Direct effects of sex steroids on prolactin release at the anterior pituitary level: interactions with dopamine, thyrotropin-releasing hormone, and isobutylmethylxanthine

When present alone for 4 or 8 days, 5 alpha-dihydrotestosterone (DHT) or the pure progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) inhibits spontaneous PRL release by 33--50% in rat anterior pituitary cells in primary culture. This inhibitory effect of DHT and R5020 can only be partially reversed by 17 beta-estradiol (E alpha). DHT and R5020 inhibit spontaneous PRL release in E2-primed cells at ED50 values of 0.5 and 3 nM, respectively. While E2 diminishes by 30--60% the maximal inhibitory effect of dopamine on PRL release and increases by 10-fold the ED50 value of dopamine action, DHT and R5020 can prevent by 30--60% the action of E2 and thus increase the potency of dopamine to inhibit PRL release. The inhibitory action of DHT and R5020 as well as the stimulatory action of E2 on spontaneous PRL release are similarly expressed on TRH- and 3-isobutyl-1-methylxanthine-induced PRL release, thus suggesting that at least part of the highly effective modulatory effects of sex steroids are exerted at a step after cAMP formation.     

The influence of chronic morphine treatment on the negative feedback regulation of gonadotropin secretion by gonadal steroids

The influence of continuous stimulation of opiate receptors with morphine (M) on the negative feedback effects of testosterone (T), 5 alpha-dihydrotestosterone (DHT), and 17 beta-estradiol (E2) on LH and FSH secretion was studied in rats that had been castrated 2 weeks previously. In the absence of gonadal steroids, 4 days of continuous M exposure did not alter LH or FSH levels. Similarly, Silastic capsules containing crystalline T (5 mm) or E2 [5 mm long (75 micrograms E2/ml) to 7.5 mm long (300 micrograms E2/ml)] alone had little effect on LH or FSH release. However, in M-exposed rats, T reduced serum LH by greater than 90%, and E2 reduced LH by more than 75%. Among the doses of DHT evaluated, only the highest dose (7.5-mm Silastic capsules packed with crystalline DHT) reduced LH secretion, and M exposure only slightly enhanced this suppression. M or gonadal steroids alone produced little change in FSH levels in castrated rats. However, the combination of M plus E2 or DHT further reduced FSH levels. Evaluation of pituitary responses to LHRH revealed that when administered alone, T did not alter, DHT reduced, and E2 enhanced the LH response to the decapeptide. Neither M treatment alone nor M plus T or DHT altered the pituitary LH response to LHRH. On the other hand, M appeared to enhance the stimulatory effects of E2 on pituitary responsiveness to LHRH. These findings suggest that the interaction of M and gonadal steroids at the level of the pituitary could not explain the observed marked suppression of gonadotropin secretion by suboptimal T or E2 during opiate receptor stimulation with M. Collectively, these observations are in accord with the view that endogenous opioid peptides may play a role in modulating the sensitivity of the hypothalamus to the negative feedback effects of gonadal steroids.     

Multihormonal control of pre-pro-somatostatin mRNA levels in the periventricular nucleus of the male and female rat hypothalamus

The influence of sex steroids as well as the possible involvement of dopaminergic pathways in the modulation of pre-pro-somatostatin (SS) mRNA levels was investigated by quantitative in situ hybridization in the hypothalamic periventricular nucleus (PeN) in adult male and female rats. In situ hybridization was performed using a [35S]-labeled cDNA probe encoding pre-proSS mRNA. Gonadectomy performed 14 days earlier decreased the mean number of silver grains/neuron corresponding to the relative pre-proSS mRNA levels by 22% in male and by 18-28% in female rats. A 14-day treatment with the nonaromatizable androgen dihydrotestosterone (DHT) increased the mean number of silver grains/neuron by 34-40% in gonadectomized animals of both sexes. Moreover, administration of 17 beta-estradiol (E2, 0.25 microgram twice daily) increased pre-proSS mRNA levels by 40% in ovariectomized (OVX) animals. Such treatment with E2 or DHT changed the frequency distribution profile of the hybridization signal intensity, thus increasing the percentage of highly labeled neurons (greater than or equal to 61 grains/neuron) by 10 to 12-fold. A 14-day treatment with the D2 dopamine receptor agonist bromocriptine (BRO) increased pre-proSS mRNA levels by 15 and 28% in intact female and OVX animals, respectively, while the dopaminergic antagonist haloperidol (HAL) decreased the value of this parameter by 20 and 30%. Furthermore, BRO increased pre-proSS mRNA levels by 10 and 20% in intact and castrated male rats, respectively, whereas HAL decreased pre-proSS mRNA levels by 25 and 14% in the same groups of animals. Administration of E2 in combination with HAL in OVX animals increased pre-proSS mRNA levels by 70% compared to those measured in OVX animals treated with HAL alone. In HAL-treated castrated male rats, administration of DHT increased the relative pre-proSS mRNA levels by 35% compared to those measured in castrated animals treated with HAL alone. The present data clearly demonstrate that androgens and estrogens as well as dopamine-mediated mechanisms could play a regulatory role in pre-proSS mRNA levels in somatostatinergic neurons in the hypothalamic PeN in both male and female rats.     

