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Previous in vivo studies have established that pituitary nitric oxide synthase type 1 (NOS1) is regulated by gonadotropin-releasing hormone (GnRH). The aim of our study was to elucidate the mechanisms of NOS1 regulation by GnRH in rat pituitary cells. Using a perifused cell system, we demonstrated that NOS1 induction was sensitive to GnRH pulse frequency and was maximally induced under continuous GnRH stimulation. In primary cultures of rat pituitary cells, sustained stimulation with the GnRH agonist triptorelin (GnRHa) increased NOS1 protein levels, whereas NOS2 and NOS3 levels were unaffected. NOS1 up-regulation occurred in gonadotroph cells only, in a time-dependent and concentration-dependent manner (maximum increase, 2.5-fold; half-maximal concentration, 0.17 nM). GnRHa effect was mimicked by cAMP pathway activators and, most importantly, was blocked by disruption of the protein kinase A (PKA) pathway using pharmacological inhibitors such as Rp-cAMP or drug phosphatase technology-protein kinase inhibitor (DPT-PKI), a cell-permeant PKI peptide. In contrast, modulation of the PKC pathway and inhibition of the MAPK cascade were ineffective. Overall, these experiments demonstrated that GnRH-induced up-regulation of pituitary NOS1 is mediated notably by the cAMP/PKA pathway. Last, in vivo administration of a GnRH antagonist markedly inhibited the pituitary cAMP rise at proestrus in addition to suppressing NOS1 increase. Altogether, our data suggest that the cAMP/PKA signaling pathway is preferentially recruited under sustained GnRH stimulation in vivo during proestrus, allowing the expression of a specific set of PKA-regulated proteins, including NOS1, in gonadotroph cells.

The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects.

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fish exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fishes exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well-documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

Daldinia concentrica and Psathyrella efflorescens are two fungi used in African traditional medicine. In the present study, their extracts were evaluated for their steroid activities in estrogen- or androgen-dependent cell lines using as endpoints steroid-dependent transcriptional activity and cell proliferation. Treatment of human breast cancer MCF-7 cells with 15 or 30 microg/ml of Daldinia concentrica or Psathyrellaefflorescens extracts in the absence of 17beta-estradiol (E2) significantly increased the transcriptional activity of an estrogen receptor (ER)-dependent reporter gene, in the same range as E2. Similar data were obtained in gonadotrope cell line alpha-T3-1. All the effects were prevented by the pure estrogen antagonist, ICI 182,780. In the absence of steroid addition, the two extracts induced cell proliferation of ER-dependent MCF-7 and Ishikawa Var-I cell lines by approximately 100% of the E2 response. Combination treatments with E2 showed no competitive or additive effects in the two latter cell lines. Interestingly, the extracts had no androgen-like response in androgen receptor (AR)-positive and ER-negative MDA-MB231 cells, suggesting that fungi effects are estrogen specific and extracts are not toxic at used concentrations. Results provided evidence that Daldinia concentrica or Psathyrellaefflorescens extracts induce estrogen-like effects in ER-positive cell lines, which could be responsible of the effects observed in vivo.

Previous works have demonstrated that stem bark extracts of Cola nitida (Sterculiaceae), Afrormosia laxiflora and Pterocarpus erinaceus (Fabaceae) provoked a blockade of female rat ovulation and estrous cycle by inhibiting pituitary LH release in vivo. In addition, these plant extracts exerted an inhibitory effect on LH release of rat pituitary cells. Therefore, these data could explain inhibitory effects of plant extracts on LH release in vivo. The present study was undertaken to examine the mechanisms by which these plants exert their antigonadotropic activities. So, we studied the biological activities of these plant extracts on pituitary cells in culture. Data show that C. nitida, A. laxiflora and P. erinaceus extracts only inhibit LH release and have no effect on FSH release. In fact, A. laxiflora, P. erinaceus and C. nitida extracts diminish LH release in culture medium without acting on rat pituitary cell content. Plant extracts form complexes with basic glycoproteins (but not with acid glycoproteins) and prevent them from entering the cells.

It is well known that the plant kingdom contains numerous bioactive substances affecting the regulation of reproduction. The present study was undertaken to examine the putative contraceptive effects of three traditional plant extracts from Côte d'Ivoire Pharmacopea. It concerns Afrormosia laxiflora (Papilionacea), Pterocarpus erinaceus (Papilionacea) and Cola nitida (Sterculiacea) stem bark. Data showed that treatment of rats with these plant extracts induced ovulation and oestrous cycle blockade at the dioestrous II stage. The analysis of the principal hormones involved in oestrous cycle regulation showed that the plant extracts decreased gonadotropin release (both LH and FSH). In fact, A. laxiflora, P. erinaceus and C. nitida extracts inhibited gonadotropin release as an antiestrogen-like substance., (Copyright 2003 John Wiley & Sons, Ltd.)

