The RNA of growing mouse oocytes and ovulated ova was labeled by injection of a solution containing tritiated uridine into the ovarian bursa. The time course of incorporation into RNA by oocytes was followed by analysis of alkali-labile acid insoluble radioactivity and by autoradiography. The results show that most of the incorporation into RNA by growing oocytes takes place within one day of bursal injection of the precursor, reflecting the rapid fall of label in the acid soluble precursor pool. The RNA of growing oocytes of all sizes is unusually stable, at least 80% of the labeled RNA present two days after bursal injection being retained until ovulation 10 to 20 days later. The fraction of heterogeneous RNA in labeled RNA of ova was estimated as 20% by sucrose gradient analysis. It is likely that egg RNA is synthesized primarily during the period of oocyte growth one to three weeks before ovulation.

Half grown oocytes were released from follicles of 7-day-old mouse ovaries during overnight culture. Naked oocytes free of attached follicular cells were cultured in MEM plus 10% fetal calf serum over the developing monolayer of ovarian cells. Oocytes increased in diameter for a period of at least a week at 1.6 micron per day, or 70% of the in vivo growth rate. The ultrastructure of cytoplasmic organelles in cultured growing oocytes was normal. During the second week, about 35% underwent spontaneous fragmentation, and at least 10-20% resumed meiotic maturation. We conclude that a significant fraction of naked oocytes reached a functional state comparable to that of normal full grown oocytes at a rate approaching the in vivo rate. Free oocytes with attached cells grew to a larger average diameter than naked oocytes, and incorporated 3H-leucine 75% more rapidly. Naked oocytes cultured in the absence of ovarian cells did not grow, and died when pyruvate was omitted from the medium. Naked oocytes did not grow when cultured over primary mouse fibroblasts, L-cells, CHO cells, or hepatoma cells. Naked oocytes separated from the monolayer of ovarian cells by a 0.7 mm layer of agar grew at 60% of the rate of control oocytes.

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A method is described for in vitro culture of naked growing mouse oocytes for at least four days. Tests for metabolic function indicate that the oocytes increase in volume, accumulate glucose-6-phosphate dehydrogenase, and maintain a steady level of 3H-uridine and 3H-leucine incorporation.

A comparison was made between a new, commercially available light microscope assay for T- and B-lymphocyte enumeration and existing procedures. The Quantigen assay employs two color-coded Immunobeads; one Immunobead binds to and labels T-cells, while the second labels B-cells. Phagocytic cells contaminating lymphocyte preparations ingest both types of beads. A high degree of correlation was found between this simultaneous assay for T- and B-cells and the E-rosette method for T-cells and FITC-labeling of B-cell surface immunoglobulin. The results of a comparison of the Quantigen assay to flow cytometric analyses are also given. Data on chronic lymphocytic leukemia samples are presented, demonstrating that these cells are double-markers with the Quantigen assay because of the use of monoclonal antibody T101, which recognizes T-cells and B-CLL cells.