Prolactin messenger ribonucleic acid concentrations in 4-day cycling rats and during the prolactin surge

Pituitary PRL mRNA concentrations were measured during the 4-day rat estrous cycle. Adult female Sprague-Dawley rats were killed at 3-h intervals throughout the cycle and hourly between 1000 and 2400 h on proestrus (n = 5-12). Serum PRL was increased on the afternoons of proestrus (P) and estrus (E), with peak concentrations at 1700 h (P, 624 +/- 126; E, 261 +/- 107 ng/ml). PRL mRNA concentrations were elevated during the evening on P and E (2300 h: P, 14.4 +/- 1.5; E, 16.1 +/- 1.3 ng cDNA bound/100 micrograms pituitary DNA) to values 2-fold higher than those at 0800 h on each respective day. On diestrus (D) PRL mRNA levels decreased abruptly during the morning (1100 h, 1.7 +/- 0.3 ng cDNA bound), followed by a 6- to 7-fold increase between 1700 and 2000 h on the same evening. In contrast, PRL mRNA levels were elevated at 0800 h on metestrus (M). The changes in PRL mRNA concentrations obtained on M and D were not associated with increased PRL secretion. A more detailed examination of P revealed that PRL mRNA levels increased during the morning (1000 h, 9.9 +/- 2.6 ng cDNA bound), then decreased abruptly at 1100 h (4.9 +/- 1.2). The morning rise in mRNA concentrations was followed by a 2-fold rise in pituitary PRL content. As serum PRL rose during the afternoon surge, a coincident decrease in pituitary PRL content and an increase in PRL mRNA levels were observed. The relationship between PRL secretion and gene expression was further examined in ovariectomized estradiol-replaced rats receiving either bromocriptine (1.2 mg/day, sc) or vehicle control sc. The vehicle-treated group expressed a characteristic afternoon PRL surge between 1500 and 2100 h. Pituitary PRL decreased during the surge to 10% of morning values, and PRL mRNA levels increased 2-fold beginning 2 h after initiation of the surge. These changes in serum PRL, pituitary PRL, and PRL mRNA levels were abolished by bromocriptine administration. These data reveal that alterations in PRL mRNA concentrations occur on a daily basis during the rat estrous cycle. Increases occur during the evenings of P and E at the time of the increase in PRL secretory activity. The effect of blocking the PRL surge in ovariectomized estradiol-replaced rats suggests a regulatory interaction between secretion and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are key factors in the brain-pituitary-gonad axis and understanding their regulation remains essential for future management of eel reproduction. In this regard, we developed quantitative real-time RT-PCR (qrtRT-PCR) assays for the expression of European eel LHbeta, FSHbeta and GPalpha subunits, using the Light Cycler system. The qrtRT-PCR was adapted to permit detection of the three gonadotropin subunit mRNAs in individual pituitaries and in dispersed pituitary cells. The validated assays were applied to investigate the effects of sex steroids (estrogens and androgens) on gonadotropin subunit expression, in vivo in steroid-injected eels, and in vitro by steroid treatments of primary cultures of eel pituitary cells.In vivo, a stimulation of LHbeta mRNA was observed after estradiol (E2) treatments, while testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT) had no effect. Concerning FSHbeta expression, slight but non-significant decreases were observed after both E2 and androgen treatments. Different results were obtained in vitro: E2 induced an increase in FSHbeta mRNA levels but had no effect on LHbeta expression. In contrast, androgens (T and DHT) stimulated LHbeta expression while no significant variation was observed on FSHbeta mRNA levels following androgen treatment. Concerning the GPalpha mRNA, no significant effect of sexual steroids was observed in vivo or in vitro. This demonstrated specific direct actions of steroids on gonadotropin subunit expression. The differences observed between in vivo and in vitro experiments may be explained by the involvement of cerebral control, including GnRH and dopamine neurons, and their specific regulation by sex steroids. The data indicate that sex steroid feedbacks on gonadotropins are exerted via multiple pathways, indirectly at the brain level and directly on pituitary gonadotrope cells.     

Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells

Roles of follicle-stimulating hormone (FSH) and sex steroids in regulating the expression of mRNA species encoding the alpha-, beta A- and beta B-subunits of inhibin were studied in cultured granulosa cells from immature rat ovaries. Inhibin subunit mRNAs were detected by Northern blot analysis of total RNA extracted from granulosa cell monolayers which had been incubated for 48 h in serum-free medium containing FSH (100 ng/ml) and/or a steroid (10(-6) M): estradiol (E), testosterone (T) or 5 alpha-dihydrotestosterone (DHT). Levels of mRNA encoding each inhibin subunit in untreated (control) cultures were low. In cultures treated with FSH alone, levels of inhibin alpha-, beta A- and beta B-subunit mRNA were approximately 60-fold, 70-fold and 66-fold greater than control, respectively. In cultures treated with E alone, levels of inhibin alpha- and beta B-subunit mRNA were elevated approximately 4-fold and 2-fold, respectively, but the level of inhibin beta A-subunit mRNA was not measurably affected. Treatment with T or DHT alone had no consistent effect on the levels of any inhibin subunit mRNA. The stimulatory effects of FSH were not consistently altered by the presence of either androgen or estrogen. These results confirm the role of FSH in regulating inhibin alpha-subunit gene expression and provide direct evidence that both inhibin beta-subunit genes are inducible by FSH in granulosa cells. All three inhibin subunit mRNAs followed the same pattern, suggesting that their expression is coordinately regulated by FSH during granulosa cell differentiation.     

Gonadotropin gene expression and secretion in gonadotropin-releasing hormone antagonist-treated male rats: effect of sex steroid replacement.

The possibility of direct pituitary effects of sex steroids on gonadotropin gene expression and synthesis was studied in male rats. The animals were treated with a potent GnRH antagonist, Ac-D-pClPhe-D-pClPhe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-+ ++NH2CH3COOH (Org 30276; 0.5 mg/kg BW, sc, twice daily) for 10 days. Groups of the antagonist-treated rats were implanted at the beginning of the injections with Silastic capsules containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), or diethylstilbestrol (DES). Groups treated with the antagonist alone or vehicle served as controls. The antagonist treatment decreased unoccupied pituitary receptors of GnRH by 93% (P less than 0.001), serum LH by 34% (P less than 0.01), and serum FSH by 30% (P less than 0.05), and serum T became undetectable (less than 0.10 nmol/liter). Compared to antagonist treatment alone, no further effects on serum or pituitary LH levels found after steroid replacements. In contrast, the antagonist-induced decreases in serum and pituitary FSH (30% and 70%, respectively; P less than 0.05-0.01) were totally reversed by the T and DHT implants, but not by DES. Pituitary levels of the LH beta-subunit mRNA were decreased by 60% (P less than 0.01) after antagonist treatment. Combination treatment with androgens had no further effect on this mRNA, whereas DES partially reversed this suppression (P less than 0.05). In contrast, the pituitary mRNA level of the FSH beta-subunit, which decreased with antagonist treatment by 90% (P less than 0.01), returned to the control level with T and DHT replacements, but only partially with DES. The pituitary mRNA level of the common alpha-subunit was significantly suppressed only by combined antagonist plus DHT treatment (P less than 0.01). However, combination of DES with the antagonist increased alpha-subunit mRNA levels 2.4-fold (P less than 0.05) compared to antagonist treatment alone. It is concluded that the suppression of gonadotropin secretion by GnRH antagonist treatment is accompanied in male rats by a parallel reduction in mRNA levels of the gonadotropin beta-subunits. Sex steroid replacement of the antagonist-treated animals selectively reverses some of the mRNA changes. Androgens (T and DHT) increase the mRNA of FSH beta-subunit, but have no effect on the LH beta-subunit. Estrogen increases the mRNA levels of common alpha- and LH beta-subunits and slightly increases that of FSH beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Additive stimulatory action of glucocorticoids and androgens on basal and estrogen-repressed apolipoprotein-D messenger ribonucleic acid levels and secretion in human breast cancer cells.

