The metabolism of 19-nor contraceptive progestins modulates their biological activity at the neuroendocrine level

In this communication, a series of studies from our laboratory dealing with the mechanism of action of 17 alpha-ethinyl derivatives of 19-nor testosterone are reviewed. The administration of norethisterone (NET) to long-term castrated female rats induces the nuclear translocation of pituitary estradiol receptors and is followed by some estrogenic-like effects at the hypothalamic-pituitary unit. It is established that an A-ring reduced metabolite of NET, the 3 beta,5 alpha-tetrahydro NET derivative, is responsible for the observed in vivo estrogenic effects of the parent compound. 3 beta,5 alpha-NET binds to the estrogen receptor and is efficient in inducing the pituitary estrogen-dependent progesterone receptor and in increasing the uterine weight in long-term castrated rats. Furthermore, administration of 3 beta,5 alpha-NET and the 5 alpha-reduced metabolite of NET (5 alpha-NET) are able to inhibit the release of gonadotropins in the castrated animal to a greater extent than NET. Moreover, pretreatment with tamoxifen, an estrogen binding site competitor, results in a significant diminution of the antigonadotropic potency of 3 beta,5 alpha-NET but not of the 5 alpha-NET, which is only inhibited by the administration of cyproterone acetate. These findings underline the importance of the metabolic rate of NET for the expression of its biological effects at the hypothalamic-pituitary unit.     

Ab initio electronic structure of the progestogen norethisterone and its 5 alpha-derivatives

The steroid 17 alpha-ethynyl-19-nor-4-androsten-17 beta-ol, 3-one (Norethisterone; NET) and its 5 alpha-dihydro (5 alpha-NET), 3 alpha- and 3 beta-tetrahydro derivatives (3 alpha,5 alpha- and 3 beta,5 alpha-NET), were comparatively studied by the ab initio quantum mechanics theory. Additionally, 5 alpha-androstan-3 beta,17 beta-diol (ADIOL) was also studied. The Hartree-Fock method and the 6-31G(*) basis set were used to obtain the lowest energy conformation, geometries, electronic structure and physicochemical properties of the steroids. The results showed bond distances and valence angles similar among all steroids, but some differences in dihedral angles in the A-B-ring system were observed. The electronic structure analysis showed that NET has both frontier orbitals that is, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) located at the C4-C5 pi-bond. In A-ring reduced derivatives, the HOMO was found at the 17 beta-OH and ethynyl groups. In the case of 5 alpha-NET, the LUMO was confined to the A-ring and its C3 carbonyl group while the two NET tetrahydro-reduced derivatives showed the LUMO at the 17 beta-OH and ethynyl groups. The energy changes of the rotational barrier of the 17 beta-OH group suggest that its movement is somewhat restricted by the 17 alpha-ethynyl group. Interestingly both groups at C17 form a single electrostatic potential with high electronic density. On the other side, the 19-nor condition increases the A-ring mobility. However, the 3 beta-OH group of 3 beta,5 alpha-NET may rotate without significant energy differences as compared to the same group in ADIOL. The electronic structure of NET and its A-ring reduced derivatives explains in some extent their interaction with androgen and progesterone receptors as well as their selectivity for the estrogen alpha-receptor.     

Stereospecificity of the intracellular binding of norethisterone and its A-ring reduced metabolites

The interaction of norethisterone (NET) and four A-ring reduced metabolites of NET with cytosol receptors for progesterone (PR), androgen (AR), and estrogen (ER) was investigated. Cytosol preparations from: uteri of adult estrogen-primed castrated rats, ventral prostates of adult castrated rats and uteri of immature rats were used as the source of PR, AR, and ER respectively. 3H-Labeled ORG-2058, R-1881, and 17 beta-estradiol were used as the radioligands. The results of competitive studies disclosed that: the most efficient competitor for PR binding sites was NET (Ki = 1.1 X 10(-7) M) followed by 5 alpha-dihydro NET (5 alpha-NET), whereas the 3 alpha,5 alpha; 3 beta,5 alpha and 3 alpha,5 beta-tetrahydro NET derivatives were ineffective the most efficient competitor for AR binding sites was 5 alpha-NET (Ki = 1 X 10(-8), immediately followed by NET, while the three tetrahydro NET derivatives were not competitors and remarkable competition for ER binding sites was only exhibited by the 3 beta,5 alpha-tetrahydro NET derivative (Ki = 4.6 X 10(-8) M) and to a lesser extent by its 3 alpha,5 alpha-epimeric alcohol, while NET and 5 alpha-NET were completely ineffective. These findings demonstrate the stereospecificity of the intracellular binding of NET and its reduced metabolites with cytosol steroid putative receptors, and provide biochemical support to the understanding of the variety of hormone-like effects observed after the in vivo administration of NET.     

