Abstract
The specific role of each oestrogen receptor (ER) isoform (α and β ) and site (nucleus and plasma membrane) in LH release was determined in ovariectomized (OVX) rats injected over 6 days (days 15–20 after OVX) with a saturating dose (3 mg/day) of tamoxifen (TX), a selective ER modulator with nuclear ERα agonist actions in the absence of oestrogen. This pharmacological effect of TX was demonstrated by the fact that it was blocked by the selective ERα antagonist methyl-piperidinopyrazole. Over the past 3 days of the 6-day TX treatment, rats received either 25 μg/day oestradiol benzoate (EB), 1.5 mg/day selective ERα agonist propylpyrazole triol (PPT) and the selective ERβ agonist diarylpropionitrile (DPN), or a single 3 mg injection of the antiprogestin onapristone (ZK299) administered on day 20. Blood samples were taken to determine basal and progesterone receptor (PR)-dependent LH-releasing hormone (LHRH)-stimulated LH secretion and to evaluate LHRH self-priming, the property of LHRH that increases gonadotrope responsiveness to itself. Blood LH concentration was determined by RIA and gonadotrope PR expression by immunohistochemistry. Results showed that i) EB and DPN potentiated the negative feedback of TX on basal LH release; ii) DPN reduced TX-induced PR expression; iii) EB and PPT blocked TX-elicited LHRH self-priming and iv) ZK299 reduced LHRH-stimulated LH secretion and blocked LHRH self-priming. These observations suggest that oestrogen action on LH secretion in the rat is exerted at the classic ERα pool and that this action might be modulated by both ERβ and membrane ERα through their effects on PR expression and action respectively.
Introduction
Luteinizing hormone (LH) secretion in the rat is dependent on the activation of gonadotrope oestrogen receptors (ERs) by ovarian-derived oestradiol-17β (E2; Fink 1988, 1995). Rat gonadotropes express both isoforms of ER, the predominant ERα (Lindzey et al. 2006) and ERβ at nuclear level (Kuiper et al. 1997, Mitchner et al. 1998, Vaillant et al. 2002, Sánchez-Criado et al. 2005a). In addition, an ER which appears to be the same protein as nuclear ERα is expressed at the plasma membrane level (Bression et al. 1986, Razandi et al. 1999, Toran-Allerand et al. 1999, Schmidt et al. 2000, Kelly & Levin 2001, Simoncini & Genazzani 2003, Toran-Allerand 2004, Levin 2005). Integration of E2 effects at different ER pools in the gonadotrope (nuclear ERα and ERβ and membrane ERα ) determines the cellular actions of E2 on basal and preovulatory LH secretion and hence on ovulation (Fink 1988, 2000). Since E2 activates the complete ER orchestra in the gonadotrope, it is difficult to chart separately the specific role of each ER pool on LH secretion using either intact cyclic or ovariectomized (OVX) rats treated with the cognate ligand. ERα and ERβ isoform knockout mice (Pelletier et al. 2003, Lindzey et al. 2006) are not useful models for the study of ER isoforms interaction either because one or the other ER isoform is lacking. The discovery of ER subtype-selective ligands, such as the ERα agonist propylpyrazole triol (PPT; Stauffer et al. 2000), the ERβ agonist diarylpropionitrile (DPN; Meyers et al. 2001) and the ERα-selective antagonist methyl-piperidinopyrazole (MPP; Sun et al. 2002, Harrington et al. 2003), affords useful tools for dissecting the biology of ER subtypes at pituitary level (Sánchez-Criado et al. 2004, 2006a) in OVX rats. A ying–yang relationship between ERβ and ERα on LH secretion has been reported in 2-week OVX rats treated with these ER-selective agonists (Sánchez-Criado et al. 2004, 2006a).
Tamoxifen (TX), a type I oestrogen antagonist with selective ER modulator (SERM) properties (McDonnell 1999, 2003, McDonnell et al. 2002, Smith & O’Malley 2004), exhibits agonist activities in the gonadotrope of OVX rats. These actions of TX include shrinkage of OVX-induced hypertrophy, induction of progesterone receptor (PR) mRNA and protein expression, and PR-dependent LH-releasing hormone (LHRH) self-priming in vitro (Bellido et al. 2003). LHRH self-priming is a phenomenon in which the magnitude of the LH response to the second of two equal exposures of LHRH separated by an interval of 60 min is significantly greater than the response to the first exposure to LHRH (Fink 1988). Evidence derived from in vitro work indicates that the agonist actions of TX in the rat gonadotrope are exerted at the nuclear ERα level exclusively. Therefore, incubated pituitaries from OVX rats injected over 3 days with pharmacological doses of TX exhibit LHRH self-priming (Sánchez-Criado et al. 2005b) and this agonistic effect of TX is inhibited when E2 or the membrane-impermeable analogue conjugated E2–BSA is added to the incubation medium. Moreover, addition of the pure type II anti-oestrogen ICI182 780 (Smith & O’Malley 2004) to the medium blocks the rapid inhibitory action of E2 on TX-elicited LHRH self-priming, whereas TX itself does not (Sánchez-Criado et al. 2005b). One emerging explanation of these in vitro data is that TX selectively binds nuclear ERα but not ERβ (Tzuckerman et al. 1994, Bellido et al. 2003, Sánchez-Criado et al. 2004, 2005a) and exhibits extremely low affinity for membrane ERα in rat gonadotropes (Sánchez-Criado et al. 2005b). In addition, these data suggest that E2 inhibition of TX-elicited LHRH self-priming is due to activation of the plasma membrane ERα (Sánchez-Criado et al. 2005b, 2006b).
