Biochemical studies of mammalian oogenesis: Synthesis and stability of various classes of RNA during growth of the mouse oocyte in vitro

Abstract

The synthesis of various classes of RNA in mouse oocytes at different stages of growth has been examined after incubating follicles in medium containing radiolabeled uridine. After fractionation on poly(U)-Sepharose of radiolabeled oocyte RNA, of which about 83% is associated with the nucleus after a 5-hr labeling period, revealed that about 40–50% of the radiolabeled RNA behaved as poly(A)-containing RNA. This value remained fairly constant during the period of oocyte growth in which oocyte diameter increased from about 35 to about 55 μm. After a 5-hr labeling, the percentage of radiolabeled poly(A)-containing RNA in either the fully grown dictyate oocyte, metaphase II oocyte, or one-cell embryo was about 20%. After a 5-hr labeling, agarose gel electrophoretic analysis of the radiolabeled species of oocyte RNA obtained after fractionation on poly(U)-Sepharose revealed the presence of a putative ribosomal RNA precursor, ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA and heterodisperse poly(A)-containing RNA. A significant fraction of the radiolabeled RNA species was quite large (>40 S). The ratios of the relative proportions of the radiolabeled ribosomal RNAs and transfer plus 5 S RNA remained essentially constant during oocyte growth. The stability of various classes of RNA was examined by incubating follicles with radiolabeled uridine, washing the follicles free of radioactivity and culturing the follicles under conditions which support oocyte growth in vitro ( Eppig, 1977). Under these conditions, total oocyte radiolabeled RNA was quite stable as determined by retention of acid-insoluble radioactive material ( t 1 2 = 28 days ). However, under conditions in which oocytes are viable but do not grow, the half-life of total RNA was about 4.5 days. Poly(A)-containing RNA was also very stable; after 8 days in culture, about 50% of the radiolabeled poly(A)-containing RNA present after 5 hr of labeling was still present. Agarose gel electrophoretic analysis of radiolabeled RNA in oocytes after 4 days of culture and after fractionation on poly(U)-Sepharose revealed the presence of ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA, and heterodisperse poly(A)-containing RNA. At this time, these RNAs are located in the oocyte cytoplasm. In addition, the molecular weight distribution of poly(A)-containing RNA was significantly lower than that after 5 hr of labeling. The ratios of the relative proportions of radiolabeled ribosomal RNAs and transfer plus 5 S RNA were quite similar to those after 5 hr of labeling.