Species and tissue distribution specificity of proteins binding 16alpha,17alpha-cycloalkane derivatives of progesterone

The binding of [3H]progesterone and [3H] 16 alpha,17 alpha-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16 alpha,17 alpha-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H] 16 alpha,17 alpha-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16 alpha,17 alpha-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17 beta-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.     

A competitive radioligand assay for danazol (17 alpha-pregn-4-en-20-yno(2,3-d)isoxazol-17-ol) using pregnant guinea pig plasma

A method is described for the radioligand assay of circulating levels of a novel, pituitary gonadotrophin inhibiting agent, danazol (17α-Pregn-4-en-20-yno[2,3-d]isoxazol-17-ol). The assay described is based upon the ability of danazol to bind avidly to pregnant guinea pig plasma proteins, a binding system hitherto considered highly specific for progesterone. Danazol is separated from progesterone and other possible interfering substances by paper chromatography. The method was used to measure danazol in a series of normal human subjects given the drug at 800 mg/day.     

Direct radioimmunoassay of progesterone in rat serum

A direct method has been described which makes possible a specific assay of progesterone in rat serum without extraction. Anti-progesterone serum was prepared in our laboratory by the immunization of three rabbits with 4-pregnen-3, 20-dione-3 CMO:BSA. This antiserum (Gunma OGP#1) displayed little or no cross reaction with 20 alpha-dihydroprogesterone (0.38%), pregnenolone (0.44%), 17 alpha hydroxypregnenolone (less than 0.1%), 20 beta hydroxyprogesterone (2.4%), 17 alpha hydroxyprogesterone (2.88%) or deoxycorticosterone (2.19%). The nonspecific inhibitory effect of serum was compensated for by adding progesterone-free serum to the standard curve tubes. The sensitivity of this assay was 1.1 pg/tube and serum progesterone could be measured by using as little as 1 microliter of serum. The working range of the standard curve was 1.25-2560 ng/ml. Under the conditions of this assay (1 microliter of serum per tube), interference from steroid binding proteins did not affect the sensitivity, precision or reliability of the assay. The intra-assay and inter-assay coefficients of variation were 5.5% and 8.7%, respectively, and the assay values correlated well with those obtained by the extraction method (R = 0.997, P less than 0.001). Analytical recovery indicates a close correlation between added and recovered progesterone concentrations (R = 0.992, P less than 0.001), and the recovery rate averaged 96%. Compared with the extraction method, the direct progesterone assay has the advantage of speed, precision and simplicity. The method described is particularly suitable for routine assays of progesterone in rat serum.     

Concentrations of progesterone,testosterone and estradiol-17β in the serum during the estrous cycle of Asian elephants (Elephas maximus)

The levels of progesterone, testosterone and estradiol-17β in serum samples from two female Asian elephants were measured for the period of 32 months from February 1987 to September 1989. Serum samples were collected weekly from unanesthetized elephants. Each elephant showed eight ovarian cycles in 32 months. Ovarian cycles, characterized by changes in concentrations of serum progesterone, averaged 16.8 ± 0.6 (mean ± SEM. n = 14) weeks in length. The changes in concentrations of testosterone in the serum showed a similar pattern to those of progesterone with a striking increase noted during the luteal phase. The highest levels of serum estradiol-17β were noted when progesterone levels showed low basal values. These results suggest that estradiol-17β may be an index of follicular maturation during the estrous cycle in Asian elephants, and that the ovaries of Asian elephants may produce testosterone in the luteal phase.     

