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The objective of the present study was to further elucidate our previous observation that beta2-adrenoceptor activation induces oxytocin receptor (OTR) expression in rat myometrium. We wanted to investigate whether the mechanism behind this effect was under the influence of gonadal steroids. Ovariectomized non-pregnant rats were treated with estrogen, progesterone or a combination of both for 3 days. Some rats were concomitantly treated with isoproterenol. Estrogen treatment increased both OTR mRNA production and maximal binding of [3H]-oxytocin to isolated myometrial plasma membranes, but it did not affect contractility of isolated uterine strips challenged with oxytocin. When the estrogen regimen was combined with isoproterenol treatment, an augmented maximal contractile response (Emax) to oxytocin was observed although no further increase in OTR mRNA and binding was seen. Progesterone treatment did not in itself alter OTR mRNA, OTR binding or Emax. However, OTRs were induced at the level of gene expression when progesterone was supplemented with isoproterenol infusion. Finally, progesterone suppressed the effect of estrogen on OTR mRNA production and binding when the two compounds were administered together. However, when isoproterenol treatment was added this effect was abolished and Emax was enhanced more than that seen following treatment with estrogen alone. These data suggest that beta2-adrenoceptor activation represents an important regulator of OTR expression/function in estrogen- and progesterone-dominated rat myometrium.

Endothelin (ET)-1 and ET-3, two peptides with a potent vasoconstrictive property, produce a variety of biological effects in different tissues by acting through two different receptors, the ET-1 selective ET(A) receptor and the non-selective ETB receptor. An increasing body of literature suggests that ET-1 acts as a paracrine/autocrine regulator of ovarian function. Indeed, ETB receptors have been identified in rat granulosa cells and ET-1 is a potent inhibitor of progesterone production. In contrast, inconsistent data have been reported about the role of ET-1 on estrogen production and the effects of ET-3 are not known. Therefore, the present study was undertaken to evaluate the effects of ET-1 and ET-3 on estrogen and cAMP production, and the receptor type involved. Given that prostanoids modulate ovarian steroidogenesis and that many actions of ETs are mediated by these compounds, we also evaluated whether the effects of ETs on estrogen and cAMP production might be prostanoid-mediated. ET-1, ET-3, and safarotoxin-S6c (SFX-S6c), a selective ETB receptor agonist, inhibited basal estrogen production by granulosa cells obtained from immature, estrogen-primed female rats, in a concentration-dependent manner. All three peptides were also capable of inhibiting the production of estrogen stimulated by a half-maximal (1 mIU/ml) and a maximally stimulatory (3 mIU/ml) concentration of FSH, ET-1 and ET-3 dose-dependently suppressed basal and FSH (1 mIU/ml)-stimulated cAMP production. ET-3 and SFX-S6c were significantly more potent than ET-1 in suppressing estrogen production, suggesting that this effect was not mediated by the ET(A) receptor. Indeed, BQ-123, a selective ET(A) receptor antagonist, did not influence the inhibitory effects of ET-1 and ET-3 on basal and FSH-stimulated estrogen release. To determine a possible involvement of prostanoids, we evaluated the effects of maximally effective concentrations of ET-1 and ET-3 on estrogen and cAMP production in the presence of indomethacin, a prostanoid synthesis inhibitor. This compound did not have any effect on the suppressive effects of ETs on basal or FSH (1 mIU/ml)-stimulated estrogen or cAMP production. In conclusion, ET-1 and ET-3 were able to inhibit estrogen and cAMP production by rat granulosa cells, indicating that the inhibitory effects of ETs on ovarian steroidogenesis are not limited to progesterone biosynthesis. This effect does not appear to be mediated by prostanoids or by the classical ET(A) and ETB receptors, at least under these experimental conditions.

The effects of in vivo treatment with estrogen and progesterone on isoproterenol-induced uterine relaxation and beta(2)-adrenoceptor (beta(2)AR) mRNA production in non-pregnant rat myometrium were investigated. Whether homologous myometrial desensitization of beta(2)AR function was dependent on or modulated by the two steroids was also examined. Estrogen treatment alone or in combination with progesterone reduced maximal relaxation (E(max)) of isolated uterine strips subsequently challenged with isoproterenol whereas progesterone alone had no effect on this parameter. The reduction was accompanied by an enhanced beta(2)AR mRNA concentration. The concentration of isoproterenol giving half-maximal relaxing response (EC(50)) increased following estrogen treatment and this effect was curbed by progesterone. Isoproterenol had no effect on beta(2)AR transcription irrespective of the steroid regimes employed. E(max) of isolated uterine strips was reduced following prolonged in vivo treatment with isoproterenol but the effect was found only when estrogen alone was administered concomitantly. Finally, in vivo treatment with isoproterenol increased EC(50) of uterine strips subsequently stimulated with isoproterenol in vitro. This effect was independent of steroid treatment. We conclude that homologous desensitization of beta(2)AR function in non-pregnant rat myometrium in terms of sensitivity (EC(50)) is independent of sex steroids but in terms of maximal response (E(max)) occurs only in the presence of estrogen. We speculate whether progesterone withdrawal in connection with the well-known estrogen dominance at rat parturition may strengthen the desensitization induced by beta(2)AR activation and thus contribute to the transformation of the uterus from a quiescent to a highly contractile organ.