Recent elucidation of the amino acid sequence of the progesterone-binding protein GCDFP-24, the major protein found in human breast gross cystic disease fluid, reveals that this glycoprotein corresponds to apolipoprotein-D (apo-D), a member of the alpha 2-microglobulin superfamily which binds small hydrophobic ligands. The present study describes the multiple hormonal control of apo-D mRNA levels, intracellular protein content, as well as secretion compared to cell proliferation in human ZR-75-1 breast cancer cells. In these cells, exposure to the synthetic glucocorticoid dexamethasone (DEX) markedly decreases basal as well as 17 beta-estradiol (E2)-induced cell proliferation while causing a maximal 10-fold stimulation of apo-D secretion in the presence or absence of E2. Incubation with 500 nM DEX or 10 nM dihydrotestosterone (DHT), alone and in combination, markedly increased apo-D mRNA/actin mRNA ratios by 16-, 22-, and 28-fold, respectively. Exposure to 1 nM E2 decreased the apo-D mRNA/actin mRNA ratio by 65%. In E2-treated cells, simultaneous exposure to DHT, DEX, and DHT plus DEX markedly increased the apo-D mRNA/actin mRNA ratios by 50-, 35-, and 105-fold, respectively. The stimulatory effect of DEX on intracellular apo-D content and secretion was also additive to that of the androgen DHT in the presence or absence of E2. The present study provides the first data describing the hormonal regulation of apo-D mRNA levels and intracellular protein content and demonstrates the effect of glucocorticoids alone as well as their interaction with androgens and estrogens on these parameters as well as on apo-D secretion. As shown in the present data, the effects of steroids on apo-D gene expression, intracellular apo-D protein content, and secretion are opposite their respective specific effects on cell proliferation in human ZR-75-1 breast cancer cells.     

Population density alters the responsiveness of GH4C1 pituitary tumor cells to 17 beta-estradiol

In this study we have examined the effect of population density on the ability of 17 beta-estradiol (E2) to induce PRL mRNA, DNA synthesis, and progesterone receptor in GH4C1 pituitary tumor cells. These parameters were examined at three subconfluent population densities that varied over a 4-fold range. The culture medium was changed daily in these experiments to reduce the possibility of nutrient depletion, medium toxification, or E2 metabolism. At the low population density, a 5-day treatment with E2 at a concentration of 1.0 nM resulted in an 8.1-fold increase in the level of PRL mRNA, as measured by the cytosolic dot blot procedure. At the intermediate density, E2 induced PRL mRNA 2.4-fold. At the high density, the level of PRL mRNA was reduced by 50% in response to 5 days of treatment with E2. The cytosolic dot blot procedure would reflect changes in the level of PRL mRNA per cell as well as changes in the number of cells per culture. Therefore, the level of beta-actin mRNA was also measured, assuming that it would remain constant on a per cell basis. When the level of PRL mRNA was normalized to the level of beta-actin mRNA in the same cytosols, E2 increased PRL mRNA 2.6-fold in the low density cultures and 1.9-fold in the intermediate density cultures, and had no effect on the level of PRL mRNA in the high density cultures. The effect of population density on the induction of GH4C1 cell proliferation by E2 was examined directly by measuring cellular DNA, thymidine incorporation by whole cells, and deoxythymidine triphosphate (dTTP) incorporation by isolated nuclei. At the low density, E2 initially stimulated GH4C1 cell proliferation, as evidenced by an increased rate of dTTP incorporation. However, this stimulatory effect was lost by day 5 of treatment, as the population density of the E2-treated low density cultures increased. At the high density, the inhibitory effect of E2 on dTTP incorporation was observed by day 2 of treatment and thereafter become more pronounced. These stimulatory and inhibitory effects of E2 were also revealed when the level of cellular DNA was measured. In contrast to the effects of population density on the induction of PRL mRNA and cell proliferation, an increase in density had no observable effect on the induction of progesterone receptor by E2. These data illustrate the critical importance of the culture environment in studies examining estrogen action in vitro.     