Further studies on the antigonadotropic mechanism of action of norethisterone.

To examine the molecular mechanisms involved in the antigonadotropic effects of norethisterone (NET) and two of its A-ring reduced metabolites the 5 alpha-norethisterone (5 alpha-NET) and the 3 beta, 5 alpha-norethisterone (3 beta, 5 alpha-NET) at the neuroendocrine level, a series of experiments were undertaken in adult castrated rats. Animals were primed either with 0.2 mg of tamoxifen (Tam) for 4 consecutive days or 1.0 mg of cyproterone acetate (CPA) for 7 days followed by a single subcutaneous injection of 0.5 mg of NET, 5 alpha-NET or 3 beta, 5 alpha-NET. Four hours later, they were sacrificed and blood obtained for the measurement of immunoreactive serum LH and FSH. The results indicated that antiestrogen (Tam) pretreatment precluded the inhibitory effects of NET and the 3 beta, 5 alpha-NET but not those of the 5 alpha-NET derivative. Pretreatment with CPA did not modified the antigonadotropic action of the 3 beta, 5 alpha-NET metabolite but it markedly reduced the inhibitory action of the 5 alpha-NET, thus indicating that in the experimental model used, the antigonadotropic effects of NET, are in part the result of its metabolic conversion to its A-ring reduced metabolites. While the 5 alpha-NET displayed an androgenic effect, the 3 beta, 5 alpha-NET exhibited estrogen-like effect at the neuroendocrine level.     

Hormonal control of uteroglobin secretion in rabbit uterus. Inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5alpha-dihydrotestosterone, either alone or concomitantly with oestradiol-17beta and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)-cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5alpha-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17beta (50mug/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5alpha-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17beta or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17beta (50mug/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.     

Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors

Neutral reduced metabolites of norethisterone (NET) specifically interact with intracellular estrogen receptors in target organs. To determine if this interaction can effectively initiate estrogen-dependent cellular responses, the effects of an A-ring-reduced NET derivative upon the induction of cytosol-located pituitary progestin receptors (PR) and uterine growth were studied in adult castrated female rats. Different doses of 17 alpha-ethynyl-5 alpha-estran-3 beta, 17 beta-diol (3 beta, 5 alpha-NET) were s.c. administered to ovariectomized animals for 6 days. 17 beta-Estradiol (E2) and oil-treated rats served as experimental controls. Pituitary PR were labeled in vitro by a post-gradient technique using [3H]ORG-2058 as the ligand. PR binding specificity was determined by the use of an excess of radioinert steroids. The results demonstrated that administration of 3 beta, 5 alpha-NET induced specific 8-9S pituitary cytosol PR in a dose-dependent manner. Binding properties of the 3 beta, 5 alpha-NET-induced progestin binding sites (Kd = 1.0 X 10(-9) M; NBS = 1.2 X 10(-9) M) appear indistinguishable from those induced by E2. In addition, 3 beta, 5 alpha-NET administration resulted in a significant increase in uterine weight at the expense of myometrium and endometrium growth in a similar fashion to that observed in the E2-treated group. When 3 alpha, 5 alpha-epimeric alcohol (3 alpha, 5 alpha-NET) was administered, induction of pituitary PR and uterine growth were also observed although to a lesser extent. Inasmuch as the results demonstrate that neutral non-aromatizable NET metabolites induce biochemical and morphological estrogenic responses, they offer an alternative explanation for the mechanism of estrogen-like action of this synthetic contraceptive progestin.     