Effect of oestradiol-17 beta on ovarian and serum concentrations of relaxin during the second half of pregnancy in the rat

Immunoactivity concentrations of ovarian relaxin, serum relaxin and serum progesterone were determined from Day 12 through Day 18 of pregnancy in rats treated with oil or oestradiol-17 beta after hysterectomy or hypophysectomy and hysterectomy on Day 12. Relaxin and progesterone concentrations increased between Days 12 and 18 in sham-operated rats but failed to increase or declined in oil-treated hysterectomized or hypophysectomized-hysterectomized animals. Oestradiol treatment increased serum concentrations of relaxin and progesterone in hypophysectomized-hysterectomized rats on Day 15 and increased the concentrations of ovarian and serum relaxin and serum progesterone in hysterectomized rats on Day 18. These data are consistent with the concept that placental support for the promotion and maintenance of relaxin and progesterone concentrations from Day 12 through Day 18 may be mediated, at least in part, through a common mechanism(s) which involves oestradiol.     

The influence of estradiol-17beta and progesterone on peripheral serum concentrations of luteinizing hormone and follicle stimulating hormone in the ovariectomized ewe

Serum gonadotropin concentrations were high and variable and fluctuated episodically in short and long term ovariectomized ewes. Treatment with solid silastic implants releasing progesterone (serum levels 1.81 +/- 0.16 ng/ml) had no consistent effect. Treatment with implants releasing estradiol-17beta significantly depressed mean serum gonadotropin concentrations and peak height to values usually seen in intact ewes. This occurred regardless of implant size and serum estradiol-17beta concentrations (range 11 +/- 0.3 pg/ml to 98 +/- 12.8 pg/ml). Progesterone and estradiol-17beta together significantly depressed the frequency of peaks in LH concentration. Following progesterone removal, 95% of the ewes treated with progesterone and estradiol-17beta implants experienced a transient increase in serum LH concentrations similar to the preovulatory surge in intact ewes. Eighty-four percent of the LH surges were accompanied by a surge in serum FSH concentrations. However, following progesterone removal, 5.1 +/- 2.1 FSH surges were observed over six days. Gonadotropin surges occurred regardless of estradiol-17beta implant size and with or without the influence of supplemental estradiol-17beta.     

3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives: synthesis and interactions with progesterone receptor and other proteins

Series of 3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives (pregna-d'-pentaranes) have been synthesized with the aim of probing the surfaces of progesterone receptor's and two other protein ligand binding pockets neighboring to 3- and 19-positions of steroid core. The same derivatives were also studied as possible intermediates for attachment to matrixes. The data on affinity constants suggest the presence of hydrophobic cavities with hydrophilic necks in the progesterone receptor and serum pentaranophylin near C19 of bound ligand and the lack of such a cavity in uterine pentaranophylin. Any of 3-oxime substitutions were found to significantly diminish the ligand affinity for the progesterone receptor. It was also found that some of these modifications, in the Z-configuration particularly, might increase the affinity for serum and uterine pentaranophylins. The latter finding suggests the presence of large cavities near C3 of bound ligand in these proteins and interchangeability between 3-keto and 3-oxime groups in ligand-protein interactions.     

Steroidogenic effect of 17 beta-estradiol on rabbit luteal cells in vitro: estrogen-induced maintenance of progesterone production

Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Inhibition of ovulation with 17-alpha-ethinyl-18-methyl-11-methylene-4-estren-17-ol plus ethinylestradiol]

A combined oral application of 30 micrograms ethinylestradiol with 50 micrograms 17 alpha-ethinyl-18-methyl-11-methylene-4-estren-17-ol (a newly developed progestagen) -- for 21 days resulted in a distinct antigonadotrophic effect, shown by hormone analysis (LH, FSH, estradiol and progesterone) in serum samples of three healthy and fertile women. A final recommendation on the daily dosage, however, depends on the results of clinical experimental investigation still in progress.     

