Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT–PCR

Molecular Human Reproduction vol.5 no.11 pp. 1034–1039, 1999 Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT–PCR Nury Steuerwald1,2, Jacques Cohen1, Rene J.Herrera2 and Carol A.Brenner1,3 1Gamete and Embryo Research Laboratory, Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, New Jersey, 07052, and 2Department of Biology, Florida International University, Miami, Florida, 33199, USA 3To whom correspondence should be addressed Rapid cycle DNA amplification is a refinement of the polymerase chain reaction (PCR) method that permits increased product specificity while reducing amplification time by an order of magnitude. Combined with the use of micro volume capillaries, minute samples can be examined by this technique. Thus, this approach is ideally suited to the analysis of gene expression in individual cells. As the current understanding of early developmental processes is still rudimentary, further characterization of transcription in single oocytes and embryos may provide additional insight into the molecular mechanisms directing these events. In this study, we examined the suitability of fluorescence monitored reverse transcription (RT)–PCR for the study of gene expression during oogenesis and embryogenesis using transcripts of the housekeeping gene, β-actin, as an experimental model. Product accumulation was monitored by either the double-stranded DNA dye SYBR Green I or sequence-dependent hybridization of reporter molecules called molecular beacons. Dyes bind generically and are economical to use. However, both specific and non-specific products are labelled. Hybridization probes permit very specific and sensitive target recognition but they can be costly to manufacture. Once molecular markers indicative of optimal development are identified, this technology could be used in a clinical in-vitro fertilization laboratory as a diagnostic tool. Key words: human oocytes/molecular beacons/preimplantation embryos/rapid cycling/RT–PCR Introduction Gaining knowledge about the physiological timetable of gene expression during oocyte maturation and preimplantation development is crucial for a better understanding of human development and refinement of assisted reproductive technology. It is necessary to understand what molecular markers are important for assessment of embryonic health, viability and genetic status of the mammalian oocyte. Once markers are chosen which reflect prognosis for development, techniques can be developed to extract minute amounts of cytoplasm or polar bodies for use as a diagnostic tool. In order to pursue a detailed analysis of the processes of oocyte and embryo development, highly sensitive investigative methodologies are required. Ideally, the strategy selected would be sensitive enough for use in single cells to avoid analysis of pooled material that could otherwise confound interpretations. Analysis of gene expression from single cells has been hindered by the limitations of classical molecular techniques such as Northern blot analysis. The advent of reverse transcription– polymerase chain reaction (RT–PCR) provided the level of sensitivity necessary to study transcription despite a scarcity of material (Rappolee et al., 1988; Brenner et al., 1989). With further refinements such as rapid cycle DNA amplification, specificity and yield has been improved (Wittwer and Gerling, 1991) precluding the need for nested amplifications. This technique allowed the examination of extremely minute samples when used in conjunction with micro volume 1034 capillaries (Wittwer et al., 1997b). Coupled with radiolabelled probes, RT–PCR has permitted the analysis of expression from a small number of embryos (Rambhatla et al., 1995). By the addition of fluorescent probes or double-stranded DNA (dsDNA) dyes, product accumulation can be monitored using a suitably equipped fluorescence temperature cycler. Sequencespecific fluorescent probes allow for highly sensitive and specific target detection. With probe systems, fluorescence is typically achieved when a fluorophore and a quencher are separated either by distance or by hydrolysis. For example, hairpin primers (Nazarenko et al., 1997) are designed in a manner to achieve fluorescence only when the primer, labelled with a fluorophore at one end and a quencher at the other, is made linear by incorporation into an amplification product. Hairpin probes or molecular beacons (Kramer and Tyagi, 1996) employ a similar scheme to obtain fluorescence. The beacon unfolds when it comes in contact with its complementary sequence within the PCR amplicon producing a fluorescent signal. Hydrolysis probes (Holland et al., 1991) achieve fluorescence by separating the fluorophore and quencher through the 59 to 39 exonuclease activity of Taq polymerase during product extension. An alternate approach uses resonance energy transfer by adjacent hybridization probes (Wittwer et al., 1997a). A pair of probes are designed such that the labelled ends are brought together during hybridization in order to obtain a fluorescent signal. In contrast, dsDNA specific dyes permit more generic product identification. © European Society of Human Reproduction and Embryology Rapid RT–PCR fluorescence of oocytes and embryos Early oocyte maturation in mammals is distinguished by active gene transcription that results in the production of vast amounts of various RNA species (Telford et al., 1990). Some transcripts are required for oocyte-specific processes and metabolism while others are presumably stored for use during early embryonic development, prior to the activation of the embryonic genome at the 4–8-cell stage (Tesarik et al., 1986; Braude et al., 1988). The early embryo undergoes rapid cell proliferation prior to the emergence of two distinct cell populations, the inner cell mass and trophectoderm, at the blastocyst stage. Undoubtedly, this differentiation is the result of differential gene expression. Although striking progress in in-vitro techniques has permitted the study of oogenesis and embryogenesis at a molecular level, these processes still remain better described than understood. The specific transcripts of the oocyte and embryo have yet to be characterized. Clearly, further research is warranted in order to decipher the role that these messages play as well as to unravel the tapestry of responses that they mediate throughout development. It is the aim of this investigation to employ rapid realtime RT–PCR fluorescent methods to analyse gene expression in oocytes and embryos. To demonstrate the suitability of fluorescence monitored RT–PCR for this purpose, transcripts of the housekeeping gene, β-actin, were used as an experimental model. Furthermore, in order to compare fluorescent probe technology, rapid cycle DNA amplification was monitored by two different techniques, one which uses the double-stranded DNA dye SYBR Green I (Molecular Probes, Eu (...truncated)