The present study was designed to ascertain whether the in vitro inhibitory effects of both nuclear ERβ-initiated signalling (Sánchez-Criado et al. 2004) and membrane ERα-initiated signalling (Sánchez-Criado et al. 2005b) upon the LH secretory actions of the nuclear ERα-initiated signalling were also operative in the whole animal. To this purpose, three groups of in vivo experiments were conducted: the first, to verify the effects of TX on LH secretion, pituitary PR expression and LHRH self-priming in 2-week OVX rats; the second, to evaluate the role of the different ER isoforms and sites in LH release in gonadotropes with TX-activated nuclear ERα using the cognate ligand E2 and the selective ERα and ERβ agonists PPT and DPN respectively and the third to verify that TX acts through ERα by studying the effects of the selective ERα antagonist MPP.
Materials and Methods
Animals, general conditions and surgery
Adult female Wistar rats weighing 190–210 g were used. Rats were housed under a 14 h light:10 h darkness cycle (light on at 0500 h) and 22 ± 2 ° C room temperature, with ad libitum access to rat chow and tap water available ad libitum. Rats were bilaterally OVX under ether anaesthesia at random stages of the oestrous cycle and assigned to experimental groups 14 days later. At the time indicated in each experiment, a right atrial cannula was implanted using a previously described procedure (Harms & Ojeda 1974, Sánchez-Criado et al. 1993), and rats received an i.v. injection of 20 IU heparin/250 μl saline. At 0900 h (time 0) the following day, the distal ends of the cannulae were attached to extension tubing (P50; Adams, Parsippany, NJ, USA) to permit blood sampling and LHRH administration. Rats were given an i.v. bolus of 25 ng LHRH, and a second one 60 min later. Blood samples (250 μl each) were taken at 0, 15, 60, 75 and 120 min. At the end of the experiments, anterior pituitaries were removed and dissected out for the determination of protein and LH contents. On day 18 (experiment 1) and day 21 (experiments 2, 3, 4 and 5), vaginal smears were taken. All experimental protocols were approved by the Ethical Committee of the University of Córdoba, and experiments were performed in accordance with the rules on laboratory animal care and with international law on animal experimentation.
Drugs and treatments
TX (Sigma) was injected at pharmacological/saturating doses of 3 mg/day (Sánchez-Criado et al. 2004, 2005b, 2006b). The selective ERα and ERβ agonists PPT and DPN respectively (Tocris Cookson Ltd, Avonmouth, UK; Stauffer et al. 2000, Meyers et al. 2001) were injected at doses of 1.5 mg/day (Sánchez-Criado et al. 2004). PPT has a 400-fold preference for ERα and does not activate ERβ (Stauffer et al. 2000). DPN has a 70-fold higher binding affinity for ERβ than for ERα (Meyers et al. 2001). Oestradiol benzoate (EB; Sigma) was injected at the pharmacological dose of 25 μg/day. The potent ERα-selective antagonist methyl-piperidino-pyrazole (MPP; Tocris) was dissolved in DMSA/olive oil (1/14, v/v) and injected at a dose of 1 mg/day. This compound displays 220-fold more affinity for ERα than for ERβ (Sun et al. 2002, Harrington et al. 2003). The PR antagonist onapristone (ZK299; Schering, Berlin, Germany; Neef et al. 1984, Bellido et al. 1999) was injected at a dose of 3 mg. All ER ligands and ZK299 were given subcutaneously in 0.2 ml oil (Table 1). Synthetic LHRH (Peninsula Laboratory, Inc., Merseyside, UK) was dissolved in saline at a concentration of 125 ng/ml, and 0.2 ml of this solution was injected intravenously. The saturating doses of steroid receptor ligands used in the present experiments were based on previously published studies (Table 1).
Experiment 1: effect of TX on LH secretion, PR expression and LHRH self-priming in OVX rats
Rats were injected either with TX or with oil vehicle and EB (negative and positive control groups respectively) on days 15–17 after OVX. Less than 0.4 ml blood was drawn by direct left jugular venipuncture under light ether anaesthesia at 0900 h on days 15, 16 and 17. On the afternoon of day 17, rats were implanted with atrial cannulae. Pituitary LH secretory response to LHRH was studied on day 18. Pituitaries from similarly treated rats (four rats/group) were processed for PR expression on day 18 after OVX. Serum and plasma samples were stored frozen until the LH RIA was run.
