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Analysis of human preimplantation embryos and aging oocytes by cDNA microarrays
by Xeragon, Inc. (Huntsville, AL) using 2-O-Triisopropylsilyl-Oxy-Methyl chemistry. These short interfering RNA (siRNA) constructs, directed toward exon 5 of the E-cadherin message, were 21 bases in length with a 2⬘-base 3⬘ overhang on each strand of the duplex (Elbashir et al., Nature 2001 411:494 – 498). Both types of constructs were microinjected individually into the cytoplasm of CB6F1 mouse zygotes. Control zygotes were injected with an appropriate diluent. Injected embryos were cultured in vitro and examined over a period of 4 days using light microscopy. Embryos from the construct injected and control groups were selected just prior to the time of cavitation and subjected to immunohistochemistry for the E-cadherin protein via confocal microscopy. Results: Survival and early development of construct injected and control embryos were satisfactory and unremarkable. At the time of compaction and cavitation, however, embryos injected with either the 587-bp dsRNA or 21-nucleotide siRNA constructs exhibited a clear phenotype associated with a loss of function of the E-cadherin gene product as previously reported by others using dsRNA and traditional gene knockout (Wianny and ZernickaGoetz, Nature Cell Biol. 2000 2:70 –75). Treated embryos failed to form an intact blastocoelic cavity and instead exhibited multiple individual cavities, a disorganized appearance and arrested. Approximately 70% of embryos injected with either construct over multiple experiments exhibited this phenotype; which was not observed in control embryos. Construct injected embryos exhibited a consistent reduction in staining for this protein in comparison with control embryos. Conclusions: This report supports prior studies demonstrating the efficacy of RNAi in mouse embryos and in addition provides direct evidence for an effect on the associated protein (Svoboda et al., Biochem Biophys. Res. Commun. 2001 5:1099 –104). Furthermore, this is the first report of successful gene silencing using a siRNA construct in mammalian embryos. This result is highly significant since the generation and use of such constructs is a relatively simple matter. Short oligoribonucleotides suitable for RNAi can be directly purchased from multiple suppliers negating the requirement for an in-house molecular biology laboratory. Therefore, any basic embryology laboratory can easily perform experiments using such constructs. This capability should allow for a simple and efficacious research strategy for analyzing the role of specific gene products in early development and may even have potential in therapeutic manipulation of gene expression in a clinical setting. Supported by: The Institute for Reproductive Medicine and Science of Saint Barnabas.
Tuesday, October 15, 2002 3:00 P.M. O-199 Analysis of human preimplantation embryos and aging oocytes by cDNA microarrays. Anthony T. Dobson, Michael J. Abeyta, Christopher Haqq, Renee A. Reijo Pera. Univ of CA, San Francisco, San Francisco, CA. Objective: To develop methods to analyze single oocytes and preimplantation embryos by cDNA microarrays 1) to define differences between oocytes from younger and older reproductive-aged women, and 2) to examine the cascade of gene expression that follows fertilization. Design: Descriptional study of human oocyte and embryo gene expression. Materials/Methods: These studies were approved by the UCSF Committee on Human Research. Consenting patients from the UCSF in vitro fertilization (IVF) program were candidates for these studies. Donated immature (germinal vesicle) oocytes from patients undergoing ICSI were stripped of granulosa cells and frozen individually in tubes. Embryos were obtained from patients who had completed their families and wished their remaining embryos be donated to research rather than destroyed. RNA was prepared using PicoPure RNA Isolation Kit (Arcturus Engineering), and amplified using a high sensitivity proprietary kit (collaborative effort, Arcturus Engineering). Cy3 (control) and Cy5 fluorescently-labeled probes were reverse-transcribed from amplified RNA. Two microarray chips (⬃22,000 genes and ESTs each) were hybridized, washed, and scanned using the GenePix 4000B scanner. To test the amplification protocol, RNA from two oocytes was mixed then divided into two tubes for independent amplification. Analysis was performed using the Pearson Correlation. Oocytes from women 30 years and less were compared to oocytes from women 40 years and older, and to embryos at day 1 (pronuclear) and day 3 of
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development. Analysis was performed using GenePix 3.0, Significance Analysis of Microarrays (Stanford), and Cluster/TreeView (Lawrence Berkeley National Lab) software. Results: RNA amplification using these techniques was 1.8 million-fold, sufficient for multiple microarray experiments. The amplification control yielded a correlation coefficient of 98.3%, demonstrating reproducibility of the amplification methods. SAM analysis identified the most differentially expressed genes; more importantly, however, it revealed that biological and experimental variability can be overcome by relatively few experiments. Few genes are differentially expressed between young and old oocytes, suggesting that any differences are likely functional or at the protein level. Striking differences are evident by day 1 of embryo development, and this pattern contrasts greatly with those at day 3. Cluster analysis clearly demonstrates unique expression profiles among the different stages of development tested and has identified potential candidate genes for further study. Conclusions: cDNA microarray analysis of extremely small samples is now technologically feasible. These studies have shown few differences in the RNA between young and old eggs, but robust changes at the pronuclear stage of embryo development following fertilization. Future studies will more fully define the differences between the oocytes of younger and older women, and the different stages of preimplantation development. Supported by: Reproductive Scientist Development Program and Arcturus Engineering (amplification kits).
