Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos
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abstract
Variation in the abundance of a specific gene transcript was assessed in single bovine oocytes and in vitro-derived blastocysts. Transcripts encoding the Na+,K+-ATPase 1 subunit were detected by reverse- transcription polymerase chain reaction (RT-PCR) and quantified relative to an exogenously supplied rabbit -globin mRNA using laser-induced fluorescence capillary electrophoresis (LIF-CE). The precision of this relative abundance (RA) calculation was predicted and shown to resolve 2- fold differences in transcript abundance between individual blastocysts and predicted in oocytes to resolve 3-fold differences. The RA of the 1 subunit transcript differed by 2- to 3-fold among blastocysts, and 3- to 6- fold among oocytes. Comparison of a general population of oocytes with blastocysts revealed little overlap in RA values between the two groups, with a 8-to 14-fold increase in the mean RA for each group with development observed in two successive experiments (P 0.05). In contrast, oocytes selected for their developmental competence on the basis of morphologic criteria exhibited only a 1.6- to 1.7-fold developmental increase when the assay was performed on cDNA generated from either embryo pools (n = 6 versus 6) or individuals (n = 7 versus 7), respectively. These results provide the first characterization of the degree of heterogeneity in the abundance of a specific mRNA transcript among individual mammalian oocytes and preimplantation embryos and demonstrate that transcript relative abundance can be correlated with bovine oocyte morphology.