Experiment 2: effects of EB, PPTand DPN on LH secretion and LHRH self-priming in TX-injected OVX rats
Rats were injected with TX (control group) on days 15–20 after OVX. Over the past 3 days (days 18–20 after OVX) of TX treatment, rats were additionally injected with EB, PPTor DPN. Less than 0.4 ml blood was taken by jugular venipuncture at 0900 h on days 18–20. On the afternoon of day 20, rats were implanted with atrial cannulae. The pituitary LH secretory response to LHRH was studied on day 21. Serum and plasma samples were stored frozen until the LH RIA was run.
Experiment 3: effects of EB, PPTand DPN on PR expression in TX-injected OVX rats
Rats were injected with TX (control group) on days 15–20 after OVX. Over the past 3 days (days 18–20 after OVX) of TX treatment, rats were additionally injected with EB, PPTor DPN. At 0900 h on day 21 after OVX, four rats in each of the four groups (TX, TX + EB, TX + PPTand TX + DPN) were decapitated and their anterior pituitaries dissected out and processed for PR immunoreactivity.
Experiment 4: effects of ZK299 on LH secretion and LHRH self-priming elicited by TX
The action of ligand-independent activation of PR on LH secretion was evaluated in 6-day TX-injected OVX rats given a single injection of 3 mg ZK299 at 0900 h on day 20. Basal and LHRH-stimulated LH secretion and LHRH self-priming were studied on day 21. Plasma samples were stored frozen until the LH RIA was run.
Experiment 5: effects of MPP on LH secretion, PR expression and LHRH self-priming in TX-injected OVX rats
Rats were injected daily from days 15 to 20 after OVX with MPP, with or without TX injections on days 18–20 after OVX. Control groups consisted of OVX rats injected with 0.2 ml oil from days 15 to 20 and TX alone from days 18 to 20. Less than 0.4 ml blood was taken by jugular venipuncture at 0900 h on days 15, 18, 19 and 20. On the afternoon of day 20, rats were implanted with atrial cannulae. On day 21, the pituitary LH secretory response to LHRH was studied. In addition, pituitaries from similarly treated OVX rats (three rats/group) were processed for PR immunoreactivity on day 21. Serum and plasma samples were stored frozen until the LH RIA was run.
Pituitary LH content determination
At the end of each experiment, either on day 18 or 21, anterior pituitaries were removed and homogenized in 1 ml RIA buffer and subjected to ultrasonic treatment. Samples were centrifuged at 2800 g for 10 min and the supernatants frozen at − 20 ° C until assayed by LH RIA. Pituitary LH content was expressed as μg/mg protein. Pituitary protein content was determined by the micro-turbidimetric method using benzethonium chloride in alkali (Iwata & Nishikaze 1979). The sensitivity of the method was 40 μg/ml.
RIA of LH
Serum or plasma LH concentrations were measured in duplicate by RIA using a double-antibody method with a kit supplied by NIH (Bethesda, MD, USA) and a previously described microassay method (Sánchez-Criado et al. 1993, Bellido et al. 1999). Rat LH-I-9 was labelled with 125I by the chloramine-T method (Greenwood et al. 1963). The intra- and inter-assay coefficients of variation were 8 and 9% respectively. Assay sensitivity was 3.5 pg/tube. LH was expressed as ng/ml of the reference preparation LH-rat-RP-3.
LHRH self-priming
The peak pituitary response of LH occurs after 15-min administration of LHRH pulse (Sánchez-Criado et al. 2005b). In the present experiments, LHRH self-priming was evaluated as the percentage increase in LH secretion to the second LHRH pulse (primed pituitary response) with respect to the first LHRH pulse (LHRH-stimulated LH secretion or unprimed pituitary response).
