242 POST-TRANSCRIPTIONAL REGULATION OF JY-1 mRNA ABUNDANCE DURING THE BOVINE OOCYTE-TO-EMBRYO TRANSITION

Abstract

Oocyte-expressed genes play key roles in folliculogenesis and early embryonic development. The function of JY-1, a novel gene specifically expressed in bovine oocytes and early embryos, is unknown. We previously reported the expression pattern of JY-1 mRNA during meiotic maturation and early embryogenesis. The objective of this study was to elucidate the post-transcriptional regulation of JY-1 mRNA during oocyte maturation and early embryogenesis. For investigation of changes in length of JY-1 transcripts during the oocyte-to-embryo transition, total RNA isolated from germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and pronucleus (PN) stage embryos (300 oocytes/embryos per time point) was subjected to Northern blot analysis. Three major JY-1 transcripts of different length (approximately 1.8 kb, 1.2 kb, and 700 bp) were detected in GV oocytes. The size of all transcripts was decreased at MII, and PN stages by approximately 100 to 200 bp. The intermediate sized transcript was predominant at GV, MII and PN stages. Based on these initial qualitative results focused solely on changes in transcript size, we then conducted more detailed quantitative studies (using real-time PCR) focused on characterization of temporal changes in abundance of polyadenylated versus total JY-1 transcripts during early development. Total RNA samples isolated from GV and MII oocytes and from embryos at PN, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stage (n = 5 pools of 10 oocytes/embryos per time point) were divided into two equal aliquots. One aliquot was reverse transcribed into cDNA with oligo dT primers for quantification of polyadenylated transcripts and the other aliquot was transcribed with random hexamers (RH) for quantification of total transcripts. Amounts of polyadenylated JY-1 mRNA decreased during meiotic maturation (P < 0.0001), were increased (P < 0.05) at the PN and 4-cell stages relative to the MII stage, and then decreased to nearly undetectable levels after the 16-cell stage of embryo development. In contrast, amount of total JY-1 transcripts gradually decreased from PN through 16-cell stages to nearly undetectable levels thereafter. To confirm that the up-regulation of polyadenylated JY-1 mRNA in early developing embryos was not due to de novo transcription, alpha-amanitin was used to block the RNA polymerase II enzyme during the window of the first (24-13 h post-fertilization) or the second (33-14 h post-fertilization) embryonic cell cycle, and embryos at the 2-cell and 4-cell stages were collected (n = 4 pools of 10 embryos per time point). No significant changes were observed in abundance of JY-1 mRNA in control versus alpha-amanitin treated embryos. We conclude that JY-1 transcripts decrease in length during meiotic maturation and that polyadenylated JY-1 mRNAs detected in early developing embryos are oocyte-derived and not due to de novo transcription in early embryos.

This work was supported by the Rackham Foundation and the Michigan Agricultural Experiment Station.