title: Identification of a β-galactosidase transgene that provides a live-cell marker of transcriptional activity in growing oocytes and embryos.
abstract: Identifying the events and molecular mechanisms that regulate oocyte growth has emerged as a key objective of research in human fertility, fuelled by evidence from human and animal studies indicating that disease and environmental factors can act on oocytes to affect the health of the resulting individual and by efforts to grow oocytes in vitro to enable fertility preservation of cancer survivors. Techniques that monitor the development of growing oocytes would be valuable tools to assess the progression of growth under different conditions. Most methods used to assess oocytes grown in vitro are indirect, however, relying on characteristics of the somatic compartment of the follicle, or compromise the oocyte, preventing its subsequent culture or fertilization. We investigated the utility of T-cell factor/lymphoid enhancer-binding factor (TCF/Lef)-LacZ transgene expression as a predictor of global transcriptional activity in oocytes and early embryos. Using a fluorescent β-galactosidase substrate combined with live-cell imaging, we show that TCF/Lef-LacZ transgene expression is detectable in growing oocytes, lost in fully grown oocytes and resumes in late two-cell embryos. Transgene expression is likely regulated by a Wnt-independent mechanism. Using chromatin analysis, LacZ expression and methods to monitor and inhibit transcription, we show that TCF/Lef-LacZ expression mirrors transcriptional activity in oocytes and preimplantation embryos. Oocytes and preimplantation embryos that undergo live-cell imaging for TCF/Lef-LacZ expression are able to continue development in vitro. TCF/Lef-LacZ reporter expression in living oocytes and early embryos is thus a sensitive and faithful marker of transcriptional activity that can be used to monitor and optimize conditions for oocyte growth.
authors.affiliation: Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
authors.forename: Nicole
authors.initials: N
authors.lastname: Edwards
authors.affiliation: Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Research Institute of the McGill University Health Centre, Montreal, QC, Canada Department of Experimental Medicine, McGill University, Montreal, QC, Canada.
authors.forename: Riaz
authors.initials: R
authors.lastname: Farookhi
authors.affiliation: Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada Research Institute of the McGill University Health Centre, Montreal, QC, Canada Department of Experimental Medicine, McGill University, Montreal, QC, Canada Department of Biology, McGill University, Montreal, QC, Canada hugh.clarke@mcgill.ca.
authors.forename: Hugh J
authors.initials: HJ
authors.lastname: Clarke
mesh_terms: D000818:Animals; D054885:Cumulus Cells / Q000378:metabolism; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006107:Granulosa Cells / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000502:physiology*; D009866:Oogenesis / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D001616:beta-Galactosidase
keywords: assisted reproduction; in vitro growth; oocyte; transcription; transgene