Immunohistochemistry of pituitary PR
The immunohistochemical study was performed on dewaxed and rehydrated 3 μm thick tissue sections of formalin-fixed, paraffin-embedded tissue samples. The commercial mouse monoclonal anti-human PR antibody clone PR10A9, raised against the recombinant hormone-binding domain of human PR located on the C-terminal domain of PR (Immunotech, Marseille, France), diluted in the ratio of 1:15 000, and the avidin–biotin peroxidase complex (ABC) technique (Vector, Burlingame, CA, USA) were used as described previously (Sánchez-Criado et al. 2004). Tissue sections from similarly processed samples of rat uterus and human breast carcinoma were used as positive controls. The specificity of the PR antibody was shown by the lack of staining after pre-incubation of tissue sections of rat uterus and pituitaries from OVX rats treated with EB with 10− 9, 10− 7 and 10− 5 M of the cognate ligand for 1 h at 37 ° C. Substitution of the specific primary antibody by mouse ascitic fluid at the same dilution as the specific primary antibody in tissue sections of the cases under study was used as negative control. Several dilutions of the PR10A9 monoclonal antibody were tested and the optimal dilution was established at 1:15 000, because it gave the highest intensity of nuclear staining with the lowest background staining in pituitary and uterus (Sánchez-Criado et al. 2004). Nuclear counterstaining was performed with Mayer’s haematoxylin in all cases. The amount of cells immunoreactive to PR antibody was expressed as the number of positive nuclei counted in five fields at a magnification of 40 × (about 240 pituitary cells/field) in each pituitary. All immunoreactive cells were considered to be gonadotropes because they are the only pituitary cells expressing PR (Fox et al. 1990, Sánchez-Criado et al. 2005a).
Statistical analysis
Statistical analysis was performed by ANOVA to check for significant differences among groups. When significant differences existed, it was followed by the Student–Newman–Keuls multiple range test for inter-group comparison. Significance was considered at the 0.05 level.
Results
Effects of treatment with different ER ligands on vaginal smears in OVX rats
OVX rats treated with the ER agonists EB, TX and TX plus EB, PPT, DPN or TX plus the PR antagonist ZK299 showed either nucleated or fully cornified epithelial cells in vaginal smears. In contrast, OVX rats injected with oil displayed vaginal smears predominantly infiltrated by leukocytes. The ERα antagonist MPP, either alone or in combination with TX, behaved as an ER agonist because it also induced cornification of vaginal smears.
Effect of TX on LH secretion, gonadotrope PR expression and LHRH self-priming in OVX rats
Treatment of 2-week OVX rats with TX over 3 days had similar (though less pronounced) effects to treatment with the cognate ligand EB over the same period of time. Therefore, TX reduced both basal serum/plasma LH concentration (Figs 1 and 2) only on days 17 and 18 and pituitary LH content (Table 2) on day 18 after OVX. In addition, TX-induced LHRH self-priming, as the magnitude of the LH response to the second LHRH challenge, was significantly increased with respect to the LH peak response to the first LHRH pulse (Fig. 2; Table 2). A similar magnitude of LHRH self-priming was also observed in OVX rats treated with EB (positive control group), but not in OVX rats injected with oil vehicle (negative control group; Fig. 2; Table 2). However, TX did not sensitize the pituitary to LHRH, which contrasted with the pituitary sensitizing action of EB to LHRH (Fig. 2).
Effect of EB, PPTand DPN upon the inhibitory effect of TX on basal LH secretion and on TX-elicited LHRH self-priming in OVX rats
The inhibitory effect of a 3-day TX treatment on serum LH concentration and pituitary LH content in OVX rats was further enhanced after 6-day treatment of OVX rats with TX (Fig. 3; Table 2). This dose-related inhibitory effect of TX on basal LH levels was potentiated by the simultaneous administration of either EB or the selective ERβ agonist DPN on days 18–20 after OVX (Figs 3 and 4). However, administration of the selective ERα agonist PPT did not potentiate this inhibitory effect of TX (Figs 3 and 4). No effect on pituitary LH content was noted after treatment with EB, PPTor DPN (Table 2). TX treatment of OVX rats over 6 days (days 15–20 after OVX) induced LHRH self-priming which was blocked by both EB and PPT, but not by DPN treatments over 3 days (days 18–20 after OVX, Fig. 4; Table 2).
Effect of ZK299 on LH secretion and LHRH self-priming in TX treated in OVX rats
The blockade of PR activity with ZK299 given on day 20 to 6-day TX-treated OVX rats reduced basal and LHRH-stimulated LH secretion and blocked LHRH self-priming on day 21 (Fig. 4). The antiprogestagen had no effect on pituitary LH content (Table 2).
Effect of EB, PPT and DPN upon TX-induced PR expression in OVX rats
Immunoreactive products to PR antibody were detected in the nuclei of gonadotropes in pituitaries from OVX rats treated over 3 or 6 days with TX (Fig. 5). PR expression was higher after 6 days of TX treatment. In these rats, PR immunoreactivity was found in either hypertrophied or shrunken gonadotropes (Fig. 5; upper panel). In contrast, oil-injected OVX rats exhibited hypertrophied gonadotropes lacking PR expression. The number of PR immunoreactive cells observed in pituitaries from 6-day TX-treated OVX rats was reduced by co-administration of the selective ERβ agonist DPN over the past 3 days of TX treatment (Fig. 5; lower panel), while the selective ERα agonist PPT and the cognate ligand EB had no significative effect on the number of PR immunoreactive cells (Fig. 5; lower panel).