Previous work has demonstrated that stem bark extracts of Combretodendron macrocarpum (Barringtoniaceae), Cola nitida (Sterculiaceae), Afrormosia laxiflora and Pterocarpus erinaceus (Fabaceae) blocked the oestrus cycle of female rats through antigonadotropic activity. Moreover, a study of the plant substances responsible for these effects revealed the presence of phyto-anti-oestrogens in these plant extracts. In order to explain the mechanism by which these substances exert their antifertility actions, the interaction of the plant extract with oestrogen and progesterone receptors was studied. All crude extracts exerted inhibition of ((3)H)-oestradiol or ((3)H)-Organon binding to their respective receptors but their relative affinities were much lower than those of oestradiol or progesterone. Respective efficiencies of plant extracts in competing for the oestrogen receptor were as follows: A. laxiflora > P. erinaceus > C. macrocarpum > C. nitida. The efficiency order of competition for the progestin receptor was different to that of oestrogen. The most potent competitor was C. macrocarpum extract, followed by P. erinaceus, C. nitida and A. laxiflora. Moreover, the interaction between oestradiol and plant extracts with the oestrogen receptor was determined to be competitive only for C. macrocarpum and A. laxiflora, whereas all compounds produced a competitive inhibition on the progestin receptor binding. These results suggest that the plant extract binding site was the same site as for the steroid. These results suggest also that crude plant extracts may interfere with natural oestrogen and/or progestagen in vivo by binding to steroid receptors., (Copyright 2003 John Wiley & Sons, Ltd.)

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

Introduction/purpose: To explain the effect of estrogen on skeletal muscle, the presence of estrogen receptor alpha mRNA (ERalpha mRNA) was investigated in human skeletal muscle., Methods: The highly sensitive technique of nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) was applied on a variety of tissue samples of both sexes: women (deltoid, pectoral, and uterus muscles) (N= 3) and men (deltoid muscle) (N= 3). The total ribonucleic acid was isolated from each tissue sample, reverse transcribed in a thermocycler, and nested PCR was then performed with specific primers. The by-products were analyzed by agarose gel electrophoresis. Internal standard 28S was simultaneously amplified. The ERalpha mRNA level was quantitated by using the ERalpha mRNA/28S mRNA ratio., Results: The expected 204-bp product corresponding to ERalpha was amplified in all tested tissue samples, i.e., deltoid, pectoral, and uterine muscles from women and deltoid muscle from men. The ERalpha mRNA/28S mRNA ratios indicating the receptor expression levels in deltoid muscle from men and women were 0.945 +/- 0.393 (mean +/- SD) (N= 3) and 0.973 +/- 0.136 (mean +/- SD) (N= 2), respectively., Conclusions: In conclusion, the nested RT-PCR technique identified the presence of transcript encoding ERalpha mRNA in human skeletal muscles. Semi-quantification did not reveal gender difference.

The aim of this study was to examine whether the expression levels of mRNA of the three estrogen receptor (ER) subtypes, ERalpha, ERbeta, and truncated ER product-1 (TERP-1) found in the rat pituitary gland were modified during gestation, lactation, and postlactation periods. By using relative quantitative RT-PCR, we found that ERalpha mRNA significantly peaked in midpregnancy. However, the ERalpha protein level remained constant. ERbeta gene expression did not change throughout pregnancy, suggesting that it was not related to estradiol levels during this reproductive period. In contrast, both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation, being faintly detectable in early pregnancy. TERP-1 expression was rapidly reversed by lactation, whereas neither pituitary ERalpha nor ERbeta relative levels were significantly altered. In addition, pup removal for 24-96 h on d 9 postpartum significantly reduced the expression of both ERalpha and ERbeta mRNA compared with that in lactating animals, but the expression of TERP-1 mRNA was no longer detected. Collectively, our data indicate that 1) TERP-1, ERalpha, and ERbeta expression levels are differentially regulated in the pituitary; 2) TERP-1 is variably expressed depending on the hormonal environment related to the estrous cycle, pregnancy, and lactation; and 3) TERP-1/ERalpha ratios dramatically change depending on reproductive periods, suggesting a critical role for TERP-1 in reproductive events.

