Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos

Abstract

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

Capabilities and Challenges of Examination of Gene Expression for Quality Assessment of Domestic Cat Embryos

“…), RT‐qPCR has become the method of choice also for very tiny sample sizes, such as single embryos (Filliers et al. ). However, the procedure of expression normalization varies and merits examination.…”

“…Filliers et al. () validated a set of reference genes, whose expression was found to be stable. The disadvantage of that method is that much of the valuable sample material has to be used for reference gene amplification and fewer genes of interest can be analysed.…”

“…OCT4 expression was reduced in in vitro ‐produced embryos compared with their in vivo counterparts (Filliers et al. ). Expression of genes involved in the maintenance of pluripotency, histone acetylation and DNA methylation was different in cloned embryos compared with in vitro ‐derived embryos (Imsoonthornruksa et al.…”

Baicalin increases developmental competence of mouse embryos <i>in vitro</i> by inhibiting cellular apoptosis and modulating <i>HSP70</i> and DNMT expression

“…Previously, it has been reported that in vitro culture conditions can lead to increased incidence of cellular stress and apoptosis [15, 16] and induce aberrant DNA methylation in mouse embryos [17, 18], both of which may result in decreased blastocyst quality and even affect the viability of offspring after transfer into a surrogate. Gene expression analysis, a preferred method over conventional criteria like embryo morphology and developmental rates [19], has commonly been used to assess embryo quality and to optimize in vitro culture conditions [20]. Therefore, in this study, a group of marker genes related to cellular stress and apoptosis, and DNA methylation were measured to explore the regulatory mechanism of baicalin in developmental competence of mouse embryos in vitro .…”

<i>In Vitro</i> Culture of Feline Embryos Increases Stress-induced Heat Shock Protein 70 and Apoptotic Related Genes

“…In feline species, expression analysis of genes associated with embryo development has become an important tool to understand the physiological response of embryos to their in vitro culture environment [24,25,26]. Though information for several genes has been provided, the change in gene expressions associated with stress-induced apoptosis is not entirely known in this species.…”