Effect of MPP on TX agonist actions in OVX rats
The agonist actions of TX in OVX rats injected on days 18–20 after OVX included: reduction of basal LH serum/plasma concentration (Fig. 6) and pituitary LH content (Table 3), induction of LHRH self-priming (Fig. 7; Table 3) and PR expression on day 21 (Fig. 8). MPP treatment blocked the TX-elicited LHRH self-priming (Fig. 7; Table 3) and reduced the TX-induced PR expression (Fig. 8). On the contrary, MPP alone injected daily from days 15 to 20 after OVX reduced basal LH serum concentration and pituitary LH content (Figs 6 and 7; Table 3), a negative feedback on LH secretion similar to the agonist effect of TX (Figs 1–3) in OVX rats.
Discussion
The results of the present experiments indicate that TX has ERα agonist actions in the gonadotrope of OVX rats. Assuming that a 6-day TX treatment also prevented the action of other ER ligands at the nuclear ERα level (Sánchez-Criado et al. 2005b), results show, in addition, that the bimodal components of gonadotrope LH release with TX-activated nuclear ERα are differentially affected by the simultaneous activation of ER isoforms and sites as follows: 1) The inhibitory effect of TX on PR-independent basal LH secretion was potentiated by activation of ERβ with DPN and EB but not by activation of membrane ERα with PPT. 2) The PR-dependent LH secretory surge elicited through nuclear ERα was inhibited by activation of ERβ with DPN and EB, and membrane ERα with PPT and EB through their effects on PR expression and action respectively.
The known in vitro agonist actions of TX on LH release have been confirmed in vivo using long-term OVX rats. The 2-week OVX rat model was used because treatment of these rats over 3 days with EB mimicks the endocrine events of pro-oestrus through augmentation of the LHRH-releasing pathway, induction of PR expression and induction of PR-dependent LHRH self-priming (Bellido et al. 2003, Sánchez-Criado et al. 2004) culminating in an ovulatory LH surge (Legan & Tsai 2003). The administration of pharmacological/saturating dose of TX to these OVX rats decreased both serum and pituitary LH levels in a negative feedback manner, induced upregulation of PR expression in the gonadotrope, and elicited LHRH self-priming. In addition, it has also been confirmed in vivo that these agonist actions of TX in the gonadotrope of OVX rats are due to the activation of nuclear ERα (Sánchez-Criado et al. 2005b). The administration of the ERα-selective antagonist MPP to TX-treated OVX rats both reduced TX-induced PR expression to a minimum and abolished TX-elicited LHRH self-priming. However, MPP did not behave as an ERα antagonist exclusively. This is because MPP reduced basal LH secretion and induced vaginal smears cornification in the absence of TX treatment. Taken together, these findings indicate that MPP behaved as a SERM.
The first and longer phase of LH release in the gonadotrope is PR-independent basal LH secretion. For most of the reproductive life of females, LH levels are kept within a relatively low range by the PR-independent (Chappell et al. 1999) negative feedback of moderate levels of oestrogen (Fink 1988). Whereas activation of ERα with the selective agonist PPTreduces LH secretion in OVX rats (Sánchez-Criado et al. 2004), PPT failed to reduce serum LH levels when administered to TX-treated OVX rats, indicating a probable competition of the selective ERα agonist for the same ER isoform in which TX acts. In addition, the present data showed that: i) activation of ERβ either with DPN or EB potentiated the TX-induced reduction of serum LH levels in OVX rats; ii) activation of both ER isoforms with EB was more effective in reducing serum LH levels than activation of nuclear ERα alone with TX and iii) the potent selective ERα antagonist MPP administered alone reduced PR-independent LH secretion in an agonistic manner. These findings indicate that the negative feedback of oestrogen on LH secretion is a genomic ERα effect potentiated by activation of ERβ .
The expression of PR in the gonadotrope is an oestrogen effect of both E2 and TX which occurs simultaneously with their negative feedback on LH secretion (Sánchez-Criado et al. 2004, 2006a). Gonadotrope PR expression gives rise to a complex phase: the PR-dependent LH surge elicited by an acute rise in E2 levels (Chappell et al. 1999). The LH surge has two components: 1) LHRH-stimulated LH secretion and 2) the LHRH self-priming. The former occurs in an oestrogen positive feedback manner, in which oestrogen increases pituitary responsiveness to the releasing effects of LHRH (Arimura & Schally 1971, Schuiling et al. 1999, Schwartz 2000) through activation of the nuclear ERα isoform (Sánchez-Criado et al. 2004, Lindzey et al. 2006). Since TX, neither in vitro (Sánchez-Criado et al. 2002, 2004) nor in vivo (present results), sensitized the pituitary to the releasing effects of LHRH, the role of the different ER pools in LHRH-stimulated LH secretion cannot be directly determined from the present experiments. However, two findings strongly suggest that PR actions are involved (Chappell et al. 1999, Sánchez-Criado et al. 2004) in ER actions in TX-treated rats: i) activation of ERβ with DPN halved pituitary PR expression levels and ii) the complete blockade of PR action with ZK299 decreased LHRH-stimulated LH secretion. The absence of a significant effect of activation of ERβ with EB on TX-induced PR levels might be due to PR synthesis-related gene transcription from the large cytosolic pool of ER as EB activates the whole ER orchestra (Levin 2001, 2005). This may have resulted in overlapping ER functions in TX-treated OVX rats. Furthermore, it is also possible that the cognate ligand could have partially displaced TX from nuclear ERα because of the pharmacological doses used. Overall, the accumulated evidence from presented and previous data (Sánchez-Criado et al. 2004, 2006a) suggests that activation of ERβ reduces the efficiency of LHRH in stimulating LH release in TX-treated rats.
