Pablo I. Szendro

Biosynthesis and transformation of microsomal and cytosol estradiol receptors.

Abstract Extracts of uterine microsomes contain a “basic” 3.5 S and an “acidic” 4.5 S estradiol receptor. The smaller “basic” molecule appears to be an early product of receptor biosynthesis. It dimerizes to a “basic” 4.5 S entity on heating. Both the artificial “basic” dimer and the extracted “acidic” 4.5 S molecule are reversibly dissociated by protonation into 3.5 S “basic” and 3.5 S “acidic” subunits respectively. The heat-accelerated formation of stable dimers requires monomer-bound estradiol. The dimers are also dissociated by 2 M urea. High-speed supernatant (cytosol) of uterine homogenates, prepared with either low ionic strength buffer, pH 7.5, or buffered 0.25 M sucrose, contains only “acidic” receptors sedimenting at 4 S and 5 S in sucrose gradients prepared with buffered 0.4 M KC1. The radioactivity sedimenting at 5 S is shifted to the 4 S position by protonation. The reverse shift by proton withdrawal is accelerated by heating. Similar to the estradiol requirement for the formation of stable microsomal 4.5 S dimers, stable cytosol 5 S-estrogen complexes are only derived from estradiolcharged 4 S molecules. In contrast to the microsomal 4.5 S dimer, the cytosol-estradiol 5 S complex is not affected by 2 M urea, which improves the resolution of the two cytosol peaks and avoids the formation of rapidly sedimenting aggregates on heating. The following sequence of receptor forms is indicated as the major in vivo pathway: “basic” microsomal 3.5 S → “acidic” microsomal 3.5 S → cytosol 4 S → cytosol 5 S. Receptor synthesis in the uterus is not necessarily dependent on estradiol since it persists in the uteri of both ovariectomized and ovariectomized/hypophysectomized rats. The administration of estradiol to hormone-deprived animals results in a “depletion-replenishment” sequence of receptor levels, which is caused by irreversible utilization and resynthesis of receptors. Estradiol thus appears to enhance the rate of receptor synthesis.

Activation of transcription-regulating proteins by steroids☆

Abstract The intracellular proteins, which bind steriod hormones with high affinity and specificity have been generally considered as instruments of hormone action. A reversal of assignments might seem a merely semantic exercise, but is indeed in better agreement with experimental evidence identifying ‘receptors’ as transcription-regulating proteins. The series of events in the presence of hormone are: 1. attachment of the steroid to the ‘receptor’ which undergoes a major conformational change when ‘enveloping’ the steroid, 2. dimerization to steroid-receptor: steroid-receptor 3. translocation of the dimer into the nucleus, 4. enhancement of transcription. One product of the latter is ‘receptor’ mRNA, the translation of which initiates within 60–90 min after pulse-administration of steroid. In the absence of hormone, ‘receptor translocation’, degradation and biosynthesis continue to proceed but at a much slower rate. Although these results have been primarily obtained with the estradiol-‘receptor’ system, all other systems seem to follow the same pattern. The molecular mechanism by which enhancement of transcription is achieved is as yet unknown. Its specificity must be quite particular since several steroid-‘receptor‘ systems occur simultaneously within the same cell.

MECHANISMS INVOLVED IN THE REGULATION OF STEROID RECEPTOR LEVELS

Abstract The turnover of steroid receptors comprises: synthesis in the cytoplasm, translocation into the cell nucleus and degradation at a still unknown site. From studies on the oestradiol/receptor system, the following conclusions can be drawn. 1. The in-vivo uptake of oestradiol by target cell nuclei is receptordependent. Steroid and receptor are translocated from the cytoplasm in a 1:1 ratio. A recycling of receptor is undetectable after pulse administration of oestradiol. Receptor replenishment in the cytoplasm is accomplished by synthesis. 2. The nuclear uptake of receptor-in contrast-proceeds also in the absence of oestradiol. Both forms of receptor, monomer and “activated” dimer are present in oestrogen-free nuclei. 3. Oestradiol enhances the “nucleotropy” and the turnover rate of receptor. 4. Oestrogenicity and antioestrogenicity are apparently linked to effects exerted on the “nucleotropy” of receptor and its ability to interact with the relevant nuclear structures.

Alterations in the subcellular distribution of 17β-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17β-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2–4 μm diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.

BIOSYNTHESIS AND TRANSFORMATION OF MICROSOMAL AND CYTOSOL ESTRADIOL RECEPTORS

Extracts of uterine microsomes contain a “basic” 3·5 S and an “acidic” 4·5 S estradiol receptor. The smaller “basic” molecule appears to be an early product of receptor biosynthesis. It dimerizes to a “basic” 4·5 S entity on heating. Both the artificial “basic” dimer and the extracted “acidic” 4·5 S molecule are reversibly dissociated by protonation into 3·5 S “basic” and 3·5 S “acidic” subunits respectively. The heat-accelerated formation of stable dimers requires monomer-bound estradiol. The dimers are also dissociated by 2 M urea. High-speed supernatant (cytosol) of uterine homogenates, prepared with either low ionic strength buffer, pH 7·5, or buffered 0·25 M sucrose, contains only “acidic” receptors sedimenting at 4 S and 5 S in sucrose gradients prepared with buffered 0·4 M KCl. The radioactivity sedimenting at 5 S is shifted to the 4 S position by protonation. The reverse shift by proton withdrawal is accelerated by heating. Similar to the estradiol requirement for the formation of stable microsomal 4·5 S dimers, stable cytosol 5 S-estrogen complexes are only derived from estradiolcharged 4 S molecules. In contrast to the microsomal 4·5 S dimer, the cytosol-estradiol 5 S complex is not affected by 2 M urea, which improves the resolution of the two cytosol peaks and avoids the formation of rapidly sedimenting aggregates on heating. The following sequence of receptor forms is indicated as the major in vivo pathway: “basic” microsomal 3·5 S → “acidic” microsomal 3·5 S → cytosol 4 S → cytosol 5 S. Receptor synthesis in the uterus is not necessarily dependent on estradiol since it persists in the uteri of both ovariectomized and ovariectomized/hypophysectomized rats. The administration of estradiol to hormone-deprived animals results in a “depletion-replenishment” sequence of receptor levels, which is caused by irreversible utilization and resynthesis of receptors. Estradiol thus appears to enhance the rate of receptor synthesis.