Uptake of three [3H]progestins by target tissues in vivo: Implications for the design of diagnostic imaging agents

We have investigated the tissue distribution of radioactivity for 0.5–4 h follwing the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [³H]progesterone shows minimal uterine uptake (<0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [³H]R 5020 (promegestrone) and [³H]ORG 2058 show highly selective uptake that reaches 4–5% ID/g by 1–3 h. The uterus to non-target tissue activity ratio at 2–4 h is approximately 12–20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.     

Effect of different estro-progesterone sequences on nidation of dormant blastocysts administered in diapause in ovariectomized rats]

The hormonal condition permitting delayed nidation in rats ovarectomized on day 4 or 5 of pregnancy was studied by administering progesterone and estradiol in differents sequences between days 9 and 14. The best result was obtained when progesterone was given 3 days before an estrogen-progesterone association. The same treatment induced delayed nidation in rats receiving Reserpine and ovariectomized day 5, but not when progesterone and estradiol were present day 4.     

Progesterone in the uterus. VI. On the question of the in vitro progesterone metabolism in human endometrium and myometrium

Human endometrial and myometrial tissue pieces were incubated with radioactively labeled progesterone in nutrient medium for 20 min., 1 hr and 2 hrs. The only compound extracted from the tissue pieces and the nutrient fluids was identified to be progesterone by TLC, chemical reactions and crystallization experiments. Radiometabolites could not be detected in the tissue pieces and in the nutrient fluids under the experimental conditions applied ( 10^?7 M 1,2-³H-progesterone in the incubation medium). This result is comparable with recent findings on the in vitro progesterone metabolism by rat uterine tissue.     

Metabolic pathways for androstanediol formation in immature rat testis microsomes

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [¹⁴C]progesterone and 17α-hydroxy[³H]progesterone resulted in a large preference of 17α-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [³H]progesterone together with [¹⁴C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low ¹⁴C/³H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17α-hydroxyl[³H]progesterone and [¹⁴C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17α-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.     

Role of prostaglandin F2α in modulation of LH-stimulated steroidogenesis in vitro in different types of rat and ewe corpora lutea

An inhibitory effect of PGF_2α at a dose of 7 × 10^?7 M on LH stimulated synthesis of progesterone was observed $\text{in vitro}$ after incubation of pseudopregnant rat ovaries for a period of 2 hours. A similar effect was seen with cyclic and gestant ewe corpora lutea at the same dose of PGF_2α. This effect was observed both in the secretion of progesterone and on the amount of progesterone present in the tissue. This inhibitory effect of PGF_2α on LH stimulated progesterone synthesis may explain the modification in the time course for gonadotropin action in luteal tissue at high and low doses.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

Hormonal regulation of collagen degradation in the uterus: Inhibition of collagenase expression by progesterone and cyclic AMP

Dibutyryl cyclic AMP markedly increases the ability of progesterone to prevent the expression of collagenase activity in cultures of post-partum rat uterus. Dibutyryl cyclic AMP itself and, to a lesser extent, native cyclic AMP, are capable of producing a partial decrease in enzyme activity, but complete abolition is not observed at high cyclic nucleotide concentrations (5 mM) in the culture medium. Theophylline, when added to cultures, mimics the effect of dibutyryl cyclic AMP. Other cyclic nucleotides were without effect on levels of collagenase activity in the uterine cultures.When non-inhibitory concentrations of either dibutyryl cyclic AMP (1 · 10^?4 M) or theophylline (1 · 10^?4 M) are added to cultures together with a non-inhibitory concentration of either progesterone (5 · 10^?6 M) or the potent progesterone analogue Provera (1 · 10^?8 M) the ability of the tissue to produce collagenase is decreased by 40–70%. Collagenase activity is consistently diminished more than additively by combinations of steroid and cyclic nucleotide. Theophylline mimics the effect of dibutyryl cyclic AMP on steroid activity in culture. In the presence of dibutyryl cyclic AMP, diminution of collagenase activity can be observed at concentrations of steroid more than two orders of magnitude lower than the normal minimally inhibitory dose. Reduction of collagenase activity is reflected in all experiments by a concomitant decrease in the normal proteolytic degradation of collagen in the tissue ex-plants. The possibility that progesterone acts in the uterus to raise cyclic AMP levels is suggested by the fact that uterine tissue, when cultured in the presence of progesterone, contains reduced levels of cyclic nucleotide phosphodiesterase.These data suggest that, in some way a cyclic AMP-mediated system is critically involved in the control of collagenase activity by progesterone in the rat uterus.     

