Dissociation of uterine eosinophilia and water imbibition from other estrogen-induced responses by nafoxidine pretreatment

We investigated the effect of pretreatment of immature rats with 5 or 50 micrograms nafoxidine (UA), or with 0.05 micrograms 17 beta-estradiol (E2) on several uterine responses elicited by treatment with a test injection of 15 micrograms E2, administered 48 h after pretreatment. Early (6 h) and late (24 h) responses were measured, including wet weight, RNA, protein and glycogen content and number of blood eosinophils per uterus. The results showed that, like a 24 h pretreatment with 5 micrograms UA, a 48 h pretreatment with either of the UA doses dissociated the early wet weight response from the late responses to E2 treatment, only the former being restored. In the case of E2 pretreatment, both types of response to E2 treatment were reinstalled. By contrast, uterine eosinophilia, induced 6 and 24 h after E2 treatment, was not only restored but even markedly amplified following any of the 3 pretreatments. This was obtained without amplification of the early wet weight response and with various levels of the other parameters at the time of administration of the test E2 injection (i.e. due to the pretreatment alone). From this it may be concluded that if the previously documented correlation between estrogen-induced eosinophilia and edema actually reflects the existence of a causal link between the 2 responses, as postulated by Tchernitchin in 1972, this would be with eosinophils controlling edema, rather than the reverse. Testable working hypotheses for the mechanism of amplification of the eosinophil response are proposed.     

Multiple forms of nuclear estrogen receptor in the hypothalamus and pituitary after in vitro exchange with [3H]estradiol or [3H] hydroxytamoxifen

We have examined the sedimentation properties of hypothalamic and pituitary estrogen receptors (ER) translocated to the nucleus by in vivo estradiol (E₂) or nafoxidine treatments in the immature female rat. Nuclear ER were extracted with 0.4 M KC1 and incubated in vitro with saturating concentrations of [³H]E₂under conditions which allowed exchange with endogenous unlabeled ligands (23°C for 1–16 h). Sucrose gradient centrifugation of pituitary extracts from rats treated for 1, 24 or 48 h with E₂ yielded two components, sedimenting at 3 and 4–5S; in hypothalamic extracts the 3S form and variable amounts of a heavier shoulder were observed. After nafoxidine treatment the 4–5S species was present in greater proportions than after E₂. Both species interacted with monoclonal antibodies prepared against cytoplasmic ER from MCF-7 cells to yield more rapidly sedimenting complexes (7–8S). The 4–5S receptor was preferentially labeled in nuclear extracts subjected to exchange with [³H]E₂ for l h in vitro. In addition, nuclear ER from rats treated with [³H]E₂for l h in vivo sedimented at about 5S. We also investigated the direct binding of [³H]4-hydroxytamoxifen to nuclear ER. A single 4.2–4.3S component was present in both the hypothalamus and pituitary; this peak was displaced to about 7.5S after incubation with antibodies to MCF-7 ER. In the same nuclear preparations the 3S receptor predominated after exchange with [³H]E₂;however, a 1000-fold excess of unlabeled nafoxidine or hydroxytamoxifen abolished the 3S peak. These results suggest that 4–5S nuclear ER may give rise to the 3S form through degradation by endogenous proteases and that binding of antiestrogens to the larger species may interfere with this conversion.     

Estrogen-induced proteins in luminal epithelium,endometrial stroma and myometrium of the rat uterus

The early effect of estrogen on the synthesis of cytosolic proteins was investigated in the luminal epithelium, endometrial stroma and myometrium of the uterus in adult ovariectomized rats. The procedure of Reiss and Kaye (1981) was followed (involving two-step fractionation of ³⁵S-labelled proteins and fluorographic analysis) except that the uteri were fractionated into their three main tissue components before homogenization. The results show that E₂ stimulates the synthesis of BB-CK (brain-type creatine kinase), the major component of IP (estrogen-induced protein), in the three tissues. This suggests that BB-CK is related to a function that is common to the estrogen responses (such as hypertrophy) of all three uterine tissues in ovariectomized adult animals. The synthesis of two unidentified proteins of 37000 and 27000 M_r was markedly stimulated in the epithelium. These proteins are probably rate-limiting in responses to estrogen treatment that are specific to the epithelium. The 27000 M_r protein has the same charge as that of the 27000 M_r nafoxidine-induced protein described previously (Mairesse et al., 1981) and is probably therefore the same protein.     

Oestrogen-induced increase in uterine cGMP content: A true hormonal action?

The dependency of the oestrogen-induced increase in uterine cGMP content towards the cytosol-nuclear receptor system was investigated. The following observations were made: (1) With oestradiol-17 beta (E2-17 beta), U11-100A (UA) or CI-628 (CI) the cGMP response elicited in the uterus of immature rats followed a course that was parallel to (yet delayed by about 1 h from it) the known time-course evolution of nuclear occupancy by the complex formed by each compound with the oestrogen-receptor. (2) While a marked (about 2-fold) increase in uterine cGMP content was obtained with 0.1 microgram E2-17 beta, oestradiol-17 alpha (E2-17 alpha) given at the same dose had no effect on uterine cGMP. (3) The 2--3 h response to E2-17 beta (or to UA) could not be obtained in animals that had received a first injection of E2-17 beta, 2 h, or of one of the anti-oestrogens UA or tamoxifen, 20--22 h prior to the test injection of E2-17 beta. Those 3 treatments have in common that, at the time indicated, they create a state of depletion in the uterine cytosolic receptor population. The cGMP response to E2-17 beta was restored 20--22 h following a first injection of E2-17 beta. This time is known, in this case, to correspond to full replenishment of the cytosol-receptor population. In all those tests, the wet weight increase, measured in the same organs, behaves exactly as did the cGMP response. These results support the conclusion that the increase in uterine cGMP after oestrogen administration to the immature rat, represents a true hormonal action which, like other uterotrophic actions of oestrogens, involves binding of the hormone by the cytosol receptor.     