The second component of PR-dependent LH surge is oestrogen-dependent LHRH self-priming (Waring & Turgeon 1980, 1992), the property of LHRH that increases gonadotrope responsiveness to itself. It depends both on de novo synthesis of priming proteins (Turgeon & Waring 1991) and on the oestrogen-induced upregulation of PR in gonadotropes (Turgeon & Waring 1994). PR is a neuro-endocrine integrator keystone in the LHRH self-priming process (Turgeon & Waring 1991, 1994, Levine et al. 2001). It has been observed that LHRH self-priming is a convincing nuclear ERα-mediated effect (Sánchez-Criado et al. 2004). Activation of ERα with PPT induces PR expression and elicits LHRH self-priming in OVX rats, whereas activation of ERβ alone with DPN induces PR expression not followed by LHRH self-priming (Sánchez-Criado et al. 2004). The fact that administration of TX to OVX rats induced both PR expression and LHRH self-priming further supports a nuclear ERα agonist action of TX at the rat pituitary level. Although DPN reduced PR expression levels in TX-treated rats, and LHRH self-priming depends on PR, the remnant PR, about one-half of that found in TX-treated rats, may have been enough to elicit LHRH self-priming once activated in a ligand-independent manner (Levine 1997, Blaustein 2004). This finding rules out the possibility of an ERβ involvement in EB inhibition of TX-induced LHRH self-priming. The finding that treatment with either EB or PPT, but not DPN, blocked TX-elicited LHRH self-priming sharply contrasted with the facilitatory action of both ERα agonists on LHRH self-priming in the absence of TX treatment (Sánchez-Criado et al. 2004). Moreover, ZK299 similarly annulled TX-elicited LHRH self-priming. These facts suggest that both EB and PPT acted on an ERα pool different from that bound to TX resulting in inhibition of PR action (Sánchez-Criado et al. 2006b).
On the basis of both the present in vivo and the previous in vitro results (Sánchez-Criado et al. 2004, 2005a,Sánchez-Criado et al. b, 2006a,Sánchez-Criado et al. b), one might hypothesize the following mechanism of E2 action on the complete ER orchestra upon LH secretion in the gonadotrope of the rat: 1) the PR-independent negative oestrogen feedback may be exerted at the nuclear ERα and ERβ complementarily; 2) the positive feedback of oestrogen on LHRH-stimulated LH secretion, a nuclear ERα action, may be modulated by the inhibitory action of ERβ on PR expression (Fig. 9) and 3) the oestrogen-dependent LHRH self-priming may be a nuclear ERα action modulated by surface ERα-initiated signalling inhibition of PR action (Fig. 9).