Ultrastructural changes in granulosa lutein cells and progesterone levels during preimplantation,implantation, and early placentation in the western spotted skunk

Summary The ultrastructure of corpora lutea obtained during the preimplantation, implantation and early postimplantation periods has been studied in 20 western spotted skunks. Fine structure of granulosa lutein cells was correlated with progesterone levels. The corpus luteum of the prolonged (7 month) preimplantation period contained undifferentiated small granulosa cells and differentiated large granulosa lutein cells. The former ranged in size between 12 and 20 and the latter between 20 and 45 . The ratio of small and large cells was about equal in an animal 2 days prior to nidation whereas only few small cells and numerous large cells were observed in an animal estimated to be 8 to 12 hours from nidation. Occasionally small cells were observed amidst large ones during the 24 hour nidation period, i.e. adhesion of trophoblast with the luminal uterine epithelium, but small cells were absent in animals after this period. Small cells had some smooth and rough endoplasmic reticulum, rod-shaped mitochondria with platelike cristae, small Golgi complex, and relatively smooth plasma membranes. Large lutein cells had abundant smooth endoplasmic reticulum, membranous whorls of smooth endoplasmic reticulum, usually round mitochondria with tubular and lamellar cristae, a well developed Golgi complex, variable amounts of lipid droplets, and highly plicated and ruffled plasma membranes. Peripheral plasma progesterone levels during the prolonged preimplantation period ranged between 1.1 and 7.9 ng/ml, but during implantation it was between 8 and 16.6 ng/ml. It is suggested that plasma progesterone levels fluctuate during the time of implantation and should not be regarded as a basis to predict actual nidation in the western spotted skunk.This research was supported in part by Grant Number HD06556 from the National Institute of Child Health and Human Development.     

Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

In vitro accumulation and saturation of 3H-progestins in selected brain regions and in the adenohypophysis, uterus and pineal of the female rat

Tissue samples from adult ovariectomized female rats were bisected, weighed and incubated in media containing ³H-progesterone with or without additional unlabeled progesterone. In brain tissue incubated without unlabeled progesterone samples from the interpeduncular region of the mesencephalon accumulated more ³H-progestins than samples from occipital cortex, posterior and anterior hypothalamus and medial-dorsal hippocampus. Within this latter group of tissues uptake was approximately equal. Adenohypophysial uptake in media without unlabeled progesterone was similar to that of cortex, while uterine uptake was less and pineal uptake more than that of cortical samples. Unlabeled progesterone reduced ³H-progestin accumulation in the interpeduncular, hippocampal, pituitary and uterine samples.     

Source of Ovarian Preovulatory Progesterone

AN increase in plasma progesterone has been detected before ovulation in several species. There is, however, no definitive information as to which ovarian compartments produce this progesterone. Previous results with the golden hamster have demonstrated a preovulatory progesterone surge after the critical period for gonadotrophin release on the afternoon of pro-oestrus (day 4)^1,2. This is paralleled by the development of 3β-hydroxysteroid dehydrogenase (3βHSD) activity in the theca interna and granulosa of pre-ovulatory follicles³, suggesting they could be a source of preovulatory progesterone. As the interstitium also contains 3βHSD activity, it could be an additional source of progesterone³. Hamster corpora lutea, however, have negligible 3βHSD activity during pro-oestrus³ and are not capable of producing progesterone when isolated and incubated in vitro (Leavitt et al., unpublished work).     

Uptake and release of H prostaglandins E2 and F2α in uterin strips isolated from ovariectomized rats. Influence of progesterone

The accumulation and output of ³H -prostaglandins (PGs), E₂ and F₂α, into and from uterine strips isolated from ovariectomized rats, either in presence or in absence of exogenous progesterone, were explored. Tissue-to-medium ratio of ³H - counts (T/M-ratio), was determined. The same was done in solutions containing ¹⁴C-sucrose. During a 60 min incubation period in a solution containing ³H -PGF₂α, a net accumulation of radioactivity was evident in control (no progesterone) uterine slices. The T/M-ratio for ³H-PGF₂α, increased with time, reaching maximal values at 45 min. Progesterone (100 ug.ml⁻¹) attenuated the uptake process, as evidenced by stable values of T/M-ratio, as time progressed. On the other hand, control T/M-ratio for fluenced by the presence of exogenous progesterone. Regarding labelled PG release from the tissue, it was observed that, during an experimental period of 60 min, most tritium from control slices was released within the first 30 min after incubation with ³H -PGF₂α, whereas, following the presence and subsequent removal of exogenous progesterone, the bulk of ³H -released took place at 6–70 min. On the other hand, the release of ³H after an incubation with ³H -PGE₂, was also maximal as that for ³H -PGF₂, α within the first 30 min and resulted not altered after a period of exposure and removal of progesterone. The foregoing results suggest an specific pharmacological effect of progesterone, attenuating the uptake and retarding the outflow of PGF₂α, but not that of PGE₂, into and from uterine slices of ovariectomized rats. Findings reported herein are discussed in terms of progesterone priming and withdrawal, in relation to PGF₂α fluxes in the rat uterus during the sex cycle, as well as in relation to PG binding to tissue receptors.     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

Relative Binding Affinity of Antiprogestins Zk 98.299 And Zk 98.734 For Progesterone Receptors in the Endometrium and Myometrium of Bonnet Monkeys