Nature and characteristics of the binding of oestradiol-17beta to a uterine macromolecular fraction

The binding of oestradiol-17β to two proteins, namely serum albumin and a uterus fraction, was studied in vitro. The former protein has a physiological function in the transport of the hormone and the latter is involved in the selective uptake of the steroid by the target organ. The uterus fraction shows a high degree of stereospecificity for the binding of the steroid. Cortisone, oestradiol-17α and testosterone are bound negligibly and progesterone to a much smaller extent than is oestradiol-17β. This property is in contrast with the wide variety of ligands bound by the serum albumin. The temperature and the presence of the steroid influence markedly the binding properties. Oestradiol binding to the uterus fraction is optimum at 37° and at pH7–8·5. It is markedly decreased at pH values above or below this range, suggesting stringent conformational requirements. The tissue `receptor' protein is a macromolecule with a minimum molecular weight of 100000. The protein moiety is essential for the binding function. The probable concentration of the total binding sites for oestradiol in the ovariectomized-rat uterus cytoplasmic fraction as determined in vitro is about 1mμm at a steroid concentration of 50mμm.     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.     

Quantitative changes in ovarian 17 alpha-hydroxylase/C17,20-lyase and aromatase activities during the estrous cycle of the hamster

Aromatase activity of the microsomal fraction of ovarian homogenates, measured by a tritium exchange assay using androstenedione (A-dione) as substrate, did not change during the 4-day estrous cycle of the hamster. In contrast, 17 alpha-hydroxylase, measured by a tritium exchange assay with progesterone as substrate, and particularly C17,20-lyase activity, evaluated by acetic acid production from progesterone, drastically decreased on the afternoon of proestrus (Day 4). The latter two activities remained low on Days 1 and 2 but increased dramatically on Day 3 and the morning of Day 4. The serum concentration of A-dione and 17-hydroxyprogesterone reached a peak at 1600 hr of proestrus and then decreased rapidly as the enzyme activities decreased. A-dione levels were undetectable on Day 1 but 17-hydroxyprogesterone levels remained elevated through Day 2. The results are consistent with the view that 17 alpha-hydroxylase and C17,20-lyase are activities of a single cytochrome P-450, as has been shown for testis and adrenal. Increases in hydroxylase and lyase activities occur concomitantly with decreases in the serum concentrations of progesterone, suggesting that the latter steroid may play a role in controlling these enzymes.     

Pentacyclic steroid analogs: interaction of 16 alpha,17 alpha-cyclopentanoprogesterone with rat uterus proteins

Using 3H-labeled derivatives, kinetic parameters of specific binding of progesterone (I) and 16 alpha, 17 alpha-cyclopentanoprogesterone (II) to proteins of the uterus soluble fraction in rats were measured. It was shown that their affinities to proteins are comparable (K 10.5 +/- 2.4 and 6.7 +/- 3.4 nM for (I) and (II), respectively, upon 22 h incubation). The unlabeled compound (II) can displace [3H]progesterone from complexes with the protein with a concentration-independent efficiency corresponding to the ratio of K values for compounds (I) and (II). At the same time, the efficiency of the unlabeled progesterone in the displacement of [3H]compound (II) from protein complexes fell with an increase in the progesterone concentration. The concentration of high-affinity sites of [3H]compound (II) exceeded by 1.5 to 2 times the concentration of sites for [3H]progesterone. Dynamics of dissociation of proteins complexes of [3H]progesterone and 3H]compound (II) had a two-phase character with a decrease in the dissociation rate constants for both phases as the times of exposition of [3H]ligands to proteins grew. The ratio of slow- and fast-dissociating ligand-receptor complexes was thereby unchanged. These data suggest the presence in the rat uterus soluble fraction of two types of proteins differing in the capacity to recognize the additional five-membered ring D' in the steroid molecule.     

Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins

Competition of a number of progesterone 16alpha,17alpha-cycloalkane derivatives with 3H-labeled ligands for the binding sites of the rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16alpha,17alpha-cyclopropanoprogesterone, and 16alpha,17alpha-cyclopent-3'-enoprogesterone and the selective ligands for serum pentaranophilin are 6alpha-methyl-16alpha,17alpha-cyclohexanopregna-1,4-diene-3,20-dione and 3beta-hydroxy-16alpha,17alpha-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D' we studied decreased the affinity of ligands for all the three proteins. The substitution of the delta5-3beta-hydroxy grouping for the delta4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3',4'-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.