Thomas A. Gorell

Hormonal regulation of cytoplasmic estrogen and progesterone receptors in the beagle uterus and oviduct

The hormonal regulation of uterine and oviductal cytoplasmic estrogen and progesterone receptors was studied in immature beagles that were untreated, treated with estradiol-17 beta, or treated sequentially with estradiol and progesterone. Estradiol treatment increased the concentration of estrogen receptors in both tissues. Progesterone receptors were not detectable in the reproductive tract of untreated animals, but increased dramatically under the influence of estradiol. Estrogen withdrawal following estrogen stimulation concomitant estrogen plus progesterone administration, and estrogen withdrawal plus progesterone administration all caused significant reductions in both estrogen and progesterone receptors in uterine oviductal cytosols when compared to estrogen treatment alone. Estrogen withdrawal resembled estrogen plus progesterone administration in reducing both estrogen and progesterone receptor levels, although estrogen withdrawal plus progesterone administration resulted in a further reduction in both receptor concentrations. The same positive and negative relationships between estrogen and progesterone receptor content were observed in uterine cytosols from cycling and ovariectomized adults. These data suggest that estrogen and progesterone regulate their respective receptors and that tissue sensitivity to both steroids may be controlled by mechanisms involving fluctuations in receptor concentration in the reproductive tract of the beagle.

Analysis of the progesterone receptor in the beagle uterus and oviduct

Abstract Uterine and oviductal cytosols from immature and cycling adult beagles contain macromolecules which bind progestins specifically and with high affinity as determined by an exchange assay utilizing the synthetic progestin, R5020. The [ 3 H]-R5020 bound to the progesterone receptor 2 to 3 times more tightly than progesterone and apparently did not bind to corticosteroid-binding globulin (CBG). Cytosols were incubated with 30 nM [ 3 H]-R5020, in the presence or absence of a 250-fold excess of unlabeled R5020, for 4 to 6 h at 4°C, conditions appropriate for exchange of labeled R5020 for endogenously bound steroid. Dextran-coated charcoal was used to separate bound from free ligand. The progesterone receptor in the female beagle, characterized using this standard assay, was tissue and steroid specific. Uterine cytosols contained two [ 3 H]-R5020 binding peaks (4S and 7–8S) on sucrose density gradients. The progesterone receptor bound [ 3 H]-R5020 with an apparent dissociation constant ( K D ) of approximately 2.5 nM according to Scatchard plot analysis. The concentration of uterine and oviductal progesterone receptors was under the inductive influence of estradiol. Measurable specific [ 3 H]-R5020 binding increased dramatically following administration of estradiol-17 β , but was absent in uteri and oviducts of untreated pups. These data have demonstrated the presence of a specific progesterone receptor in the beagle oviduct and uterus which displayed characteristics similar to those reported for progesterone receptors in other mammalian species.

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

Abstract The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [3H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.

A cytoplasmic estradiol receptor in the immature beagle uterus.

Abstract A cytoplasmic protein has been detected in the uteri of immature beagles by its ability to bind [ 3 H]-estradiol-17β. This estrogen binder exhibits characteristics of an estradiol receptor (E 2 R), namely specificity, high affinity binding, and saturability. Cytosols were prepared in a buffer containing 10 mM Tris and 1.5 mM EDTA, pH 7.4, and incubated with [ 3 H]-estradiol in the presence or absence of unlabeled estradiol in order to determine specific binding. Separation of bound steroid from free was accomplished by exposure of the cytosol incubation mixture to dextran-coated charcoal for 15 min at 0 to 4°C. Specific binding of [ 3 H]-estradiol was measured at various temperatures and times of incubation. Incubation at 30°C for 3 to 4 h resulted in equilibrium binding enabling the measurement of the total number of receptors present. At 37°C there was an apparent breakdown of the [ 3 H]-estradiol binding ability of the cytosol. A [ 3 H]-estradiol binding fraction was observed in the 37°C incubation that was stable for up to 24 h. Storage of the cytosol at −80°C for 30 days had no adverse effect on the ability of the cytosol to specifically bind [ 3 H]-estradiol. The beagle uterine E 2 R was shown to be saturated at concentrations of 20 to 30 nM [ 3 H]-estradiol. Proteolytic enzyme treatment of the cytosol abolished [ 3 H]-estradiol binding suggesting that the receptor is a protein. A dissociation constant ( K D ) of 1.4 × 10 −9 M was obtained from Scatchard plot analysis. The uterine E 2 R binds specifically to the estrogenic steroids and to DES, with little affinity for progesterone, testosterone, hydrocortisone, or dexamethasone. The highest amount of specific binding of [ 3 H]-estradiol was observed in the reproductive tissues (uterus and oviduct), with little or no binding present in non-target tissues such as liver, kidney, diaphragm, heart, or serum. E 2 R levels were measured in cytosols from uteri of immature beagles treated with estradiol from silastic implants for 0, 4, 6, 10 or 14 days, or from a single injection. Uterine cytoplasmic E 2 R levels increased with time of exposure to estradiol. Thus, a cytoplasmic receptor for estradiol exists within the uterus of the immature beagle, the levels of which change depending upon the hormonal milieu within the animal.