Tuesday, October 15, 2002 3:45 P.M. O-200 Assessment of parthenogenetic activation of human metaphase II oocytes for stem cell derivation. J. David Wininger, Steve Huang, Joe B. Massey, Jinqui Lei, Helen Lin. Reproductive Biology Assoc, Atlanta, GA; Stemron Corp, Gaithersburg, MD. Objective: Human stem cells derived from embryos provide potential cell based therapies for repair of degenerating or damaged tissue. However, there are many ethical concerns surrounding the use of human embryos for stem cell research. Parthenogenetic activation has been studied extensively in invertebrates, amphibians, and mice. Activation occurred in these models following an increased intracellular calcium transient. Inhibitors of protein kinases have also been used sequentially with calcium ionophores to suppress the second meiotic reductional division allowing production of diploid parthenones. The aim of this study was to assess parthenogenetic activation and blastocyst development of human donor oocytes using calcium ionophore and protein kinase inhibitors. Design: Prospective, experimental study utilizing donor oocytes. Materials/Methods: Consented oocyte donors underwent follicular stimulation with rFSH following treatment with either a GnRH agonist or antagonist. Oocyte retrieval occurred 36 hours after HCG (10,000 IU) injection and the cumulus cells were removed with hyaluronidase(80 IU/ml) one hour later. Metaphase II oocytes were treated with 5 uM calcium ionophore A23187 for 5 minutes at 33 degrees Celcius followed by a 3 hour incubation in 1 mM 6 dimethyl -aminopurine (6-DMAP) at 37 degrees. Activated oocytes with one pronucleus were cultured in IVC-1 media for 72 hours and were then moved to Gen-X Blastocyst media for 48 hours. Blastocysts which displayed a large blastocoel cavity with the clear presence of an ICM were hatched with acidified tyrodes and treated with anti-human trophoectoderm antibody followed by guinea pig complement. Treated cells were moved to 4-well dishes which were previously coated with mitotically inactivated murine embryonic feeder cells (ATCC) in Dulbeco’s modified Eagle’s medium supplented with 20% FBS. Attachment of the inner cell mass cells was evaluated after 24 hours. Results: Seventy-six oocytes were retrieved from five donors. 89% (68/ 88) were mature, metaphase II oocytes. 59% (40/68) activated following treatment with calcium ionophore and DMAP. 37% (15/40) of the parthenotes progressed to the blastocyst stage and underwent assisted hatching (see photo). Following immunosurgery, 60% (9/15) of the treated blastocysts attached to the feeder cells. Cell masses remained attatched to the feeder cells for 3–7 days before their growth arrested.
