Method to isolate polyribosomal mRNA from scarce samples such as mammalian oocytes and early embryos

The proposed method is based on mixing the extracted oocyte cytoplasm with a preparation of polyribosomes obtained from a non-homologous source (Drosophila) and using sucrose density gradient ultracentrifugation to separate the polyribosomes. It involves cross-linking the non-homologous polyribosomes and neutralizing the cross-linking agent. Using this method, we show that certain stages of oocyte maturation coincide with changes in the abundance of polyribosomal mRNA but not total RNA or poly(A). We also show that the abundance of selected sequences matched changes in the corresponding protein levels.We report here the successful use of a method to profile mRNA present in the polyribosomal fraction obtained from as little as 75 mammalian oocytes. Polyribosomal mRNA fractionation thus provides a new tool for studying gametogenesis and early development with better representation of the underlying physiological status.Gametogenesis and embryonic development in mammals involve several major cellular events marked by an unusual mode of messenger RNA management. In nearly all animal species, mRNA molecules are stored in the developing oocyte until use during maturation or after fertilization [1-8]. These stored mRNAs direct protein synthesis during the period of transcriptional silence, which begins when the germinal stage oocyte reaches its full size [9-13] and lasts until embryonic genome activation [14-16]. In cattle, this size is approximately 120 μm within a follicular antrum 3-5 mm in diameter [10,15,17]. During the period covering the remaining follicular development (i.e. from 3 to 25 mm in antral diameter), the post-LH-surge oocyte maturation, fertilization and the onset of embryonic genome activation, very little genomic transcription occurs. It is generally believed that transcript storage begins in the early stages of oogenesis and may thus last for several weeks. It is also believed that the transcripts are stored in a particulate form [18] and lack the pol