Withdrawn Chicks Research Articles

Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5′-flanking segment of the ovalbumin gene.

Four DNase I-hypersensitive regions (I-IV) occur in the 5'-flanking region of the ovalbumin gene in hen oviducts. One is centered close to the cap site (position +1) of transcription and the others at -0.8, -3.3 and -6.0 kb. The correlation of hypersensitivity with transcription was determined for each region in oviducts of chicks, where expression of the ovalbumin gene can be controlled by administration and withdrawal of steroid hormones. DNase I-hypersensitive regions were mapped by the indirect end-labeling technique and ovalbumin mRNA levels were determined by the dot blot assay. Diethylstilbestrol (DES) induces the appearance of hypersensitive regions I-IV whereas progesterone induces regions I, II and IV, but not region III. Upon withdrawal, regions II and III, and most of regions I and IV, disappear. A weak zone of hypersensitivity in region I near the cap site persists during withdrawal and a new zone of hypersensitivity appears between regions I and II. There is strong correlation between hypersensitivity at regions I-IV and gene transcription; ovalbumin mRNA levels are high in stimulated chicks, where hypersensitive regions are present, and drop to very low levels in withdrawn chicks, where the hypersensitivity is absent. We suggest that proteins, perhaps hormone receptors acting together with tissue-specific factors, induce DNAse I-hypersensitive regions I-IV of the ovalbumin gene.

Effects of progesterone and tamoxifen on glucocorticosteroid-induced egg-white protein synthesis in the chick oviduct.

A single injection of either natural (cortisol, corticosterone) or synthetic [dexamethasone (DEX), triamcinolone acetonide] glucocorticosteroids to estradiol-primed, withdrawn chicks, resulted in a dose-dependent increase in the relative rates of ovalbumin and conalbumin synthesis. The simultaneous injection of equal doses of DEX and progesterone resulted in an additive effect on the relative rate of ovalbumin synthesis at all doses tested (range: 0.05-15 mg/chick), even when the induction of ovalbumin synthesis was maximal at 6 h, for either hormone injected alone. Moreover, the simultaneous injection of DEX and progesterone yielded an additive effect on the relative rates of ovalbumin and conalbumin gene transcription. The nonsteroidal antiestrogen tamoxifen does not increase ovalbumin synthesis and only slightly increases conalbumin synthesis. The simultaneous injection of tamoxifen and DEX potentiated the effect of DEX on the relative rates of ovalbumin and conalbumin synthesis, and amplified the DEX-induced increase in the relative rates of ovalbumin and conalbumin gene transcription. These results were supported by morphological studies carried out after 4 days of stimulation, which showed an increased accumulation of secretory granules in the magnum cells of the oviducts of chickens treated by tamoxifen plus DEX, as compared to that observed in chickens injected with DEX alone. In conclusion, these results suggest that glucocorticosteroids likely act through a mechanism distinct from that of sex steroids, and may modulate the effects of the latter on egg-white protein synthesis.

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation.

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.

Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription.

Estrogen (diethylstilbesterol) was administered in vivo to chicks for various time periods. Chromatin was then prepared from oviduct nuclei and assayed for its capacity to support initiation of RNA chain synthesis in vitro in the presence of saturating levels of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). These same nuclei were also assayed by a [3H]estradiol exchange assay for their endogenous receptor content. The number of available initiation sites for RNA synthesis on chromatin was shown to correlate with the endogenous levels of nuclear estrogen receptor. A decrease in the nuclear concentration of estrogen receptor molecules and the concentration of initiation sites for RNA synthesis occurred during withdrawal of estrogen from previously stimulated chicks. Both parameters declined with a similar half-life. When estrogen was readministered to withdrawn chicks, the number of initiation sites increased 2-fold as early as 30 min and approached a maximal level (3-fold) by 1 hr. During the same period of restimulation with estrogen, the number of estrogen receptor molecules bound to nuclei increased to a maximum at 20 min and then declined at 1 hr to a steady-state level 2-fold higher than the withdrawn chicks. Simultaneous measurements of RNA chain length and RNA chain propagation rate demonstrated that parameters remained relatively constant throughout estrogen withdrawal as well as secondary stimulation. The temporal correlation between changes in the levels of nuclear-bound estrogen receptor and the number of RNA chain initiation sites on chromatin prepared from these same nuclei strongly suggested that the hormone receptor complexes act on chromatin to mediate these changes in genetic transcriptional activity.