It is well known that oestrogens exert muscle anabolic and metabolic effects. Oestrogens act via specific oestrogen receptor (ER) proteins. The mainly represented oestrogen receptor alpha messenger ribonucleic acid subtype (ER(alpha) mRNA) was described in various tissues including the skeletal muscle. Moreover, it has been shown that endurance training significantly increases ER(alpha) mRNA levels in the female rat gastrocnemius muscle. The aim of this study was to determine if this training programme also modifies ER(alpha) mRNA levels in muscles with different typology, the soleus (slow twitch muscle), extensor digitorum longus (fast twitch muscle) and gastrocnemius (intermediate muscle). So far, two groups of Wistar female rats were set up: untrained (u) (n = 7), and trained (e) (n = 7). The endurance training programme was performed for 7 weeks, 5 days per week and consisted of 1 h of continuous running on an adapted motor-driven treadmill involving progressive intensity and gradient of the treadmill. Three different skeletal muscles, extensor digitorum longus (E), gastrocnemius (G) and soleus (S), were isolated and weighed in the untrained (Eu, Gu and Su) and trained group (Ee, Ge and Se). Semi-quantification of ER(alpha) mRNA levels was performed by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. In order to attest the efficiency of our endurance training programme, the citrate synthase activity (CS) of each muscle was measured by a fluorimetric method. The CS activity was significantly increased with training in the gastrocnemius [100.00 +/- 4.99% in Gu (n = 6) vs. 138.10 +/- 8.82% in Ge (n = 6), P < 0.01] and in the soleus [100.00 +/- 2.92% in Su (n = 7) vs. 115.90 +/- 3.71% in Se (n = 7), P < 0.01] but not in the extensor digitorum longus [100.00 +/- 1.87% in Eu (n = 7) vs. 96.90 +/- 1.55% in Ee (n = 7)]. Concerning the influence of muscle type on ER(alpha) mRNA level (1) in the untrained group, the ER(alpha) mRNA level was significantly higher in soleus muscle compared with gastrocnemius and extensor digitorum longus muscles [0.43 +/- 0.04 in Su (n = 7) compared with 0.31 +/- 0.03 in Gu (n = 6) and 0.21 +/- 0.03 in Eu (n = 7), P < 0.05; P < 0.05); 2] in the trained group, the ER(alpha) mRNA level was significantly higher insoleus and gastrocnemius muscles compared with extensor digitorum longus muscle [0.43 +/- 0.06 in Se (n = 7) and 0.49 +/- 0.05 in Ge (n = 6) vs. 0.12 +/- 0.01 in Ee (n = 7), P < 0.05; P < 0.05]. Indeed, after training, the ER(alpha) mRNA level significantly increased in gastrocnemius muscle [0.31 +/- 0.03 in Gu(n = 6) vs. 0.49 +/- 0.05 in Ge (n = 6), P < 0.01], significantly decreased in extensor digitorum longus [0.21 +/- 0.03 in Eu (n = 7) vs. 0.12 +/- 0.01 in Ee (n = 7), P < 0.01] and was not significantly modified in soleus [0.43 +/- 0.04 in Su (n = 7) vs. 0.43 +/- 0.06 in Se (n = 7)]. The differences in ER(alpha) mRNA level between trained and untrained animals indicate training-induced effects that are specific to the skeletal muscle type.

Endurance training induces, in female rats, alterations of oestrous cycle with decrease in plasma oestradiol levels. Moreover, it is well known that oestradiol concentrations modify oestrogen receptor levels. In order to further explain the effects of oestrogens on skeletal muscles, we hypothesized that endurance training modifies the levels of oestrogen receptor alpha messenger ribonucleic acid (ER alpha mRNA) in rat gastrocnemius muscle. Wistar rats were separated into four groups: male controls (C(m)) (n=7), female controls (C(f)) (n=6), male trained (E(m)) (n=7) and female trained (E(f)) (n=6). The endurance training programme was performed for 7 weeks, 5 days week-1 and consisted of 1 h of continuous running on an adapted motor-driven treadmill. At the end of the training session, the gastrocnemius muscle was isolated, weighed and semiquantification of ER alpha mRNA was performed using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The citrate synthase (CS) activity of the gastrocnemius muscle was measured by a fluorimetric method. The CS activity of the male and female gastrocnemius muscle, respectively, 100 +/- 7% in C(m) (n=7) vs. 120 +/- 14% in E(m) (n=6, P < 0.01) and 100 +/- 13% in C(f) (n=6) vs. 138 +/- 23% in E(f) (n=6, P < 0.01) was significantly increased after 7 weeks of training. The ER alpha mRNA levels were significantly increased in E(f) compared with C(f) (0.49 +/- 0.15 vs. 0.31 +/- 0.11, P < 0.01) but not in E(m) compared with C(m) (0.37 +/- 0.15 vs. 0.37 +/- 0.13). In conclusion, these results demonstrate that 7 weeks of endurance training increased the level of transcripts encoding ER alpha in rats with the increase restricted to the females.

To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells.

In the rat pituitary gland the mechanism responsible for ERalpha regulation has not been fully elucidated. Using transient transfection assays in alphaT3-1 cells, a cell line of gonadotrope origin, we show that GnRH stimulates estrogen response element-containing promoters in an estrogen-independent manner. This effect was strictly ER and GnRH receptor dependent, as no activation of the reporter gene was observed in presence of the anti-estrogen ICI 182,780 or a GnRH antagonist. These data suggest that the GnRH-triggered signaling pathway results in 17beta-estradiol-independent trans-activation of the ERalpha in alphaT3-1 cells. Furthermore, an additive activation was achieved when cells were treated with both GnRH and 17beta-estradiol. In primary pituitary cells, GnRH alone (100 nM) did not cause a significant stimulation of reporter gene activity, presumingly due to the low amount of gonadotropes. Interestingly, the combination of 17beta-estradiol and GnRH resulted in a significant increase in ERalpha trans-activation compared with that in cells treated with 17beta-estradiol alone. This enhancement was prevented by ICI 182,780, showing an ERalpha requirement. Moreover, we show that the effects of GnRH on ERalpha transcriptional activity in gonadotrope cell lines are mediated by the PKC/MAPK pathway. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of ERalpha trans-activation in gonadotrope cells.