Role of dopamine in the regulation of gonadotropin-releasing hormone in the male rat brain as studied by in situ hybridization.

The effects of the dopaminergic antagonist haloperidol (HAL) as well as the D2 dopamine receptor agonist bromocriptine (BRO) on GnRH messenger RNA (mRNA) levels in the male rat were investigated by quantitative in situ hybridization. Since it had already shown that androgens could induce a decrease in GnRH mRNA levels in castrated rats, the involvement of the dopaminergic system in the inhibitory effect of dihydrotestosterone (DHT) was also investigated. In situ hybridization was performed on paraformaldehyde-fixed cryostat sections through an area extending from the preoptic area to the anterior hypothalamus using a 35S-labeled 48-base oligodeoxynucleotide complementary to the GnRH coding region of the GnRH DNA. The corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In intact animals, a 14-day treatment with BRO increased by 67% the number of silver grains per neuron while HAL decreased by 31% the value of this parameter. Hypophysectomy which induced a 22% decrease in the hybridization signal could not prevent the effects of BRO or HAL. Gonadectomy performed 14 days earlier increased by 54% the mean number of silver grains while a 14-day treatment of gonadectomized animals with DHT decreased the hybridization signal by 48%. On the other hand, the concomitant administration of HAL and DHT did not prevent the inhibitory effect of DHT but rather resulted in a decrease of the hybridization signal which was more marked than that induced by DHT or HAL alone. The present data clearly demonstrate that GnRH mRNA levels are positively regulated by dopamine and that the effects of BRO and HAL on GnRH mRNA are not mediated by variations in pituitary hormone secretion. Moreover, our results suggest that the effect of DHT on GnRH gene expression is probably not mediated by the dopaminergic system.     

Absence of direct regulation of prolactin cells by estradiol-17 beta in rainbow trout (Oncorhynchus mykiss).

The effects of estradiol-17 beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17 beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17 beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17 beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of rat pituitary prolactin messenger ribonucleic acid and hormone content with serum levels during the estrogen-induced surge

The model of the serum PRL surge generated in the ovariectomized rat after estradiol benzoate (EB) treatment was used to study the relationship between serum and pituitary PRL levels and pituitary PRL mRNA levels. Adult ovariectomized rats were injected sc with 7 micrograms EB or vehicle at noon on day 0. Three days later (day 3), the rats were decapitated every 4 h over a 24-h period (0800 h on day 3 to 0400 h on day 4) for determination of serum and pituitary PRL and GH levels by RIA. In addition, PRL and GH mRNA content was determined using dot blot hybridization with cDNAs. Administration of EB resulted in a significant rise in serum PRL levels at 1200, 1600, and 2000 h on day 3 compared to control values. At other times, serum PRL levels in the EB group were the same as control values. EB treatment also elicited a marked increase in pituitary PRL content at all time periods examined except during (1600 and 2000 h) and after the PRL surge (2400 h on day 3) when there was a significant reduction in stored pituitary PRL. The pituitary PRL mRNA content in the EB-treated group was significantly elevated (4- to 6-fold) over control levels throughout the study. Furthermore, PRL mRNA levels in EB-treated rats were significantly higher at 2000 and 2400 h on day 3 than at other time periods. In contrast to its effects on PRL, EB treatment had a slight inhibitory effect on pituitary GH content at 2000 and 2400 h on day 3 compared to control values; otherwise, this steroid had no effect on serum GH levels and pituitary GH mRNA content. Interestingly, serum GH levels and pituitary GH mRNA content in both treatment and control groups fluctuated in a pattern consistent with circadian rhythms, with peak values occurring during the lights-on hours. These data show that estrogen has a stimulatory effect on pituitary content of PRL and its corresponding mRNA in the rat 3 days after injection. These elevated PRL mRNa levels may be necessary for the occurrence of PRL surges. Furthermore, the facts that serum PRL levels were elevated only at certain times (1200-2000 h on day 3) while PRL mRNA content was increased at all times in the EB-treated rats suggest a differential regulation between PRL release and biosynthesis.     