Hormonal control of testicular protein synthesis in developing Tenebrio molitor

Abstract During differentiation, the testes of Tenebrio molitor were found to exhibit increases in biosynthetic capacity reflected by changes in the testicular protein content. A gradual increase in testicular protein content was observed during the prepupal stage. The observed increase was more dramatic in the pupal stage and reached its maximum level between days 4 and 7 of pupal development. During the adult stage, the biosynthetic processes for producing protein were apparently reduced following the first few days after adult emergence. The incorporation of radioactive leucine into TCA-precipitable testicular protein was not affected by the administration of exogenous juvenile hormone alone (JH I, 1 μg/animal) during the pupal stage. However, the administration of exogenous ecdysterone (0.5 μg/animal) to pupal T. molitor resulted in an increase in radioactive leucine incorporated into TCA-precipitable testicular proteins, particularly during the first five days after pupal ecdysis. Simultaneous administration of both JH and ecdysterone to mealworm pupae at specific ages indicated that no apparent interaction, synergistic or antagonistic, occurred between these two hormones with respect to [3H]-leucine incorporation. Furthermore, the amount of [3H]-leucine incorporated approximated to that obtained following injection of ecdysterone alone in all the pupal ages studied.

Analysis of progesterone receptor binding in the ovine uterus

The appropriate conditions for the measurement of ovine uterine cytoplasmic progesterone receptors (PR) have been determined to be 20 nM 3H-progesterone (3H-P4) with and without a 100-fold excess of non-radioactive progesterone (P4) 0-4 degrees C and 4 h of incubation. Under these conditions PR readily exchanged bound progesterone for progesterone added during the assay. This exchange occurred even when saturating concentrations of P4 were present. The progestins, R5020 and P4, effectively competed for the ovine uterine PR binding while non-progestin steroids and diethylstilbestrol failed to compete for the PR binding. The dissociation constant (Kd) measured for the 3H-P4 binding was 1.60 x 10-9 M indicating that the 3H-P4 binding was of high affinity. The levels of PR and the dissociation constant measured using 3H-R5020 in place of 3H-P4 were similar indicating a lack of corticosteroid binding globulin (CBG)-like binding in the ovine uterus.

Electrophoretic analysis of testicular protein components in developing Tenebrio molitor

Abstract The testicular protein products of prepupal, pupal and adult Tenebrio molitor were analyzed by gel electrophoresis. The 27 testicular protein components differed in their molecular weights and charge properties. Since these components were present at different developmental ages and persisted for distinct lengths of time, some of these proteins may be necessary for the formation of specific germ cell types during differentiation. In addition, a variety of these testicular proteins incorporated [ 3 H]-leucine at measurable levels throughout development, particularly during the pupal stage. 20-Hydroxyecdysone administration (1.5 μg per animal) stimulated the incorporation of [ 3 H]-leucine into specific testicular protein components that were not radioactively labelled when testes from non-treated animals or animals injected with a lower dose of 20-hydroxyecdysone (0.5 μg per animal) at the same age of normal development were analyzed. Thus, the higher dose of 20-hydroxyecdysone appeared to alter the testicular differentiation programme by enhancing the incorporation of [ 3 H]-leucine into not only the age-specific testicular proteins but also into new proteins not normally present at these specific ages.

Histochemical localization of 17β-hydroxysteroid dehydrogenase activity in rat uterus

Abstract The histochemical localization of 17β-hydroxysteroid dehydrogenase (17β-HSD) in the uteri of mature cycling rats and immature rats primed with estradiol-17β (E 2 ) was determined. Enzyme activity appeared localized within the glandular and luminal epithelial cells although some activity was also evident in the stromal cells. Uteri from proestrus, estrus, and metestrus rats possessed greater 17β-HSD activity than did uteri from rats at diestrus. The immature rat uterus lacked 17β-HSD activity, but activity was induced following three days of priming with E 2 . Administration of E 2 and progesterone to immature rats resulted in a slight depression of uterine 17β-HSD activity when compared to rats receiving E 2 alone. The localization of uterine 17β-HSD activity was dependent on the hormonal state of the animal, particularly on the presence of E 2 .

Nuclear progesterone receptors in the beagle uterus

Abstract Receptor-progesterone complexes in the uterine nuclei of adult beagles were demonstrated by a nuclear exchange assay using the synthetic progestin, promegestone (R5020). The radioactive R5020 ligand totally exchanged with previously bound progesterone when uterine muclei were incubated at 4°C for 6 h or 15°C for 2 h. The beagle uterine nuclear progesterone receptor was saturable with respect to the concentration of ligand exchanged, was highly specific for R5020 and progesterone, and bound R5020 with high affinity. In vivo injections of progesterone to an estrogen-primed, ovariectomized adult resulted in a rapid loss of cytoplasmic progesterone receptors with a concomitant increase in nuclear occupation by these receptors. Two forms of nuclear progesterone receptors were demonstrable following incubations of beagle uterine nuclei with 0.4–0.5 M KCl: a salt-extractable receptor, representing approximately 70% of total nuclear binding, and a salt-resistant receptor fraction which possessed a slightly lowered K d than observed for total nuclear binding. The significance of multiple nuclear progesterone receptors in the beagle uterus remains to be elucidated.