Quantitative Estimates of Cytoplasmic and Nuclear Oestrogen Receptors in Chick Oviduct

Abstract

[3H]Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with [3H]oestradiol during a 24-h incubation at 20 'C, 2 h at 30 "C or 45 min at 37 "C. Both 'soluble' and 'insoluble' nuclear receptors were stable for at least 6 h at 30 "C and 37 "C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 "C.The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10 x 1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3 -6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell).Oviducts from immature and withdrawn chickens had the majority (73 -77 %) of their oestrogen receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus.A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 yg oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 pg oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 pg oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h.The administration of oestrogenic compounds to the immature chicken induces growth, cytodifferentiation and the onset of egg-white protein synthesis in the magnum region of the oviduct [l]. Subsequent withdrawal from oestrogenic treatment results in cessation of protein synthesis and a quiescent, but partially differentiated oviduct which is capable of responding to secondary oestrogenic stimulation by the rapid onset of egg-white protein synthesis [2,3]. The response of the chick magnum to secondary oestrogenic stimulation offers a useful model for monitoring the early steps in oestrogenic hormone action, and may be useful in evaluating the role of changes in the concentration and subcellular distribution of specific oestrogen receptor molecules, in the ex...

Steroid Receptors and Effects of Oestradiol and Progesterone on Chick Oviduct Proteins

“…During the first hour after a single secondary injection of oestradiol, the cytosoluble receptor falls and this is accompanied by a comparable increase in the concentration of receptor in the nuclear compartment [4] as in other oestrogen target tissues (review in [6]). One hour after an injection of oestradiol or oestradiol benzoate within the range of 0.1 -1 mg/kg, the same amount of oestradiol-receptor complex is phosphate ~ Ahhrrviution Cyclic AMP or CAMP, adenosine 3',5'-monofound in the nucleus, while during the following hours the concentration of the nuclear receptor is dosedependent [4].Current theories propose that the nuclear receptor may be decisively involved in the control of oestrogenic expression. In accordance with these theories it has been observed that the synthesis of specific proteins such as ovalbumin and conalbumin is a function of nuclear receptor levels [7,8].…”

“…During the first hour after a single secondary injection of oestradiol, the cytosoluble receptor falls and this is accompanied by a comparable increase in the concentration of receptor in the nuclear compartment [4] as in other oestrogen target tissues (review in [6]). One hour after an injection of oestradiol or oestradiol benzoate within the range of 0.1 -1 mg/kg, the same amount of oestradiol-receptor complex is phosphate ~ Ahhrrviution Cyclic AMP or CAMP, adenosine 3',5'-monofound in the nucleus, while during the following hours the concentration of the nuclear receptor is dosedependent [4].…”

“…In the oestrogen-withdrawn chicken [5] where levels of endogenous oestrogen are low, the oestrogen receptor is found predominantly in the cytosoluble fraction of oviduct cells [4]. During the first hour after a single secondary injection of oestradiol, the cytosoluble receptor falls and this is accompanied by a comparable increase in the concentration of receptor in the nuclear compartment [4] as in other oestrogen target tissues (review in [6]).…”

Synergistic and antagonistic effects of progesterone and oestrogen on oestrogen receptor concentration and DNA polymerase activity in chick oviduct

“…The total oestrogen receptor concentrations in cytosol and crude nuclei were assessed by a previously reported [3H]oestradiol exchange technique [8]. Concentrations were expressed as oestrogen binding sites/cell assuming a DNA content of 2.3. pgldiploid genome [9].…”

“…The final pellet was resuspended in 5 vol. buffer 1 by suction in and out of a Pasteur pipette and 0.1 ml aliquots of this suspension were dispensed into tubes for the nuclear oestrogen receptor assay [8].…”