Regulation of galanin gene expression in the rat anterior pituitary gland by the somatostatin analog SMS 201-995.

In addition to inducing pituitary tumors in rats, estrogen (E2) markedly increases galanin and PRL gene expression. We previously showed that galanin secretion from pituitary cells in vitro is inhibited by dopamine and somatostatin and stimulated by TRH. The objectives of these in vivo studies were to assess whether the long-acting somatostatin analog SMS 201-995 alters 1) immunoreactive galanin or PRL levels in the anterior pituitary, neurointermediate lobe, hypothalamus, or plasma, 2) pituitary galanin and PRL mRNA levels, and 3) the development of E2-induced pituitary tumors. Ovariectomized Fischer 344 rats were implanted with E2-filled or empty Silastic capsules and treated with or without SMS 201-995 (1.5 mg) via Alzet miniosmotic pumps. Two or 6 weeks later, immunoreactive galanin and PRL levels were determined by RIA. In ovariectomized rats, the somatostatin analog lowered the anterior pituitary content of galanin by 50%, but had no effect on PRL concentrations. E2 increased galanin and PRL levels in the anterior pituitary by 220- and 4-fold, respectively. Concomitant E2 and SMS 201-995 treatment further increased galanin and PRL in the anterior pituitary by 60-80%, but decreased plasma galanin and PRL levels. Likewise, the administration of SMS 201-995 for 2 and 6 weeks inhibited the E2-induced growth of the anterior pituitary. Galanin and PRL mRNA levels were quantified by solution hybridization. Galanin mRNA levels were reduced to undetectable levels in ovariectomized rats treated with SMS 201-995. Furthermore, a 10-fold increase in galanin mRNA levels seen in the presence of E2 was inhibited 80% by SMS 201-995. PRL mRNA levels in E2-treated rats were unchanged by SMS 201-995. We conclude that SMS 201-995 1) lowers plasma galanin and PRL levels in E2-treated rats, 2) elevates the anterior pituitary contents of galanin and PRL in E2-exposed rats, probably through decreased secretion of the hormones, and 3) reduces galanin mRNA levels in E2-treated and untreated ovariectomized rats. Overall, these results establish the differential regulation of galanin and PRL gene expression in vivo by SMS 201-995. Moreover, the data demonstrate that somatostatin receptor agonists may have therapeutic potential for some prolactinomas.     

Differential involvement of adrenal and gonadal steroids in anterior and intermediate pituitary pro-opiomelanocortin mRNA expression induced by the endogenous benzodiazepine, octadecaneuropeptide, in adult male rats.

The involvement of the endogenous benzodiazepine, octadecaneuropeptide (ODN), in the regulation of proopiomelanocortin (POMC) mRNA expression at the pituitary level, and the influence of adrenal and gonadal steroids, have been studied using a quantitative in situ hybridization technique. I.c.v. injection of ODN (4 micrograms/kg) in sham-operated rats induced a 17 and 7% decrease in the POMC mRNA expression in anterior and intermediate pituitary lobes respectively. To determine the reciprocal involvement of adrenal and gonadal steroids in this regulation, animals were adrenalectomized and/or castrated. Adrenalectomy significantly increased POMC mRNA expression by 48% at the anterior pituitary level, but induced a 10% decrease of hybridization signal at the intermediate pituitary lobe (vs control sham-operated). Adrenal ablation reversed the effect induced by ODN and increased POMC mRNA expression at the anterior and intermediate pituitary levels by 60 and 10% respectively, compared with control sham-operated. By contrast, castration, which produced a decrease in POMC mRNA in the anterior pituitary and an increase in the intermediate lobe, did not modify the negative influence of ODN observed in sham-operated animals. When rats were adrenalectomized and castrated, the adrenalectomy influence was predominant at the anterior pituitary level, since ODN increased significantly the hybridization signal (+68% vs control sham-operated), while the castration influence was predominant at the intermediate pituitary level, since ODN induced an 11% decrease in POMC mRNA signal compared with control sham-operated. These studies indicate that, in vivo, the decrease in POMC mRNA expression in the anterior and intermediate pituitary induced by an endogenous benzodiazepine is differently modulated by adrenal and gonadal steroids, with a predominant influence of adrenal steroids at the anterior pituitary level and gonadal steroids at the intermediate pituitary level.     