List of steroid receptor ligands used indicating their action and relevant references
| Action | |||||
|---|---|---|---|---|---|
| nERα | nERβ | mERα | nPR | References | |
| nERα , nuclear oestrogen receptor α ; nERβ , nuclear oestrogen receptor β ; mERα , membrane oestrogen receptor α ; nPR, nuclear progesterone receptor; + , activation; − , blockade. | |||||
| Name | |||||
| Tamoxifen (TX) | + | Tzukerman et al.(1994) and Sánchez-Criado et al. (2005b) | |||
| Oestradiol benzoate (EB) | + | + | + | Sánchez-Criado et al. (2004, 2006b) | |
| Propylpyrazole triol (PPT) | + | + | Stauffer et al.(2000) and Sánchez-Criado et al.(2004) | ||
| Diarylpropionitrile (DPN) | + | Meyers et al.(2001) and Sánchez-Criado et al.(2004) | |||
| Methyl-piperidino-pyrazole (MPP) | − | − | Sun et al.(2002) and Harrington et al.(2003) | ||
| Onapristone (ZK299) | − | Neef et al.(1984) and Bellido et al.(1999) | |||
Luteinizing hormone (LH)-releasing hormone (LHRH) self-priming and LH pituitary content (μg/mg protein) in ovariectomized (OVX) rats injected daily with 0.2 ml oil (oil-3), 25 μg oestradiol benzoate (EB-3) and 3 mg tamoxifen (TX-3) over 3 days (days 15–17 after OVX), or with 3 mg TX alone (TX-6) over 6 days (days 15–20 after OVX) or in combination with 25 μg EB (TX-6 + EB), 1.5 mg PPT (TX-6 + PPT) or 1.5 mg DPN (TX-6 + DPN) injected on days 18–20 after OVX. A single 3 mg ZK299 injection was given on day 20 to TX-treated rats (TX-6 + ZK299). See legend of Figs 2 and 4 for additional details of treatments. LHRH self-priming was expressed as the percentage increase of peak LH response to a second 25 ng LHRH pulse (primed pituitary response) with respect to the LH peak response to the first 25 ng LHRH pulse (unprimed pituitary response) after 1 h. Values are means ± s.e.m. of eight to ten determinations
| LHRH self-priming | LH (μg/mg) | |
|---|---|---|
| *P < 0.05 versus oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test. | ||
| Groups | ||
| Oil-3 | 101.9 ± 8.9 | 54.6 ± 3.1 |
| EB-3 | 152.4 ± 13.7* | 43.3 ± 3.9* |
| TX-3 | 146.4 ± 7.7* | 39.7 ± 3.6* |
| TX-6 | 143.7 ± 3.4* | 35.2 ± 2.4* |
| TX-6 + EB | 100.8 ± 11.0 | 38.7 ± 3.9* |
| TX-6 + PPT | 103.7 ± 10.6 | 38.0 ± 4.2* |
| TX-6 + DPN | 150.5 ± 11.6* | 37.7 ± 4.3* |
| TX-6 + ZK299 | 104.9 ± 8.9 | 31.6 ± 2.3* |
LHRH self-priming and LH pituitary content (μg/mg protein) in OVX rats injected over 6 days (days 15–20 after OVX) with 0.2 ml oil or 1.0 mg selective ERα antagonist MPP alone, or combined with 3 mg TX on days 18–20 after OVX
Serum LH concentrations in ovariectomized (OVX) rats injected on days 15, 16 and 17 after OVX with 0.2 ml oil, 25 μg oestradiol benzoate (EB) or 3 mg tamoxifen (TX). Values are means ± s.e.m. of ten rats. aP < 0.05 versus oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Plasma LH concentrations in ovariectomized (OVX) rats injected on days 15, 16 and 17 after OVX with 0.2 ml oil, 25 μg oestradiol benzoate (EB) or 3 mg tamoxifen (TX). At 0900 h on day 18 (time 0), rats received an i.v. bolus of 25 ng LHRH (first arrow), and a second one 60 min later (second arrow). Blood samples (250 μl each) were taken at 0, 15, 60, 75 and 120 min through a right atrial cannula implanted on the afternoon of day 17 after OVX. Values are means ± s.e.m. of eight to ten rats. aP < 0.05 versus LH values 15 min after the first challenge with LHRH. bP < 0.05 versus oil-injected rats. cP < 0.05 versus LH concentration (time 0) in oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Serum LH concentrations on days 18, 19 and 20 in ovariectomized (OVX) rats injected daily on days 15–20 after OVX with 3 mg tamoxifen (TX) alone, or in combination with 25 μg oestradiol benzoate (TX + EB), 1.5 mg PPT (TX + PPT) or 1.5 mg DPN (TX + DPN) injected over the past 3 days (days 18–20 after OVX) of TX treatment. Values are means ± s.e.m. of ten rats. aP < 0.05 versus TX-injected rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Plasma LH concentrations in ovariectomized (OVX) rats injected on days 15–20 after OVX with 3 mg tamoxifen (TX) alone, or combined with 25 μg oestradiol benzoate (TX + EB), 1.5 mg PPT (TX + PPT) or 1.5 mg DPN (TX + DPN) on days 18, 19 and 20 after OVX. A single 3 mg ZK299 injection was given on day 20 to TX-treated rats (TX + ZK). At 0900 h on day 21 (time 0), rats received an i.v. bolus of 25 ng LHRH (first arrow), and a second challenge 60 min later (second arrow). Blood samples (250 μl each) were taken at 0, 15, 60, 75 and 120 min through a right atrial cannula implanted on the afternoon of day 20 after OVX. Values are means ± s.e.m. of eight to ten rats. aP < 0.05 versus LH levels 15 min after the first LHRH challenge. bP < 0.05 versus LHRH-stimulated LH secretion in TX-injected rats. cP < 0.05 versus LH concentration (time 0) in TX-treated rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Immunohistochemical progesterone receptor (PR) expression in the pituitary of 15-day ovariectomized (OVX) rats. The