Mechanisms of hormonal and antihormonal action of contraceptive progestins at the molecular level

19-Nor synthetic progestins undergo extensive metabolism at the target cells. The resulting metabolic conversion products interact with putative steroid receptors within the cells, and through those interactions, they may exert either agonistic, synergistic and antagonistic hormonal effects. Studies conducted in our laboratories have disclosed that norethisterone (NET) and D-(l) norgestrel (LNG), two widely used contraceptive progestins, are biotransformed to several A-ring reduced (dihydro and tetrahydro) derivatives. The resulting metabolites 5-dihydro NET (5-NET) and 5-dihydro LNG bind with relative high affinity to the progesterone and androgen receptors. To gain insight into the underlying molecular events mediating the mode of action of NET and its neutral metabolites, we have examined the expression of their biological effects at target organs by using the rabbit uteroglobin gene model and the ß-glucuronidase activity of the mouse kidney. The results of a series of experiments seem to indicate that the enzyme-mediated formation of the 5 (trans A/B ring junction) NET derivative results in a significant diminution of its progestational and androgenic potencies. Furthermore, 5-NET acquire a potent anti-progestational/ contragestational effect as assessed in the female rabbit. These results demonstrated that 5-reduction of 19-nor progestins exerts a paradoxical effect, at least in terms of their hormone-like effects. The overall data are in line with the concept that metabolism of synthetic progestins at hormone-sensitive organs modulates their mechanisms of action.     

Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its a-ring reduced metabolites

Norethisterone (NET) has been used as a contragestational postcoital agent. It is biotrans-formed to 5α dihydro-NET (5α-NET) and 3β,5α tetrahydro-NET (3β,5α-NET) in target tissues. The participation of these metabolites in NET effects is unknown. We have examined the antiimplantation and antiprogestational effects of NET and its metabolites, in adult mated female rabbits, by assessing the number of implantation sites and the expression products of the uteroglobin (UTG) gene in the uterus, and by comparing them with those of RU-486 and estradiol. Steroids were daily administered s.c. at several doses for 7 consecutive days, starting 24 hr after coitus. To assure that fertilization occurred in all animals, the presence of early pregnancy factor was determined. The results demonstrated that high doses (5 mg/kg) of NET reduced both implantation and the expression of the UTG gene. On the other hand, lower doses (1.5 mg/kg) of 5α-NET produced an antiimplantation effect and suppressed UTG synthesis and its mRNA. These effects were similar to those of RU-486. At lower doses (1 mg/kg), both estradiol and the estrogenic metabolite 3β,5α-NET were also effective in inhibiting implantation and UTG gene expression. The overall results suggest that NET metabolites exert antiimplantation and antiprogestational effects through their interaction with progesterone and estrogen receptors, and provide an explanation for the molecular mechanisms involved in the postcoital contraceptive action of NET. © 1995 Wiley-Liss, Inc.     

Prolactin enhances uteroglobin gene expression by uteri of immature rabbits

The effect of prolactin on uteroglobin production by immature rabbits was studied with neonatal (1 day old) and juvenile (14 days old) does. The animals were divided into 11 treatment groups for each age category and exposed to a 9-day injection protocol. Each day the animals received a subcutaneous injection of oestradiol-17 beta and/or ovine prolactin and/or progesterone, or were sham-injected. Juvenile animals, which received 100 micrograms oestradiol/kg 24 h-1, plus progesterone or plus prolactin and progesterone, produced detectable amounts of uteroglobin in the uterine secretions (0.034 +/- 0.010 mg uteroglobin/mg total protein and 0.098 +/- 0.03 l mg uteroglobin/mg total protein, respectively). None of the animals in the other juvenile treatment groups or any of the neonatal groups produced uteroglobin. From this survey it was apparent that uteroglobin secretion could be induced by exogenous oestradiol and progesterone in rabbits treated as early as 14 days of age, and that the added supplementation of prolactin enhanced the response to the ovarian steroids. As a result, additional juvenile animals were injected with 100 micrograms oestradiol +/- prolactin + progesterone and the effects of these two treatments were quantitated as follows: uteroglobin mRNA levels by slot-blot hybridization; endometrial surface area by computerized image analysis; and oestrogen, progesterone and prolactin receptors by immunocytochemistry. Prolactin modified the response of the juvenile rabbit uterus to oestradiol + progesterone for all parameters tested.     

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.     

