 1 h incubation at 37°C with 10 µg proteinase K was performed. The reaction was terminated by the addition of lysis buffer, which consisted of 4 mol/l guanidine thiocyanate, 0.4 mol/l sodium acetate, 0.1 mol/l 2-mercaptoethanol and 0.7% sodium laurylsarcosinate (pH 4.0). External controls of 1 pg of rabbit globin mRNA per tube were treated simultaneously and were used to asses the efficiency of the nucleic acid recovery. The nucleic acid was extracted with 100 µl of phenol–chloroform– isoamylalcohol, and separated by centrifugation at 1200 g for 15 min. The RNA was precipitated from the aqueous phase using isopropanol. Further purification was accomplished by treatment with deoxyribonuclease, repeat precipitation and 70% ethanol wash. The isolated pure RNA was dissolved in 10 µl of 0.1% dimethlypyrocarbonate-treated water and was stored at –80° until ready for use.
Reverse transcription
Reverse transcription was conducted on RNA isolated from single blastomeres of embryos at various stages of cleavage. The reactions were carried out in 22 µl of 20 mmol/l Tris–HCl (pH 8.4), containing 50 mmol/l KCl, 2.5 mmol/l MgCl2, 1 unit/µl RNasin and 1.5 mmol/l of all four 2'-deoxyribonucleoside 5'-triphosphates. Random hexanucleotides or oligo(dT) were used as primers for the M-MLV reverse transcriptase. The reaction was executed at 37°C for 1 h, heated for 5 min at 99°C, and then cooled to 5°C for 5 min. External controls of 1 pg of rabbit globin mRNA were utilized during this step of the reaction.
Polymerase chain reaction
The cDNA synthesized by the reverse transcriptase was used as a template for PCR amplification. The reactions were performed in 50 µl of reaction mixture containing 2.5 IU of Taq DNA polymerase (Promega Biotech), 10 mmol/l Tris–HCl (pH 8.40, 50 mmol/l KCl, 1% Triton X-100, 1.5 mmol MgCl2, 200 µmol/l of each 2'-deoxyribonucleoside 5'-triphosphates and 0.5 µmol/l of specific primers. Table I lists the amplicon primer sequence and amplicon size, annealing temperatures and cycle number for each of the genes studied. The basic PCR programme used consisted of denaturing at 94°C for 1 min, annealing at 58–62°C for 45 s and extension at 72°C for 1 min for 38–42 cycles. The number of cycles for each of the products was determined using semi-log plots of the amount of labelled product as a function of cycle number, in order to maintain the reactions in the linear phase of the curve. Using this approach, the intensity of fluorescence staining of the final products was proportional to the number of cDNA templates in the initial reaction mixture. The amplified products were separated on 3% agarose gel and stained with ethidium bromide. The intensity of the fluorescence of each of the products was measured with a photographic imaging system (Fotodyne, Madison, WI, USA). Given that the transcripts for IGF-1, IGF-1R and actin were amplified in different amplification conditions and that the amplicons are a different size, only the relative amounts of same-gene transcripts can be compared.
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The use of external controls of rabbit globin mRNA confirmed the mRNA recovery rate to be in excess of 90% during all reactions, and the inter- and intra-assay coefficients of variation were <10% and 7% respectively.
Statistical analysis
The mean level of IGF-1and IGF-1R mRNA expression between embryos of different cell number was compared using the analysis of variance. The statistical association between IGF-1 expression and embryo growth in culture and between blastocyst grade and cell number was compared using 
2-test.
Results
Eighty-seven biopsies of mouse embryos at various stages of cleavage were performed by removing a single blastomere from each embryo. Simultaneous amplification of IGF-1, IGF-1R and B-actin mRNA was performed on biopsied blastomeres (Figure 1). There was a failure of amplification of B-actin signal in six of those blastomeres (6.9%), suggesting that a blastomere was lost during processing.
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IGF-1 and IGF-1R mRNA detection in various stages of cleavage
IGF-1R mRNA was detected in isolated blastomeres of the majority of embryos tested at all cleavage stages (Figure 1
). IGF-1 mRNA was detected in 34 out of 81 blastomeres (42.0%), and the proportion of embryos in which it was detected increased with advancing stages of cleavage. Table II demonstrates the number of embryos biopsied and the rates of IGF-1 mRNA detection at each cleavage stage. IGF-1 transcripts could be detected in ~ a third of the embryos at the 4- or 5-cell stage. The rates of expression increased considerably thereafter. The mean level of expression within the positive blastomeres did not change significantly regardless of the cleavage stage. However, because of the greater cell numbers at the later stages of cleavage, the total number of transcripts within the entire embryo would be expected to increase markedly as the cell number increased.
