Analysis of gene expression in uniparental mouse blastocysts produced in different culture media

In the field of in vitro embryo production it is generally thought that the culture media are not as good as the oviductal fluid, resulting in detrimental changes of embryonic gene expression. Yet in vitro embryo culture (IVC) is one of the pillars of the assisted reproductive technologies. Therefore, unintended effects on gene expression may impact on the health of ART babies (PMID 27554442). Granted, human ART has many more components than just the culture media, but, the practice of embryo culture superimposes with the phase of life when cellular totipotency is present - that's why embryo culture media receive special attention. Discerning what culture media do is difficult because several confounders are present, including but not limited to oocyte heterogeneity, sperm heterogeneity as well as time of fertilization. We propose to take advantage of experimentally produced parthenogenetic embryos, and thereby remove two confounders (sperm variability, time of fertilization), in order to assess the effects of specific culture media of embryonic gene expression, in the mouse model of human reproduction. After synchronous parthenogenetic activation of MII oocytes, pronuclear oocytes were cultured in parallel in ART medium vs KSOM(aa) medium. Resultant blastocysts were compared and contrasted for gene expression among the parthenotes, as well as against zygotic blastocysts. High embryo uniformity ensured by the time of activation (same for all, chemical parthenogenesis) and by the chromosomal sex (same for all parthenotes, XX) allows to ascribe gene expression differences to the culture media applied from the pronuclear stage onward. After superovulation and collection of oocytes from oviduct, MII oocytes were activated using SrCl2 (10 mM) and latrunculin B (5 microM) in Ca-free alpha-MEM medium, so as to generate diploid parthenotes. These were cultured individually in 75 microliters Continuous Single Culture®Complete, Global®total®LP, GM501 CULT, G-TLTM, SAGE 1-StepTM, or KSOM(aa), using 96-well plates. At the blastocyst stage, samples of 5 parthenogenetic embryos were collected in duplicate from each medium (except for zygotic embryos, 3 samples from KSOM(aa) only), and lysed in lysis buffer. Samples were subjected to Affymetrix microarray analysis, performed by Anika Witten c/o Institut fuer Humangenetik, Universitaetsklinikum Muenster.