Abstract

Progesterone bound with high affinity to the endometrial and myometrial cytosol of ovariectomized bonnet monkeys pretreated with estradiol benzoate and progesterone. The equilibrium dissociation constant (K_d) of ³H-progesterone was 4.5 nM and 5.5 nM and the binding capacity was 1.7 nM and 1.4 nM for the endometrial and myometrial receptors, respectively. This experimental ‘model’ was used to assess the relative binding affinity (RBA) of progesterone, ZK 98.299 and ZK 98.734. The tested compounds showed competitive binding to cytoplasmic progesterone receptors. The RBF of progesterone in the endometrium (100) was more than that of ZK 98.299 (25.1) and ZK 98.734 (17.8). A similar RBA pattern was observed in the myometrial cytosol. Both ZK 98.299 and ZK 98.734, like progesterone, displaced the ³H-progesterone bound to the receptors. The administration of ZK 98.299 or ZK 98.734, during the mid-luteal phase, has been reported to shorten the cycle length in bonnet monkeys and marmosets, respectively. These compounds, therefore, appear to intercept the progesterone action by blocking progesterone binding sites in the target tissue. Since ZK 98.299 has higher binding affinity than ZK 98.734, it may be a more potent progesterone antagonist.     

Corpus luteum size and plasma progesterone concentration in cows

It is often assumed that a larger corpus luteum will produce more progesterone and generate higher circulating plasma concentrations. The aim of the study was to determine whether the size of the corpus luteum does actually determine circulating plasma progesterone concentrations. Data were collated from a number of studies on various aspects of luteal function in non-lactating dairy cows to allow comparisons to be made between corpus luteum weight and plasma progesterone concentration across the luteal phase. In these studies oestrous cycles had been synchronised and animals slaughtered on day 5, day 8 or day 16 following oestrus. Both corpus luteum weight and plasma progesterone concentration increased between day 5 and day 8. Plasma progesterone concentration but not luteal weight also increased between day 8 and day 16. On day 5 there was a strong relationship between corpus luteum weight and plasma progesterone (R² = 0.64; P < 0.001). However, no such relationship was present on day 8 or day 16. These results indicate that while during the early stage of corpus luteum development a relationship between size and progesterone is present, by day 8 of the cycle, the size of the corpus luteum is no longer of importance in determining circulating progesterone concentrations.     

Rapid block of gonadotropin uptake by corpora lutea in vivo induced by prostaglandin F2α

Intravenous administration of ¹²⁵I-hCG to 7–8 day pseudopregnant rats resulted in maximum uptake of radioactivity to corpora lutea 2 hours after treatment. At this time tissue/plasma radioactivity ratios on an equal weight basis were: corpora lutea, 70.2 ± 12.8; ovarian interstitium, 4.6 ± 0.2; kidney, 2.2 ± 0.1. No appreciable uptake was seen by adrenals or liver. Radioactivity in corpora lutea was associated primarily with membranes which sedimented at 2000g and when released by heat it was more than 63% bound to luteal LH receptor preparation in vitro. Radioactivity in renal tissue was associated primarily with the 100,000g supernatant fraction and was bound less than 1% to luteal LH receptors in vitro.PGF₂α significantly reduced uptake (p<.001) of ¹²⁵I-hCG by corpora lutea within 30 minutes (?63%) as well as at 1 (?64%), 2 (?75%), 4 (?68%) and 24 hours (?85%). No clear effect of PGF₂α on uptake of ¹²⁵I-hCG by ovarian interstitial tissue was seen. Plasma progesterone was significantly decreased (p<.001) within 30 minutes (?47%; p<.01) after PGF₂α treatment and also at 1 (?65%), 2 (?82%), 4 (?68%) and 24 hours (?92%). Two hours after PGF₂α treatment the content of progesterone in corpora lutea was depressed (?46%; p<.001). It is suggested that the rapid inhibition of luteal progesterone production induced by PGF₂α in vivo occurs through a block in gonadotropin uptake by corpora lutea.     

Progesterone receptor in the rat anterior pituitary: Effect of estrogen priming and adrenalectomy

The present study was done to determine if a progesterone receptor is present in rat pituitary. Cytosol was labeled with ³H-progesterone (3HP) or ³H-RS020 (³HR) and subjected to sucrose-glycerol density-gradient centrifugation. Serum progesterone was measured for correlation with progesterone receptor levels. Two ³HP-binding peaks (4S + 6S) were evident in uterine and pituitary cytosols. The 4S peak was eliminated by competition with unlabeled cortisol leaving a single 6S peak (progesterone receptor). Estradiol (E) priming of the male or female rat increased progesterone receptor levels in pituitary cytosol as demonstrated using ³HP and ³HR, and pituitary progesterone receptor bound ³HR with a higher affinity than ³HP. Following adrenalectomy of gonadectomized rats, progesterone receptor levels were increased in pituitary and uterine cytosol of both E-primed and unprimed groups. An inverse relationship was established between serum progesterone and progesterone receptor levels in the uterus and pituitary suggesting that stressinduced adrenal progesterone secretion significantly influences progesterone receptor levels in the rat. These results demonstrate an estrogen-inducible progesterone receptor in the rat pituitary with properties similar to those of the uterine progesterone receptor.