The effects of estrogen and progesterone on corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid levels in the paraventricular nucleus and supraoptic nucleus of the rhesus monkey

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17beta-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 microm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density + SEM, 0.38 +/- 0.06, 0.13 +/- 0.08, and 0.14 +/- 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.     

Prolactin suppresses luteinizing hormone secretion and pituitary responsiveness to luteinizing hormone-releasing hormone by a direct action at the anterior pituitary

In pathological or experimental hyperprolactinemia, the elevated circulating levels of PRL are the usual cause of the impairment in gonadotropic function. The present study was undertaken to determine whether PRL could suppress basal LH secretion and LHRH-stimulated LH release by a direct action at the anterior pituitary. Anterior pituitaries from ovariectomized rats were incubated in medium 199 alone or in medium 199 containing ovine PRL, and basal and the LHRH-stimulated LH release were followed for 2 or 3 h in vitro. Ovine PRL at 40 and 80 micrograms/ml suppressed basal LH release by 41% and 72%, respectively, at 2 h of incubation. This suppressive effect of both concentrations of PRL continued to the third hour of incubation. LHRH at 5 ng/ml increased the release of LH from pituitaries incubated in medium alone by 57%, 61%, and 107% at 1, 2, and 3 h of incubation, respectively. However, in the pituitaries treated with 40 micrograms/ml ovine PRL, the stimulatory effects of LHRH were diminished at all time points measured. Pretreatment of anterior pituitaries with ovine PRL for 6 h significantly inhibited by 81% the LHRH (5 ng/ml) stimulation of LH release at 2 h of incubation. On the other hand, inhibition of endogenous PRL release by 10(-6) M bromocriptine enhanced the stimulatory effects of 5 ng/ml LHRH by 2.5-fold at 2 h of incubation. The inhibitory effects of PRL on basal and stimulated LH secretion appeared unique, since neither BSA nor vasopressin could elicit similar suppressive effects on LH. These results suggest that in anterior pituitaries exposed to elevated levels of PRL, LH secretion and pituitary responsiveness to LHRH could be impaired. This phenomenon may contribute in part to the antigonadotropic effects of PRL.     

Effects of estrogens on the ultrastructural characteristics of female rat prolactin cells as evaluated by in situ hybridization in combination with immunogold staining technique

We have recently reported that, in the rat pituitary, prolactin (PRL) is synthesized and stored in two different cell types: the typical PRL cell characterized by the presence of large irregular secretory granules and another cell type containing small round secretory granules. Since the secretion of PRL is stimulated by estrogens, we decided to investigate the effect of gonadectomy and estradiol (E2) replacement therapy in female rats on both cell types using a combination of in situ hybridization and immunostaining. Gonadectomy induced a marked decrease in hybridization signal and an almost complete disappearance of the classical PRL cells. The most abundant PRL cells were the small-granule-containing cells and other PRL cells characterized by the presence of both small and large granules. These changes in the PRL cell population were completely prevented by E2 administration to castrated animals. These studies indicate that E2 cannot only regulate PRL mRNA but also induce important morphological changes in PRL cell types.     

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats

We have examined the changes that occur in neuronal expression of LHRH mRNA in response to castration and hyperprolactinemia in male rats. Single cell levels of LHRH mRNA were determined by quantitative in situ hybridization histochemistry using an 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Nine days postcastration, a 10.4-fold increase in mean plasma LH titers was observed which was associated with significantly increased LHRH mRNA in rostral hypothalamic neuronal cell bodies. Both increases were blocked in rats rendered hyperprolactinemic by the presence of the 7315a PRL-secreting pituitary tumor. The location and number of neurons expressing LHRH mRNA were unchanged, indicating that these differences were attributable to amounts of mRNA expressed per neuron. Experimental differences occurred in LHRH perikarya situated throughout the rostral hypothalamus from the organum vasculosum of the lamina terminalis to the caudal extent of the medial preoptic nucleus. These results suggest that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of LHRH in the rostral hypothalamus.