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep.

Analysis of the mechanism of action of estrogen receptor shows protein and mRNA polymorphism within distinct pituitary receptor-positive cells. The lactotropes exhibit unique properties in these mechanisms that distinguish them from gonadotropes. Therefore, this cell type constitutes an especially interesting model in the male as well as in the female for estrogen receptor studies.

We have prepared an ovine pituitary cDNA library, isolated a clone containing the full-coding sequence of estrogen receptor (ER) cDNA, and determined its primary structure. This cDNA encodes a protein of 596 amino acids which shows great homology to other mammalian ER sequences, the highest degree being 95% with the porcine receptor. Northern blot analysis of ovine pituitary RNA revealed a 6.3 kb transcript. This receptor was showed to bind a consensus ERE and to be transcriptionally activated by E2. Studies investigating the pattern of expression of the ovine ER mRNA were also carried out, using the reverse transcription/PCR technique. Expression of ER mRNA was analyzed in ram pituitary and hypothalamus after contrasted light regimen and castration. Results showed that the light regimen had no effect on ER mRNA expression whereas castration induced a slight (approximately 20%) but significant increase of ER mRNA expression at both the hypothalamic (P < 0.05) and pituitary (P < 0.01) levels, indicating a negative regulation of ER gene expression by testicular steroids. Since we have previously shown no variations in ER protein levels after castration, data suggest the activation of a complex pattern including both transcriptional and post-transcriptional regulatory mechanisms in the ram hypothalamo-pituitary axis.

1. We examined the potential effect of GnRH pulses on pituitary estrogen receptor mRNA level. 2. The treatment of perifused pituitary cell aggregates with four hourly pulses of GnRH (10nM/1 min/h) resulted in a marked increase in the steady-state level of ER mRNA (25% vs unstimulated control, n = 3). 3. No changes were observed for the LH beta mRNA. Data suggest, for the first time, that a cross-talk between the GnRH and nuclear ER may occur in the gonadotrope cells.

The presence of multiple monomeric forms has been described for the estrogen receptor (ER) in the pituitary gland. We analyzed ER mRNA forms in male and female rat pituitary. A single 6.2-kb ER mRNA species was detected in the male rat pituitary, whereas the female rat pituitary exhibited two ER mRNA forms of 6.2 and 5.5 kb, respectively. The 6.2-kb mRNA was present throughout the different stages of the estrous cycle, while the 5.5-kb mRNA appeared to be restricted to proestrus, suggesting an acute regulation of ER transcription at this stage. The 5.5-kb ER mRNA could be rapidly induced either by 17 beta-estradiol replacement in ovariectomized adult female rats or by priming immature rats with pregnant-mare serum gonadotropin. Using enriched cell populations, an inverse and strong correlation was established between the presence of the 5.5-kb ER mRNA form and the number of gonadotropes. Conversely, the localization of the 5.5-kb mRNA form was demonstrated in lactotrope populations. In order to elucidate the structural modifications in the transiently expressed ER mRNA, a series of reverse-transcriptase polymerase chain reaction amplifications was carried out using several pairs of primers corresponding to the entire ER-coding region. The data showed that no alternative splicing was occurring in the ER-coding region involving a potential role of either 3'- or 5'-untranslated regions. Thus, ER presents a 17 beta-estradiol-dependent transcriptional mechanism triggered on proestrous day and specific to the female lactotropes.

The presence of multiple monomeric forms of estrogen receptor (ER) has been described in different target tissues. Using [3H]tamoxifen aziridine (TA) to covalently label ER and SDS-PAGE to analyze labeled products, ER forms were investigated in ram pituitary and hypothalamus. A major labeled protein of M(r) 60,000-65,000 and a minor species of 50,000-55,000 were found in the pituitary cytosol covalently labeled with [3H]TA. In the hypothalamic cytosol, the major TA-labeled species was the M(r) 50,000 form while the 65,000 ER was difficult to detect. Comparison of ER forms after in vitro translocation of the ER complex in purified nuclei of ram pituitary or hypothalamus again showed major ER forms of M(r) 65,000 and 50,000 for the glandular and nervous tissue respectively, suggesting a biological significance for the M(r) 50,000 species. A similar heterogeneity was also observed in male rats used as controls. Moreover, covalent labeling of cytosol from the pars tuberalis/median eminence area showed the presence of ER in this part migrating with a pattern between those of the hypothalamus and the pituitary. The ER heterogeneity was thus demonstrated in the hypothalamo-pituitary axis. The source of this heterogeneity could be: (1) different ER mRNAs according to tissue type; (2) a specific posttranslational processing such as a specific proteolytic activity within the nervous tissue.