Interrelationships between thyroid hormone and estrogen actions have been documented with regard to a variety of physiological functions. Both hormones stimulate transcription of target genes by binding to their nuclear receptors that interact with specific responsive elements (estrogen and thyroid hormone response elements, i.e ERE and TRE, respectively) in the regulatory regions of the gene. In vitro studies have suggested that interplay between the two hormones might be due to cross-talk at hormone responsive elements, with the respective hormone receptors and ligands able to interact, although physiological relevance has yet to be proved. We have proposed a simpler mechanism for thyroid hormone effects on estrogen responses via increase in estrogen receptor alpha (ERalpha) with resultant increase in progesterone receptors, prolactin production and tumor growth. A pituitary cell line, GH3, has been widely used to investigate the function of mammo-somatotropic cells, especially regarding regulation of GH and prolactin production. In the present study, an ERE-luc reporter was transfected into GH3 cells and the responses to endogenous ERalpha were examined. We demonstrated that: (1)l -3,5,3'-triiodothyronine (T3) induces mRNA expression of ERalpha; (2) T3 alone is able to induce ERE-luc activity and this is inhibited by OH-tamoxifen; (3) T3 synergistically acts on estradiol (E2)-induced ERE responses; and (4) ERE-luc activity is enchanted by co-transfection of an ERalpha expression vector. These results support the hypothesis that estrogen responses are potentiated by T3 through up-regulation of ERalpha levels.

It is well known that progesterone and estrogen are essential hormones for maintaining pregnancy in most mammals. Some specific roles of progesterone for the maintenance of pregnancy have been clarified, but the role of estrogen is not well known. This study examines the effects of the aromatase inhibitor, fadrozole hydrochloride (Fad), on fetuses, uterine physical properties and the mRNA expression of the uterine enzymes that are related to collagen metabolism during late pregnancy in rats. Continuous s.c. infusion with 300 micro g/day Fad from day 14 of pregnancy (day 1=the day of sperm detection) reduced the concentration of plasma estradiol-17beta (E(2)), and did not change that of plasma progesterone, compared with controls. The treatment increased the intrauterine pressure and reduced the size and compliance of the uterine tissue framework. It also caused injuries (hematomata on the extremities) in about one-quarter of fetuses by day 20. The collagen content of the uterine ampullae was not changed by the treatment. Uterine mRNA expressions of matrix metalloproteinase-1 (MMP-1), which degrades collagens, and of lysyl oxidase (LO), which is necessary for the formation of intra- and inter-molecular cross-links of collagen, were examined by quantitative RT-PCR. The treatment with Fad had no effect on the expression of MMP-1 mRNA and increased that of LO mRNA. Daily s.c. injection with 0.2 micro g E(2) restored the changes in uterine physical properties and the mRNA expression of LO caused by the Fad treatment, and prevented fetal injury, indicating that the influences of Fad treatment are due to estrogen deficiency but not to toxicological effects of Fad. These results imply that estrogen deficiency during late pregnancy in rats obstructs development of the uterine tissue framework so as to cause fetal injury. It is possible that an increase in the uterine expression of LO gene may be involved in this obstruction.

The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of both progesterone and 17beta-estradiol. However, it remains unclear whether estrogens alone or progestins alone can cause insulin resistance, or whether it is a combination of both which produces this effect. We attempted to determine the role played by progesterone and/or 17beta-estradiol on the phenomena of sensitivity to insulin action that take place during pregnancy in the rat. Ovariectomized rats were treated with different doses of progesterone and/or 17beta-estradiol in order to simulate the plasma levels in normal pregnant rats. A euglycemic/hyperinsulinemic clamp was used to measure insulin sensitivity. At days 6 and 11, vehicle (V)- and progesterone (P)-treated groups were more insulin resistant than 17beta-estradiol (E)- and 17beta-estradiol+progesterone (EP)-treated groups. Nevertheless, at day 16, the V, EP and E groups were more resistant to insulin action than the P group. On the other hand, the V, EP and E groups were more insulin resistant at day 16 than at day 6, whereas the P group was more insulin resistant at day 6 than at day 16. Our results seem to suggest that the absence of female steroid hormones gives rise to a decreased insulin sensitivity. The rise in insulin sensitivity during early pregnancy, when the plasma concentrations of 17beta-estradiol and progesterone are low, could be due to 17beta-estradiol. However, during late pregnancy when the plasma concentrations of 17beta-estradiol and progesterone are high, the role of 17beta-estradiol could be to antagonize the effect of progesterone, diminishing insulin sensitivity.