Vol. 78, No. 3, Suppl. 1, September 2002
Tuesday, October 15, 2002 3:00 P.M. O-199 Analysis of human preimplantation embryos and aging oocytes by cDNA microarrays. Anthony T. Dobson, Michael J. Abeyta, Christopher Haqq, Renee A. Reijo Pera. Univ of CA, San Francisco, San Francisco, CA. Objective: To develop methods to analyze single oocytes and preimplantation embryos by cDNA microarrays 1) to define differences between oocytes from younger and older reproductive-aged women, and 2) to examine the cascade of gene expression that follows fertilization. Design: Descriptional study of human oocyte and embryo gene expression. Materials/Methods: These studies were approved by the UCSF Committee on Human Research. Consenting patients from the UCSF in vitro fertilization (IVF) program were candidates for these studies. Donated immature (germinal vesicle) oocytes from patients undergoing ICSI were stripped of granulosa cells and frozen individually in tubes. Embryos were obtained from patients who had completed their families and wished their remaining embryos be donated to research rather than destroyed. RNA was prepared using PicoPure RNA Isolation Kit (Arcturus Engineering), and amplified using a high sensitivity proprietary kit (collaborative effort, Arcturus Engineering). Cy3 (control) and Cy5 fluorescently-labeled probes were reverse-transcribed from amplified RNA. Two microarray chips (⬃22,000 genes and ESTs each) were hybridized, washed, and scanned using the GenePix 4000B scanner. To test the amplification protocol, RNA from two oocytes was mixed then divided into two tubes for independent amplification. Analysis was performed using the Pearson Correlation. Oocytes from women 30 years and less were compared to oocytes from women 40 years and older, and to embryos at day 1 (pronuclear) and day 3 of
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Abstracts
development. Analysis was performed using GenePix 3.0, Significance Analysis of Microarrays (Stanford), and Cluster/TreeView (Lawrence Berkeley National Lab) software. Results: RNA amplification using these techniques was 1.8 million-fold, sufficient for multiple microarray experiments. The amplification control yielded a correlation coefficient of 98.3%, demonstrating reproducibility of the amplification methods. SAM analysis identified the most differentially expressed genes; more importantly, however, it revealed that biological and experimental variability can be overcome by relatively few experiments. Few genes are differentially expressed between young and old oocytes, suggesting that any differences are likely functional or at the protein level. Striking differences are evident by day 1 of embryo development, and this pattern contrasts greatly with those at day 3. Cluster analysis clearly demonstrates unique expression profiles among the different stages of development tested and has identified potential candidate genes for further study. Conclusions: cDNA microarray analysis of extremely small samples is now technologically feasible. These studies have shown few differences in the RNA between young and old eggs, but robust changes at the pronuclear stage of embryo development following fertilization. Future studies will more fully define the differences between the oocytes of younger and older women, and the different stages of preimplantation development. Supported by: Reproductive Scientist Development Program and Arcturus Engineering (amplification kits).
Tuesday, October 15, 2002 3:45 P.M. O-200 Assessment of parthenogenetic activation of human metaphase II oocytes for stem cell derivation. J. David Wininger, Steve Huang, Joe B. Massey, Jinqui Lei, Helen Lin. Reproductive Biology Assoc, Atlanta, GA; Stemron Corp, Gaithersburg, MD. Objective: Human stem cells derived from embryos provide potential cell based therapies for repair of degenerating or damaged tissue. However, there are many ethical concerns surrounding the use of human embryos for stem cell research. Parthenogenetic activation has been studied extensively in invertebrates, amphibians, and mice. Activation occurred in these models following an increased intracellular calcium transient. Inhibitors of protein kinases have also been used sequentially with calcium ionophores to suppress the second meiotic reductional division allowing production of diploid parthenones. The aim of this study was to assess parthenogenetic activation and blastocyst development of human donor oocytes using calcium ionophore and protein kinase inhibitors. Design: Prospective, experimental study utilizing donor oocytes. Materials/Methods: Consented oocyte donors underwent follicular stimulation with rFSH following treatment with either a GnRH agonist or antagonist. Oocyte retrieval occurred 36 hours after HCG (10,000 IU) injection and the cumulus cells were removed with hyaluronidase(80 IU/ml) one hour later. Metaphase II oocytes were treated with 5 uM calcium ionophore A23187 for 5 minutes at 33 degrees Celcius followed by a 3 hour incubation in 1 mM 6 dimethyl -aminopurine (6-DMAP) at 37 degrees. Activated oocytes with one pronucleus were cultured in IVC-1 media for 72 hours and were then moved to Gen-X Blastocyst media for 48 hours. Blastocysts which displayed a large blastocoel cavity with the clear presence of an ICM were hatched with acidified tyrodes and treated with anti-human trophoectoderm antibody followed by guinea pig complement. Treated cells were moved to 4-well dishes which were previously coated with mitotically inactivated murine embryonic feeder cells (ATCC) in Dulbeco’s modified Eagle’s medium supplented with 20% FBS. Attachment of the inner cell mass cells was evaluated after 24 hours. Results: Seventy-six oocytes were retrieved from five donors. 89% (68/ 88) were mature, metaphase II oocytes. 59% (40/68) activated following treatment with calcium ionophore and DMAP. 37% (15/40) of the parthenotes progressed to the blastocyst stage and underwent assisted hatching (see photo). Following immunosurgery, 60% (9/15) of the treated blastocysts attached to the feeder cells. Cell masses remained attatched to the feeder cells for 3–7 days before their growth arrested.
Vol. 78, No. 3, Suppl. 1, September 2002