The presence of multiple monomeric forms has been described for estrogen receptor (ER) in different target tissues. Using [3H]tamoxifen aziridine ([3H]TA) to covalently label ER and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze labeled products, ER forms were investigated in pituitary cytosol and purified nuclei from male rats. ER forms were also compared in cellular extracts from gonadotrope-enriched populations (GP), prepared using the fast method of centrifugal elutriation, and from lactotrope-somatotrope fractions (LSP), obtained by sequential use of both elutriation and Percoll gradient sedimentation. A major labeled protein of 60,000-65,000 mol wt (M(r)) and a minor species of 50,000-55,000 M(r) were found in the pituitary cytosol and nuclear extracts covalently labeled with [3H]TA. The same results were obtained after ER covalent labeling from cellular extracts or intact dispersed cells. In gonadotrope-enriched cell population (greater than or equal to 50% LH-immunoreactive cells), the 65,000 M(r) species is the single unique ER form; in the LSP (80% PRL- and GH-immunoreactive cells), the major TA-labeled species is the 50,000 M(r) form, while the 65,000 M(r) ER is hardly detectable. Thus, the prevalence of 65,000 M(r) protein in the initial cell population can be explained by the higher number of binding sites per gonadotrope than per lactotrope cell. In conclusion, ER heterogeneity is demonstrated in pituitary cell populations. The source of this heterogeneity could be due to 1) different ER mRNAs according to cell type, or 2) a specific posttranslational processing, such as proteolytic activity within lactotrope cells.

Aqueous extracts from stem bark of Petersianthus macrocarpus contain substances exhibiting both estrogenic and antiestrogenic potency. Triterpenic saponins were identified and extracted as a bulk. Their action on the in vitro LH released by cultured rat pituitary cells was investigated. P. macrocarpus saponins stimulated the LH release in a dose-dependent manner (from 10 micrograms/ml to 300 micrograms/ml). When added simultaneously, saponins and LHRH exerted initial additive effects on LH release, demonstrating independent mechanisms of stimulation. If cells were pre-treated with saponins for 15 min, the amount of LH released under a subsequent LHRH stimulation was lowered, presumably due to a partial depletion of the cells in hormone (data not presented). However, the action of saponins on LH release did not appear specific since a general permeabilizing effect of the cell membrane was evidenced both by trypan blue exclusion and by analysis of the total protein output. When using low concentrations of saponins (10 micrograms/ml), scanning electron microscopy did not reveal any significant alteration of the cell structure, which explains why the cells remain responsive to LHRH after withdrawal of saponins. With higher concentrations (greater than 30 micrograms/ml), the same analytical studies evidenced numerous perforations of the cell membrane, with subsequent cell death. Two highly purified saponin species were tested on LH release by cultured cells; one of them (petersaponin I) appeared responsible for the observed biological effects in vitro. But as cells were shown to be efficiently protected from saponin effects by the presence of serum, it may be concluded that saponins of P. macrocarpus extracts are probably not candidate molecules promoting the in vivo estrogenic and antiestrogenic effects.

Ethanolic extracts from stem bark of Tetrapleura tetraptera exerted an inhibitory effect on the luteinizing hormone (LH) released by cultured rat pituitary cells. These extracts contained triterpenic saponins, tannins, and flavonoids as estimated through phytochemical screening. Saponins were extracted. They apparently inhibited the luteinizing hormone-releasing hormone (LHRH)-induced LH release, the inhibition level being dose-dependent. Yet, the intracellular LH content remained constant whatever the saponin concentration, which demonstrated a lack of effect on the true release process. Accordingly, an interaction between saponins and LH released into the medium was demonstrated, which led to a decrease in the amount of immunoassayable hormone. This decrease was both time- and dose-dependent. It occurred even in the presence of serum in the medium, which suggests that the inactivation process may occur in vivo. Taken together, these results could explain the anti-gonadotrope properties of T. tetraptera extracts that are used as natural contraceptives in Ivory Coast pharmacopoeia.

An aqueous extract from the stem bark of Combretodendron africanum was prepared to investigate its potency as a regulator of fertility. The intraperitoneal LD50 in mice was 95.2 +/- 4.3 mg/kg. Daily i.p. injections of 50 mg/kg promoted a significant increase in the uterine weight of 25 day-old female rats while pituitary weight remained unaffected. Similar injections for 21 days to mature females blocked the estrous cycle in the luteal phase with a mean length of efficiency of 15 days and decreased plasma LH and FSH levels. Regular cycles were restored 10 days following the last injection. C. africanum extract was shown to compete with estradiol and with progesterone on uterine receptors. Consequently, it is thought to contain substances exhibiting estrogenic (and possibly anti-estrogenic) potency. The effects of the extract on LHRH-induced release were investigated on cultured pituitary cells. Although gonadotropin release was amplified, the extract itself appeared as to be a potent secretagogue not requiring LHRH receptors. It follows that the active molecules contained in C. africanum extracts may be different from classical steroid estrogens.

