The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

Synthesis and secretion of ovalbumin by mouse-growing oocytes following microinjection of chick ovalbumin mRNA

Mouse-growing oocytes were injected with chick ovalbumin mRNA. The oocytes were cultured for 18 h in the presence of [³H]leucine and the labeled ovalbumin was measured by immunoprecipitation. Two types of ovalbumin were precipitated by antibody to ovalbumin; one co-migrated with authentic, glycosylated ovalbumin in an 18% polyacrylamide gel and was estimated to be 45 000 D, whereas the other migrated faster with an apparent MW of 41 500 D. Both types of ovalbumin were also detected in the culture medium. This study demonstrates that mouse-growing oocytes can translate exogenous mRNA coding for a secreted protein and secrete two forms of the product.     

Characterization of the effects on ovalbumin mRNA of aminomethyl-trimethylpsoralen photoreaction with hen oviduct mRNA.

We have described the reaction of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with hen oviduct mRNA and have investigated the specific effects of AMT photoaddition on ovalbumin mRNA which constitutes 60-70% of oviduct mRNA. The photoreaction of AMT with hen oviduct mRNA appeared to occur in two phases - a rapid monoaddition followed by a slower conversion of monoadducts to diadducts (i.e. crosslinking). Both nondenaturing and denaturing gel electrophoresis revealed a photoreaction time dependent increase in ovalbumin mRNA electrophoretic mobility indicating the formation of a progressively more compact molecular structure. Identical analysis of photoreacted rabbit globin mRNA revealed no change in electrophoretic mobility suggesting that AMT was stabilizing the AU rich secondary structure of ovalbumin mRNA but having no similar effect on the relatively GC rich secondary structure of globin mRNA. Ovalbumin-specific DNA primer extension was used to demonstrate the selective and secondary structure specific photoaddition of AMT to uracil bases at the 5' end of ovalbumin mRNA.     

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterne. The cytodifferentiation of the oviduct cells was induced by 17β-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.     

Regulation of protein synthesis in hen's oviducts. II. Extracellular ovalbumin as an inhibitor of ovalbumin synthesis in the oviducts.

Washed, preincubated minced hen's oviducts, which contained low levels of extracellular and intracellular proteins, synthesized egg-white proteins actively. The addition of ovalbumin to the incubation medium resulted in inhibition of the synthesis of egg-white proteins by the washed, preincubated oviduct cells, while the addition of bovine serum albumin seemed to stimulate protein synthesis and hen's egg-white lysozyme had no effect. The inhibitory or stimulatory effect on protein synthesis was proportional to the amount of protein added to the medium. The inhibitory effect of added ovalbumin was shown not to be due to the incorporation of ovalbumin into the oviduct cells from the incubation medium. Egg-white proteins added to the medium also inhibited protein synthesis inside the cells and the extent of the inhibition appeared to correspond to the amount of ovalbumin present in egg-white.