Isolation of a plasma protein observed in patients with essential hypertension

Daily progesterone administration (1.33 mg/kg body weight) to immature rabbits brought about an initial increase in the uterine content of uteroglobin which, however, subsided when progesterone treatment was continued for 10 days. During this treatment period progesterone did not lose its own uterine receptors nor did it lose its inhibitory effect on the accumulation of occupied nuclear estrogen receptors. Since immature rabbits were used, the decrease of uteroglobin concentration cannot be explained by inhibitory effects of endogenous estrogens. The results suggest that termination of uteroglobin secretion may be a selective and inherent effect of progesterone itself.     

Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit

The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0–5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1–4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4–5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period. © 1993 Wiley-Liss, Inc.     

Servomechanism of prolactin and progesterone in regulating uterine gene expression

To investigate the interaction of PRL and progesterone in regulating uterine gene expression, we have quantitated the concentration of PRL receptor and of uteroglobin (UG) mRNA in the endometrium of rabbits of different ages and after treatment with different hormones. During uterine differentiation in 2- to 4-week old rabbits, a marked increase in unoccupied uterine PRL receptor number was observed, presumably increasing uterine sensitivity to PRL. Receptor values for 4-week old rabbits were comparable to values for sexually mature, estrous females, but were lower than in 5-day pseudopregnant (PSP) animals. When total PRL receptor was determined by Scatchard analysis after in vitro desaturation with MgCl2, PSP animals again expressed the highest receptor concentration with no changes in the dissociation constant (Kd) values. To determine whether progesterone regulates uterine PRL receptor, long term ovariectomized rabbits (greater than 12 weeks) were treated with various combinations of hormones, and unoccupied and total uterine PRL receptors were determined. Progesterone treatment resulted in the highest concentration of both unoccupied and total PRL receptor after desaturation and removal of anti-ovine PRL antibodies with MgCl2. The value for total uterine PRL receptor was equivalent to the value for mammary gland, and the Kd values (2-4 x 10(-10) M) were similar. Treatment of long term ovariectomized rabbits with progesterone, with or without estradiol, produced an increase (P less than 0.05) in the UG mRNA content, which also occurred in PSP animals. PRL alone had no effect on UG mRNA but PRL plus progesterone increased (P less than 0.05) UG mRNA in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

Variability in the response of the rabbit uterus to progesterone as influenced by prolactin

Ovariectomized rabbits from different breeders were treated at different times of the year with prolactin alone or with progesterone and the production of uteroglobin by the uterus was studied. There were seasonal, strain and dose variables in the uterine response to prolactin and progesterone. Treatment with prolactin (at 1 mg/day) plus progesterone generally induced higher levels of uteroglobin production than did treatment with progesterone alone. The differences were greatest in the winter for Tennessee animals and in the spring for animals from the New Mexico and North Carolina colonies. Ovariectomy produced a decrease (P less than 0.01) in the concentration of cytosolic oestrogen and progesterone receptors, and prolactin treatment restored the concentration to oestrous control values. However, there were no seasonally dependent changes in the concentration of the receptors for any of the treatment groups. Increased doses of prolactin (2 mg/day) induced high levels of uteroglobin production and new proteins to appear in uterine secretions of long-term ovariectomized rabbits but much lower levels (10-11%) when given to pregnant does. Additional ovulations were also noted plus adverse effects on the embryos.     

The androgenic effect of norethisterone and 5alpha-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.     

Binding of progesterone to the proteins of the uterine luminal fluid. Identification of uteroglobin as the binding protein.

1. The uterine luminal fluid of rabbits treated with estradiol and progesterone contains a protein factor with high affinity for [3-H] progesterone which is not present in the uterine secretion of control rabbits treated with estradiol. 2. This progesterone dependent factor is shown by gel filtration and polyacrylamide gel electrophoresis to be identical with the uterus specific protein uteroglobin, which seems to be required during the preimplantation phase. Uteroglobin specific antiserum, prepared in guinea pigs, completely inhibits the progesterone binding activity of the proteins of the uterine fluid. 3. Progesterone binding to uteroglobin is dependent upon millimolar concentrations of dithioerythritol. At saturation, one molecule of progesterone binds per uteroglobin molecule and the apparent association constant is 2 x 10-6 M-1 at 0 degrees C. 4. The progesterone binding species of uteroglobin exhibits a molecular weight of around 12 000 on polyacrylamide gels containing dodecylsulfate, and of 15 000 upon gel filtration, indicating a non-globular shape. This molecule is compased of two subunits of similar molecular size which are held together by a disulfide bridge among other forces.