The gonadotrope polypeptide (GP87), a secretogranin II form, has previously been found to be co-released with luteinizing hormone (LH) by rat gonadotrope cells under luteinizing hormone-releasing hormone (LHRH) stimulation. Nothing is hitherto known concerning its function and regulation. In the present paper, the modulatory effects of steroids on GP87 release were investigated using cultures of rat pituitary cell aggregates. As steroid effects are dependent on the presence of receptors, we firstly demonstrated that the steroid receptors are maintained with their typical characteristics in this culture system. In steroid-free medium, the estrogen receptor concentration increased significantly (P < 0.01) within 7 days in culture (646 +/- 122 fmol/mg DNA, n = 3) when compared to the level in freshly dispersed cells (355 +/- 59 fmol/mg DNA, n = 4) suggesting that cells can synthesize the estrogen receptor and that this cell culture system is suitable for studying regulatory effects of steroids. Pituitary cell aggregates from 14 day-old female rats were maintained for 24 h in the absence or presence of 10 nM estradiol (E(2)) or 50 nM 5?-dihydrotestosterone (DHT) and incubated for 2.5 h with [(35)S] methionine. Perifusion was initiated and 4 min pulses of 20 nM LHRH occurred every 1 h. The effects of steroids were investigated on LH, total and labeled GP87. The results indicated that: (1) DHT pretreatment reduced the amplitude of LHRH-stimulated GP87 and LH release responses; (2) by contrast, E(2) pretreatment enhanced the effects of LHRH on total or labeled GP87 release whereas the LH release was not significantly modified; and (3) when E(2) treatment started only 60 min before the first LHRH pulse, no differences between responses of E(2)-treated and control aggregates were observed. The present data show for the first time that sex steroids can modulate the GP87 release through a direct effect on the pituitary. E(2) could either stimulate the GP87 synthesis or increase its intracellular trafficking. That LH and GP87 do not strictly exhibit the same release response further suggests either some heterogeneity of GP87 localization in gonadotrope sub-types or within secretory granules.

5 alpha-androstane-3 beta,17 beta-diol (Adiol) binds to cytosol proteins from male rat pituitary with a relatively high affinity (KD = 15 +/- 6 nM) and a low capacity (n = 92 +/- 8 fmol/mg protein). These saturable proteins which bind Adiol are characterized as estrogen receptor. This conclusion was based on the binding characteristics, the binding stereospecificity and the sedimentation coefficient in sucrose linear gradients. Moreover. Adiol induces, in vivo, the nuclear translocation of estrogen receptor and some effects of estrogen action. It is efficient to induce progesterone receptor and to increase pituitary protein content but inefficient to increase DNA synthesis. Results suggest a mechanism of Adiol action in the male rat pituitary similar to that observed with androgens in other target tissues. Moreover, the study of Adiol and 17 beta-estradiol binding suggests two forms of estrogen receptor in the cytosol from male rat pituitary. The maximal concentration of binding sites was observed at 22-30 days of age for E2 and at 37-42 days of age for Adiol. On the other hand, the nuclear ontogenic pattern suggested a single class of binding sites for E2 and Adiol in the pituitary nuclei.

5 alpha-DHT is bound with a high affinity (Kd = 0.8 nM) and a limited capacity (80 fmol/mg proteins) in the cytosol of the preen gland of quails, an analogue of sebaceous glands. The complex can be precipitated by protamin-sulfate and is sedimented in the 8 S region after ultra-centrifugation in a sucrose gradient. The occurrence of this binding that exhibits the main properties of an androgen receptor, supports the use of the preen gland of quails as a tool for the study of the mechanism of action of androgens in birds and as a model for the study of the androgen control of sebaceous-like glands.

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.

The concentrations of estradiol (E2) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-Adiol) receptor were quantified in the pituitaries of male rats, aged 15--100 days. The available binding sites were measured directly in pituitary cytosol. E2 receptor levels reached a peak between days 22 and 30 and then declined slightly. 3 beta-Adiol-binding sites increased between days 15 and 37, reached a maximum level at the time of puberty (37--42 days of age), and then declined markedly to become undetectable on day 75. Occupation of cytosol binding sites by endogenous steroid was investigated by means of an exchange assay. No occupation was seen for E2 binding. On the other hand, 3 beta-Adiol-binding sites were partly occupied on day 43 and completely occupied on day 75. The E2 and 3 beta-Adiol association constants remained relatively constant throughout the entire period studied. Total E2- and 3 beta-Adiol-binding sites were measured by an exchange technique in nuclear extracts. The ontogenic patterns of E2- and 3 beta-Adiol-binding sites were similar at all ages. The number of E2- and 3 beta-Adiol-binding sites reached a maximum on days 28--30 and remained constant up to day 75. These data demonstrate the developmental appearance of pituitary E2 and 3 beta-Adiol receptors in the male rat. They suggest two forms of estrogen receptor in pituitary cytosol; one could be involved in the onset of puberty. Moreover, the nuclear ontogenic pattern suggested a single class of binding sites for E2 and 3 beta-Adiol in the pituitary nuclei.

Radioactive testosterone is very quickly collected by the mullerian ducts, and then metabolized in androstanolone and androstanediols. According to these investigations, the mullerian duct shows an usual feature of the effectors of androgens. The possible rôle of the different metabolites is discussed.

