Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos.
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 alpha 1 subunit were detected by reverse-transcription polymerase chain reaction (RT-PCR) and quantified relative to an exogenously supplied rabbit alpha-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 alpha 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 < or = 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.
De Sousa
P A
PA
Department of Obstetrics and Gynaecology, University of Western Ontario, London, Canada. pdesousa@physiology.uwo.ca
Westhusin
M E
ME
Watson
A J
AJ
D000199:Actins / Q000235:genetics; D000818:Animals; D001755:Blastocyst / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D002417:Cattle; D004622:Embryo, Mammalian / Q000201:enzymology; D014644:Genetic Variation; D005914:Globins / Q000235:genetics; D009865:Oocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014158:Transcription, Genetic
D000199:Actins; D012333:RNA, Messenger; D005914:Globins; D012194:RNA-Directed DNA Polymerase; D000254:Sodium-Potassium-Exchanging ATPase