Nafoxidine-responsive uterine protein: further characterization and distinction from the "estrogen-induced" protein (IP)

In a previous paper, we reported that nafoxidine (UA) stimulated the synthesis of a uterine protein showing the same electrophoretic mobility as the "estrogen-induced protein" (E2-IP) first described by Notides and Gorski. In the present work, we analyzed the IP-containing electrophoretic zone by SDS polyacrylamide-gel electrophoresis, and found that estradiol-17 beta (E2) and nafoxidine stimulated the synthesis of different proteins. As expected, estradiol-17 beta stimulated the synthesis of the E2-IP of 46 000 Mr. On the other hand, UA stimulated the synthesis of 27 000 and 30 000 Mr proteins (UA-IP). These UA-IP were not precipitated by an antiserum raised against E2-IP. Therefore, UA-IP appear to be independent entities and not degradation or precursor products of E2-IP. Both UA-IP are constitutively present in the uterus and even in higher relative amounts in rat brain. The present finding, that an "anti-estrogen", such as nafoxidine, stimulates the synthesis of different proteins than estrogen, provides a new approach to the study of the molecular mechanism of estrogen action.     

Time-related effects of a triphenylethylene antiestrogen on estrogen-induced changes in uterine weight, estrogen receptors, and endometrial sensitivity in rats

Time-related estrogen antagonistic action of a single oral contraceptive (1.25 mg/kg) dose of the triphenylethylene antiestrogen centchroman was determined in ovariectomized immature rats. Tamoxifen and nafoxidine were used for comparison. A single oral administration of centchroman followed by three doses of estradiol-17β (1 μg/d, s.c.) caused significant dose-dependent inhibition in estradiol-17β-induced increase in uterine weight and nuclear and cytosolic estrogen receptors. But the inhibition at anti-implantation dose was evident only if estradiol-17β treatment was initiated not later than 48 h post-antiestrogen. Alternatively, when antiestrogen treatment was followed by a single dose of estradiol-17β between days 2–7, a synergistic action, typical of antiestrogens possessing weak estrogen agonistic activity, was observed. In immature rats in which a condition mimicking preimplantation was produced by estradiol-17β (0.5 μg/d, s.c.) priming on days -2 and -1, followed by progesterone (1 mg/d, s.c.) and an endometrial sensitizing dose (0.5 μg/d, s.c.) of estradiol-17β at 1600 h on day 4, anti-implantation dose of centchroman administered on day 1, too, failed to inhibit uterine weight gain induced by sensitizing dose of estradiol-17β, but caused marked inhibition in endometrial sensitivity to a deciduogenic stimulus and decidualization and weight gain of traumatized uterine horn 96 h post-traumatization over non-traumatized horn was only about 150% (725% in controls). Inhibition in endometrial sensitivity and decidualization was evident when the interval between antiestrogen treatment and sensitizing estradiol was <126 h. Pinopods were present on endometrial surface on day 5 whether or not priming and/or sensitizing doses of estradiol were administered, but decidual response was mild if either of these doses of estradiol-17β was deferred. Findings suggest that: (a) duration of antiestrogenic action of single anti-implantation dose of centchroman in rat was about 126 h, which in ovariectomized immature rats was evident only when a condition mimicking preimplantation was produced and the antiestrogenic response was based on inhibition in estradiol-induced endometrial sensitivity and not uterine weight gain; (b) priming as well as sensitizing estrogen were essential to get optimal decidual responses; (c) appearance of pinopods on endometrial surface may not be related to endometrial sensitivity; and (d) tamoxifen and nafoxidine appear slightly longer acting with duration of antiestrogenic action of 150 h.     

Multiple forms of nuclear estrogen receptor in the immature rat uterus after in vitro exchange with [3H]estradiol or [3H] antiestrogens

We have compared the properties of salt-extracted uterine nuclear estrogen receptors (ER) labeled with [³H]estradiol (E₂) or [³H]antiestrogens after in vivo exposure of immature rats to these compounds or after in vitro exchange. As expected, nuclear ER labeled during a l h in vivo treatment with [³H]E₂ migrated predominantly at 5S, although this peak was skewed towards the lighter side. [³H]4-Hydroxytamoxifen ([³H]OH tamoxifen, the active metabolite of tamoxifen)-nuclear ER complexes showed comparable sedimentation rates (4.6 ± 0.05S) after injection of the [³H]antiestrogen. On the other hand, when nuclear extracts from rats treated with unlabeled E₂ in vivo were subjected to exchange with [³H]E₂ or [³H]antiestrogens for 16 h at 23°C, a marked difference. in sedimentation profiles was observed. While the [³H]E₂ -nuclear ER complex sedimented primarily as a 3S species with variable amounts of a heavier (4.0–4.5S) shoulder, complexes between nuclear ER and [³H]tamoxifen, [³H]OH tamoxifen or [³H]CI628M formed sharp, symmetrical peaks at 4S. Both the 3S and 4S components represented forms of ER as they were eliminated in the presence of excess unlabeled DES, and they were displaced to 7–8S after reaction with specific anti-ER antibodies. The 3S peak was also abolished by addition of excess nonradioactive nafoxidine or OH tamoxifen during exchange with [³H]E₂. These results suggest that more rapidly sedimenting forms of uterine nuclear ER (4–5S) may be converted to a 3S species by the action of endogenous proteases and that association of these large forms with antiestrogens may stabilize them in a conformation less or differentially susceptible to cleavage.