The proteins co-released with gonadotropins were analyzed using perifusion of pituitary cell aggregates from 14-day-old female rats, after a pre-labeling period with [35S]methionine. Radioimmunoassays of hormones and electrophoretic analysis were performed on each 4 min effluent. Gonadotropin-releasing hormone (GnRH) pulses increased significantly (P less than 0.01) the release of several proteins (Mr range from 140,000 to 28,000). The main stimulation appeared for -1, a 87 kDa species, previously characterized as gonadotrope polypeptide 87 (GP87) in monolayer cultures and identified as a secretogranin II (SgII) form; -2, a second species of 80 kDa designated as B2. Secretory patterns of radiolabeled GP87 and B2 paralleled the luteinizing hormone (LH) ones. The release of these species was -1, GnRH dose dependent; -2, monophasic for short pulses but complex when the duration of GnRH pulses increased to 16 min, suggesting different pools of GP87 and B2 as for LH; -3, induced by thyrotropin-releasing hormone (TRH). A slight output was also elicited by corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), but this release was partly impaired in the presence of a potent anti-GnRH ([Ac-D-(2)-NAL1,pF-D-Phe2,D-Trp3,D-Arg6]-LAF) suggesting a non-specific effect of these two factors. GP87/SgII thus appeared mainly associated with the release of hormonal glycoproteins. In conclusion, perifusion of pituitary cell aggregates allows a precise minute-to-minute kinetic analysis of the various proteins co-released with hormones. The similar timing in output of LH, GP87 and B2 suggests that these three proteins co-exist in the same secretory granules inside gonadotropes.

The effect of castration on pituitary androgen and estrogen nuclear receptors was examined in eleven 3-year-old 'Préalpes du Sud' rams which were either intact (N = 5) or surgically castrated (N = 6) 11 months before. To avoid a receptor loss, pituitaries were frozen less than 1 min after slaughter. Specific androgen and estrogen bindings were measured upon extracts from purified nuclei. Neither the concentration of estrogen receptors (30.5 +/- 3.0 vs 28.1 +/- 3.0 fmol/mg protein) nor the affinity constant (ka = 1.21 X 10(9) mol-1 in both groups) differed between intact and castrated rams. Conversely, androgen receptor concentrations differed markedly between groups and were found significantly higher in intact rams (11.3 +/- 1.1 fmol/mg protein) than in castrated rams where nuclear receptors were undetectable in 5 out of 6 animals. This result keeps open the possibility that the decrease of LH sensitivity to testosterone negative feedback observed after long-term castration, could be related to the quasi absence of nuclear receptors, at least at the pituitary level.

The in vitro action of hydro-alcoholic extracts of plants from Ivory Coast pharmacopoeia was analyzed on cultured rat pituitary cells. Cells were treated for 24 hours with various doses of extracts and then stimulated for 4 hours with 10(-8) M LHRH. Extracts from Afrormosia laxiflora, Cola nitida, Pterocarpus erinaceus and Tetrapleura tetraptera inhibit the LHRH-induced release of LH. On the contrary, extract from Combretodendron africanum stimulates the basal release of LH and this increase is added to the LHRH-induced release. Therefore, the natural substances contained in these plants may in vitro exert a regulation of the gonadotropin release.

The distribution of 5 alpha-dihydrotestosterone (DHT)-binding sites within specific pituitary cell populations, as compared with the estradiol (E2)-binding sites distribution, could be estimated after gonadotrope cell enrichment. Centrifugal elutriation allowed the purification of large gonadotropes from 42-day-old male rat pituitaries; this population contained 5.3 +/- 1.0% of the cells, 54 +/- 3% of the LH, and less than 5% of PRL. A gonadotrope-depleted fraction was also defined that contained more than 92% of the total PRL and was thus called the lactotrope population though it still contained small gonadotropes and most somatotropes. DHT and E2 receptors were quantified by multidose saturation analysis. The association constants (Ka) ranged from 0.2-0.5 X 10(9) M-1 both for E2 and DHT independent of the cell population. The binding of DHT was mainly observed in the gonadotropes (50-100 fmol/10(6) cells), while it was very low in the other cells (0.9-3 fmol/10(6) cells). E2-binding sites measured within the same cell populations showed a broader distribution than DHT-binding sites. Whereas the ratio of binding DHT/E2 averaged 0.86 +/- 0.01 in the gonadotropes, it reached only 0.35 +/- 0.07 in the lactotropes. Thus, using enriched cell populations, our results demonstrate for the first time a quantitative selective distribution of androgen-binding sites as compared with the estrogen sites.