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Association of IGF-1 expression with embryo growth in culture
Given that IGF-1 mRNA was not detected in all embryos tested, we examined whether there was an association between early expression and subsequent embryo development in culture. This was done to test the hypothesis that early activation of embryonic genome and thus early IGF-1 transcript detection may be used as a predictor of embryonic developmental potential. Table III
demonstrates the association between IGF-1 gene expression and in-vitro embryo growth. Significantly more embryos in which early IGF-1 mRNA expression was detected formed blastocysts in culture (85 versus 61%, P = 0.01). Furthermore, significantly more of the blastocysts formed were grade 1 (61.8 versus 14.9%, P < 0.001). This suggests that the detection of IGF-1 mRNA in early cleavage stages may be associated with improved developmental potential of such embryos.
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In order to validate the accuracy of our embryo grading system, the trophectoderm and the inner cell mass nuclei were labelled in situ, utilizing polynucleotide specific fluorochromes, and counted (Handyside et al., 1984) (Figure 2
). The accuracy of our scoring system was confirmed, as the embryos of worse grade were found to have a decreased total cell number and fewer inner cell mass nuclei (Table IV).
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Discussion
As previously reported by us (Liu et al., 1997), utilization of RT–PCR for the study of gene expression in the individual preimplantation embryos provides a sensitive method for the semiquantitative comparison of gene expression between embryos of different cleavage stages. Although it can only be used to compare relative amounts of the same gene transcript between samples, it can help to elucidate changes in gene expression at various stages of preimplantation development and further our understanding of various cleavage-stage-related events.
Studies of temporal patterns of IGF-1 expression during mouse preimplantation embryogenesis have demonstrated the presence of IGF-1 transcript during all stages of preimplantation development from oocyte to blastocyst (Doherty et al., 1994). The relative amounts of transcripts detected have been shown to decrease from oocyte to 8-cell stage and then increase as the development progresses. The intermediate cleavage stages between the 2-cell and 8-cell stage were not previously studied. Furthermore, conclusions regarding overall patterns of gene expression were limited by the need to study pooled samples of as many as 75 same-stage embryos in order to exceed detection limits of the techniques utilized. Ours is the first description of the variability in IGF-1 gene expression and its receptor in the early stages of cleavage between isolated blastomeres of different mouse embryos. We demonstrate that the IGF-1 mRNA is detectable with increasing frequency in the more advanced cleavage stages; however, in some embryos even at the 8-cell stage IGF-1 mRNA could not be detected. Furthermore, we detected IGF-1R transcripts at all the cleavage stages. This suggests that the zygotic gene activation which in the mouse is thought to be initiated by the 2-cell stage (Flach et al., 1982; Bensaude et al., 1983) may be somewhat variable and may occur at different times in the cleavage process for different embryos. Additionally, activation of zygotic gene transcription occurs at different stages of cleavage for different genes.
The importance of IGF-1 in embryogenesis has been highlighted by in-vitro demonstration of improved embryo growth when IGF-1 is added to the culture media (Harvey et al., 1992; Smith et al., 1993). Identification of IGF-1 transcripts in the preimplantation embryo suggests a possible role for the endogenously produced IGF-1 to act in an autocrine fashion and influence embryonic development. We were able to demonstrate differential levels of expression of IGF-1 between individual embryos, and furthermore we were able to demonstrate improved preimplantation development in the embryos in which early expression was detected. This, in agreement with previous studies, highlights the importance of the IGF-1 gene product in early embryonic development.
Earlier investigations have demonstrated the presence of IGF-1R transcripts only after the 8-cell stage of cleavage was reached in the mouse (Smith et al., 1993). We were able to detect the presence of transcripts at much earlier stages than previously shown. This supports the autocrine role for the endogenously produced IGF-1 in regulation of preimplantation development. The fact that maternally derived IGF-1 may also play a role in the regulation of early embryonic development in vivo cannot be discounted however, and may be one of the factors contributing to the improved embryonic development consistently demonstrated in vivo.
In addition to the study of the role of various growth factors in embryonic development, indicators of developmental potential are being sought. Thus far embryonic grading at the blastocyst stage has been utilized to determine variations in developmental potential between individual embryos. It has been indirectly suggested that protein synthesis or relative amounts of mRNA produced could be used to that same purpose (Ho et al., 1994, 1995; Liu et al., 1997). Our study demonstrates that growth factor mRNA expression at the early stages of cleavage is significantly associated with subsequent development to the blastocyst and the blastocyst grade. This suggests that it may be possible to predict embryonic developmental potential by detecting gene expression of critical genes in the preimplantation embryo.
In summary, the improved techniques in the study of gene expression will further our understanding of the role of various gene products in embryonic development. This can subsequently be used in the design of improved culture media, and may ultimately lead to improved ability to predict the developmental potential of individual preimplantation embryos.
Notes
1 To whom correspondence should be addressed at: University of North Carolina at Chapel Hill, CB#7570 MacNider, Chapel Hill, NC 27599, USA 
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Submitted on November 12, 1998; accepted on June 8, 1999.
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