Use of a Specific Probe for Ovalbumin Messenger RNA to Quantitate Estrogen-Induced Gene Transcripts

DNA complementary to purified ovalbumin messenger RNA (cDNA_ov) was synthesized in vitro using RNA-directed DNA polymerase from avian myeloblastosis virus. This cDNA_ov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (cDNA_ov) molecules per tubular gland cell of the chick oviduct. The changes in mRNA_oV were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNA_ov per tubular gland cell was also determined for egg-laying hen. Daily estrogen administration to the immature chick resulted in the growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNA_oV in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNA_ov concentration decreased to a level of 0–10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNA_ov. Within 30 min, the mRNA_ov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNA_ov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messenger RNA should be on the order of 40–60 hr and that the steady-state concentration of mRNA_0V per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothesis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascent mRNA molecules which in turn code for synthesis of hormone-induced proteins.