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

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.

Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts.

Isolation of pure inner cell mass (ICM) and trophectoderm (TE) samples from a single blastocyst is necessary to obtain accurate information on the transcriptomes of these cells. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. However, the small size of blastocysts hampers tissue processing prior to LCM. This article describes a protocol for the application of LCM to isolate homogeneous ICM and TE cell samples from single bovine blastocysts for downstream gene expression analysis.

Laser capture microdissection for gene expression analysis of specific cell populations in single blastocysts.

Laser capture microdissection (LCM) allows for the isolation of small tissue fractions from heterogeneous tissue sections, for downstream genetic or proteomic analysis without contamination by the surrounding tissue. This technique can also be successfully used for the isolation of small tissue fractions from developing embryos, such as expanding blastocysts. However, the small size of early-stage embryos hampers tissue processing prior to LCM. The present protocol describes the application of LCM to isolate specific cell fractions from blastocysts for downstream gene expression analysis with RT-PCR.