Synthesis of estrogens in progenitor cells of adult fish brain: evolutive novelty or exaggeration of a more general mechanism implicating estrogens in neurogenesis?

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.

Mechanisms underlying antigonadotropic effects of some traditional plant extracts in pituitary cell culture.

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.

Effects of some traditional plant extracts on rat oestrous cycle compared with Clomid.

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.

Interaction of some traditional plant extracts with uterine oestrogen or progestin receptors.

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.

Effect of endurance training on oestrogen receptor alpha expression in different rat skeletal muscle type.

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.

Effect of endurance training on oestrogen receptor alpha transcripts in rat skeletal muscle.

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.

Novel intronic promoter in the rat ER alpha gene responsible for the transient transcription of a variant receptor.

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.

Steroid-independent activation of ER by GnRH in gonadotrope pituitary 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.

Estrogen receptor protein and mRNA expression in the ovary of sheep.

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.

Cell-specific mechanisms of estrogen receptor in the pituitary gland.

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.

The sheep estrogen receptor: cloning and regulation of expression in the hypothalamo-pituitary axis.

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.

Sex- and cell-specific expression of an estrogen receptor isoform in the pituitary gland.

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.

Effect of gonadotropin-releasing hormone on estrogen receptor messenger ribonucleic acid level in perifused pituitary cells.

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.

Size heterogeneity of affinity-labeled estrogen receptor in the ram hypothalamo-pituitary axis.

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.

Multiple forms of affinity-labeled estrogen receptors in rat distinct pituitary cells.

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.

Stimulation of LH release from cultured pituitary cells by saponins of Petersianthus macrocarpus: a permeabilizing effect.

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.

Inhibitory effects of saponins from Tetrapleura tetraptera on the LH released by cultured rat pituitary cells.

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.

Combretodendron Africanum bark extract as an antifertility agent. I: Estrogenic effects in vivo and LH release by cultured gonadotrope cells.

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.

Steroid receptors and regulation of LH and secretogranin II (GP87) secretion in pituitary cell aggregates.

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.