5 alpha-Dihydrotestosterone (DHT) metabolism into 5 alpha-androstane-3 alpha, 17 beta-diol (alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (beta-diol) was studied in monolayer cultures of distinct cell populations from prepubertal male rats pituitaries. Cells were characterized through immunocytochemistry with the various antihormone antisera. Centrifugal elutriation was used to prepare a gonadotrope-enriched population "G" and a gonadotrope-depleted population "L", containing most lactotropes and somatotropes. Using centrifugation on Percoll gradient, two sub-populations, P1 and P2, were prepared by further fractionation of the "L" population. Cells were incubated for 48 h with [3H]DHT (1 microM, sp. act. 0.9 Ci/mmol) and metabolites extracted from the whole cell and medium. DHT was metabolized to about the same extent (30-40%) in all cell fractions. Compared with unfractionated population, the conversion of DHT into alpha-diol increased significantly in the P1 fraction, consisting of lactotropes, somatotropes and highly depleted in gonadotropes. This increase was lower in the somatotrope-enriched P2 fraction in which the amount of lactotropes was similar to P1 but that of gonadotropes slightly higher. In contrast, the conversion of DHT into alpha-diol decreased significantly in the "G" population compared with total or "L" fractions, whereas androstanedione formation, low in every population, increased significantly. The increase in alpha-diol formation could be related either to the decrease of gonadotropes or to a role of non-immunoreactive cells. As the beta-diol formation was constant in all cell types, the beta-diol/alpha-diol amount increased significantly in gonadotropes. Then, beta-diol and DHT could be both active steroids in gonadotrope regulation inasmuch as specific binding sites were identified for these two steroids. It can be concluded that DHT action at the pituitary level is subject to complex control mechanisms involving a specific balance of its metabolites in each particular cell type.

Dispersed pituitary cells from 42-day old male rats were separated using centrifugal elutriation. Based on LH and PRL cellular contents, fractionated cells were pooled into two fractions: "Lactotrope++ population" and "gonadotrope++ population". Estradiol and 5 alpha-androstane-3 beta, 17 beta-diol binding was measured in these fractions. Results revealed that: (1) The steroid receptors are not destroyed by cell dispersion and elutriation. (2) The estradiol receptor content is higher in gonadotrope++ cells than in lactotrope++ cells. (3) The number of binding sites for the two steroids changes in the different fractions: whereas it is exactly similar in "lactotrope++ population", it is much higher for estradiol than for 5 alpha-androstane-3 beta, 17 beta-diol in "gonadotrope++ population". These results suggest two different species--or conformations--of receptor binding sites for estradiol in the male rat pituitary; the first one could link both steroids, the second one would be specific for estradiol.

The methodology to fully characterise nuclear receptor for oestradiol-17 beta (E2) in the ram pituitary has been investigated. Purified nuclei, clean under the electron microscope, were obtained from 2.4 M sucrose ultracentrifugation and were extracted for 2 h at 0 degrees C with 0.6 M NaCl. After centrifugation, the supernatant was incubated with [3H]E2 with or without a 100-fold excess of unlabelled E2. The main results were: the specific binding was maximum at 20 degrees C in 2-3 h and remained constant up to 19 h without significant metabolism; an incubation temperature of 25 degrees C reduces the binding, while at 0 degrees C maximum binding was attained at a much slower rate; the binding was linearly related to the dose of nuclear proteins; the binding was not affected by DNase and RNase but was suppressed by trypsin, pronase or a temperature of 56 degrees C; binding was specific for oestrogens; preincubation of cytosol with [3H]E2 and then coincubation with nuclei showed an uptake of the [3H]E2 receptor complex by nuclei; such a transfer was inhibited if cytosol was previously heated; after a prelabelled cytosol-nuclei coincubation, a specific binding peak was found in the nuclear extract submitted to sucrose gradient sedimentation (4.1S); in vivo injection of 100 micrograms E2 resulted in a sharp increase in nuclear receptor numbers 30 and 60 min later, with a concomitant drop in cytosolic receptor numbers. These results indicate that E2 can bind to pituitary nuclei in the ram.

When anterior hypophysis from male rats were incubated in the presence of tritiated 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol), the major part of recovered pituitary radioactivity was constituted by 5 alpha-dihydrotestosterone (5 alpha-DHT). This steroid represented an average of 72% of total tissular radioactivity. In cytosol, the bound hormonal fraction was constituted by about 47% 5 alpha-DHT and 35% 3 alpha-diol, whereas free radioactivity contained 67% 5 alpha-DHT and 22% 3 alpha-diol. These results demonstrate an intensive conversion of 3 alpha-diol into 5 alpha-DHT and consequently that the transformation of 5 alpha-DHT into 3 alpha-diol is reversible. Thus it is reasonable to think that the biological action of 3 alpha-diol one can observe following in vivo or in vitro experiments is due, in fact, to its conversion into 5 alpha-DHT.

The in vivo and in vitro metabolism of (3H)-5alpha-androstane-3lapha,17beta-diol by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5alpha-androstane-3alpha, 17beta-diol into 5alpha-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73% of the recovered radioactivity were constituted by 5alpha-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5alpha-dihydrotestosterone was the main steroid recovered except from the 105,000 X g pellet. From in vivo and in vitro experiments it was concluded that the transformation of 5alpha-dihydrotestosterone into 5alpha-androstane-3alpha, 17beta-diol was a reversible process, and that this last steroid could exert its biological action mainly via 5alpha-dihydrotestosterone.