upper panel shows representative examples of PR expression in OVX rats injected over 3 days (days 15–17 after OVX) with 0.2 ml oil (oil-3), 25 μg oestradiol benzoate (EB-3) or 3 mg tamoxifen (TX-3). An example of the PR expression in the pituitaries of 6-day (days 15–20 after OVX) TX-treated rats is also shown (TX-6). Hypertrophied gonadotropes with (white arrows) or without (arrowhead) PR expression and shrunken gonadotropes expressing PR (black arrows) are shown. Avidin–biotin peroxidase complex immunohistochemical technique, counterstaining with Mayer’s haematoxylin, × 40. The lower panel represents the number of anterior pituitary cells expressing PR in pituitaries from OVX rats injected daily over 3 days with 0.2 ml oil (oil-3), 25 μg EB (EB-3) or 3 mg TX (TX-3) and studied on day 18 after OVX, or over 6 days with TX alone (TX-6) or in combination, over the past 3-day TX treatment, with 25 μg oestradiol benzoate (TX-6 + EB), 1.5 mg PPT (TX-6 + PPT) or DPN (TX-6 + DPN) and studied on day 21 after OVX. Values are means ± s.e.m. of 20 fields (five fields × four rats). aP < 0.05 versus oil-injected OVX rats. bP < 0.05 versus TX-6. cP < 0.05 versus TX-3. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Serum LH concentrations on days 15, 18, 19 and 20 in ovariectomized (OVX) rats injected on days 15–20 after OVX with 0.2 ml oil or 1.0 mg MPP alone or in combination with 3 mg TX on days 18–20. Values are means ± s.e.m. of eight rats. Serum LH concentration on day 15 after OVX is the mean of 32 rats. *P < 0.05 versus oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Plasma LH concentrations on day 21 after ovariectomized (OVX) rats injected on days 15–20 after OVX with 0.2 ml oil or 1.0 mg MPP alone or with 3 mg TX on days 18–20. At 0900 h on day 21 (time 0), rats received an i.v. bolus of 25 ng LHRH (first arrow), and a second challenge 60 min later (second arrow). Blood samples (250 μl each) were taken at 0, 15, 60, 75 and 120 min through a right atrial cannula implanted on the afternoon of day 20 after OVX. Values are means ± s.e.m. of seven to eight rats. aP < 0.05 versus LH levels after the first LHRH challenge at time 0 in TX-injected OVX rats. bP < 0.05 versus oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Immunohistochemical progesterone receptor (PR) expression in the pituitary of 15-day ovariectomized (OVX) rats. The upper panel shows representative examples of PR expression in OVX rats injected over 6 days (days 15–20 after OVX) with 0.2 ml oil or 1.0 mg selective ERα antagonist MPPalone, or combined with 3 mg tamoxifen (TX) on days 18–20 after OVX. Hypertrophied gonadotropes with (white arrows) and without (arrowhead) PR and shrunken gonadotropes expressing PR (black arrows) are shown. The lower panel represents the number of anterior pituitary cells expressing PR in pituitaries from OVX rats injected with oil, MPP, TX or MPP + TX and studied on day 21 after OVX. Values are means ± s.e.m. of 15 fields (five fields × three rats). aP < 0.05 versus oil-injected OVX rats. bP < 0.05 versus TX-injected OVX rats. See legend of Fig. 5 for additional technical and morphological details.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
Proposed integrated action of oestrogen receptor (ER) isoforms and sites with progesterone receptor (PR) in the rat gonadotrope. E2, oestradiol-17β ; P4, progesterone; mERα , plasma membrane ERα ; nERα , nuclear ERα ; nERβ , nuclear ERβ ; LHRH, luteinizing hormone-releasing hormone; LH, luteinizing hormone; GnSI/AF, putative non-steroidal ovarian gonadotrophin surge inhibiting/attenuating factor (Byrne et al. 1996); mis, membrane-initiated signalling; nis, nucleus-initiated signalling. 1. Activation of nERα by granulosa cells E2 transcriptionally induces PR expression and the simultaneous activation of ERβ modulates this action in a ying–yang mode. 2. The E2-dependent LHRH surge activates PR in the absence of P4 in a ligand-independent manner. 3. P4 from luteinized granulosa cells in response to LH phosphorylates and activates PR. 4. Ovarian E2 activation of mERα stimulates intracellular phosphatases (Sánchez-Criado et al. 2006b), which results in non-transcriptional reduction of PR phosphorylation and decreased LHRH self-priming. 5. The putative ovarian GnSI/AF decreases PR action through membrane-initiated signalling. Broken arrow indicates that PR activation is not the only cause involved in eliciting oestradiol-augmenting and LHRH self-priming factors.
Citation: Journal of Endocrinology 193, 1; 10.1677/JOE-06-0214
(J C Garrido-Gracia and A Gordon contributed equally to this work.)
This study was subsidized by grants (BFU2005-01443 and AGL2006-09016/GAN) from the DGICYT (Spain). The authors are grateful to the National Hormone and Pituitary Program (Baltimore, MD, USA) for the LH RIA kit and to Teresa Recio for technical assistance. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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