Our overall aim is to analyse gene expression during a key and unique phase of the mammalian reproductive life cycle, the oocyte and early embryo. Our first specific aim focuses on control of translation of maternal mRNA of mouse oocytes. Actin and HPRT mRNAs are regulated differentially during meiotic maturation and early development, and we intend to determine what regions of the mRNAs are responsible, looking first at the 3' untranslated region (3'UTR). We will examine translation, changes in adenylation, and degradation of (a) endogenous actin and HPRT and mRNAs after injection of RNA complementary to the 3'UTR, and (b) injected hybrid mRNAs in which the 3'UTR of globin mRNA is exchanged with that of actin or HPRT mRNAs. Injected oocytes will be allowed to proceed through maturation; newly synthesized proteins will be labeled and displayed by electrophoresis, or Northern blots of total RNA will be hybridized to appropriate probes and examined for the amount of mRNA and for changes in molecular weight due to changes in the length of the poly(A) tail. The second and third specific aims focus on two types of small RNAs derived from repetitive sequences (B1 and B2) in the mouse genome. They are abundant in ooctyes and change during meiotic maturation. Ultimately, we hope to define their relationship to the ongoing developmental program in oocytes and early embryos. First, we intend to characterise the sequence heterogeneity of small B2 RNAs of oocytes and other tissues. Northern blots reveal several discrete size classes of B2 RNAs with different patterns in oocytes, eggs, liver, and brain. We will analyse the similarity of their sequences to that of a typical B2 probe using an RNase protection assay. The labeled probe will be incubated with excess total small RNA from each tissue or with isolated RNA bands, and RNase-resistant hybrids displayed by electrophoresis on acrylamide gels. The patterns obtained will suggest the number of sequences involved, and if, as expected, the number is small, will define sequence heterogeneity within and among tissues. This analysis constitutes an important step in characterizing these newly discovered RNAs. Finally, the role of small 4.5S RNA will be explored by injecting the RNA or its complement into maturing oocytes. The amount of 4.5S RNA is normally high in oocytes and low in eggs; the injection protocol is designed to modify this pattern. We will examine the effects on the maturation process and on the pattern of protein synthesis.
