Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells GABHELA T P ~ R EAND Z MARTAE. APFELBAUM* Ciitedra hie Fisiologia, Departamento de Famaco/ogia, Facultad de Ciencias Qui'micas, Universidad National de Cbrdoba and Chtedra de Farmcokogia, Facubfad de Ciencias MPdicas, Unkvessidad Nacionab de Cbrdoba, Cbsdoba, Argentina
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Received September 24, 1991 PEREZ,G. T., and APPELBAWM, M, E. 1992. Modulatory effect of steroid hormones on CnRH-induced LH secretion by cultured rat pituitary cells. Can. J . Physiol. Pharmacol. 70: 363 -969. The purpose of the present experiments was to examine the short- and long-term effects of estradiol-119B (E,), progesterone (P), and 5a-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 Ea in steroid-free medium, pituitary cells were further cultklred for 24 h in medium with or without E2 (1 nM), P (100 nM), s r DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of l nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show ghat the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E, resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E,. Conversely, B caused an acute stimulatsry action (118%) on the LH released in response to GnIZH and a slightly inhibitory effect (90%)after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) kH were observed in pituitary cells treated with E2 or BHT. While the stimulatory effect of E, was evident after both acute (133 %) and chronic (1 19%) treatment, that of DHT appears to be exerted mainly after long-term priming (1 18 %). These results suggest that the steroids modulate GnRH-induced LH secretion by acting on both synthesis and release sf LH. On the other hand, total hormone content was not affected by P. The acute (5 h) effects of E2, P, and BHT on the GnRH response in E,-primed (24 h) cells during a short-term incubation, were also tested. Addition of P to the pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of E2 on GnRH-induced LH release and total content. Conversely, the augmentative E2 effect on pituitary responsiveness to GnRH was abolished by DHT. Taken together, these findings suggest that the physiological significance of the stimulatory action of progesterone could be to define the final magnitude of the LH preovulatory surge, while the inhibition by DHT could be required to limit the LH surge to that day of proestms. Key words: luteinizing hormone, gonadotrophin-releasing hormone, steroid hormones, cultured pituitary cells.
P ~ R E ZG. , T., et APFELBAUM, M. E. 1992. Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells. Can. J . Phy siol. Pharmacol . 78: 963 - 969. Le but des pr6sentes expkriences a 6tC d'examiner les effets B court et 2 long terme de 190estradiol-1'7b(E,), de la progestCrone (P) et de la 5a-dihydroxytestosthne (BHT), seuls ou csmbinCs, sur la sCcr6tion de I'hormone 1utCinisante (LH) induite par l'homone de liberation des gonadotrophines (GnRH), en utilisant un modkle de culture de cellules hypophysaires de rats ovariectomises. Aprbs une incubation de 72 h dans un milieu d 6 p o u m d'hormones stCroYdes, les cellules hypophysaires ont CtC cultivCes pedant une autre pCriode de 24 h avec ou sans E, (1 nM), P (1W nM) ou DHT (10 nM). Ensuite, les cellules cultivCes ont CtC incub6es pendant 5 h en l'absence ou en prCsence de 1 nM de GnRH, avec saa sans homones stCroides. h i s , la LH a CtC CvaluCe dans le milieu de culture et les extraits des cellules par radio-immunodosage. Les rksultats rnontrent que les homones stkroides exercent des effets opposks sur la 1ibCration de LH irnduite par la GnRH, libkration qui semble &re dCpendante de la longueur de la phiode d'exposition des cellules hypophysaires auw hormones stCroYdes. En effet, l'action A court terms (5 h) de I'E, a provoquk une inhibition partielle (64% des valeurs tCmoins) de la libkration de LH en rCponse 2 la GnRH, alors que I'exposition de longue dur6e (24 h) a stimulk sa libCration (158%). Bes 5Csultats similaires ont CtC obtenus avec la DHT, bien que l'amplitude de l'effet ait 6t6 infkrieure 2i celle obtenue avec l'E2. A l'oppos6, la P a eu un effet stimulateur aigu (1 18%) sur la libkration de LH en rCponse B la GnRM et un effet l6gkement inhibiteur (90%)aprks un traitement chronique. La biosynthbse de la LH stimulCe par la GnRH a aussi CtC influencke par le traitement a m hormones stCroTdes. Des augmentations signifieatives de la teneur totale en LH (cellules plus milieu) ont Ctk observkes dans les cellules hypophysaires traitees avec l'E2 ou la BHT. Alors que l'effet stimulateur de l'E, a dtC manifeste tant aprks le traitement chronique (1 19 %) qu'aprks le traitement aigu (133 %), celui de la DWT semble s7Ctreprincipalement exerck aprks une sensibilisation 8 long terme (118%). Ces rksultats suggtrent que les hormones stkroi'des modulent la skcretion de LH induite par la GnRH en agissant autant sur la synthkse que sur la libkration de LM. Par ailleurs, le teneur hornonale totale n'a pas kt6 influencke par la B. Les effets aigus (5 h) de 17E2,de la P et de la DHT sur la rCponse 8 la GnRH dans les cellules sensibiliskes (24 h) B 19E,, durant une incubation de couae duke, ont aussi kt6 examinks. L'addition de P aux celldes hyp'~physairessensibiliskes avec 1'E2 a\ entrain6 une potentialisation aigue Be l'effet stimulateur de l'E, sur la teneur totale en LH et aa libkration par la GnRH. A I'opposC, 19effetstimulant de 19E2sur la rCactivitC pituitaire B la GnRH a Ctd supprim6 par la BHT. Ensemble, ces rCsultats suggkrent qu'en termes physiologiques, le sens de l'action stimulatrice de la progestkrone poumait etre de dCfinir l'amplitude finale de la bouff6e prkovulatoire de LH, alors que I'inhibition par la DHT pourrait &re nkcessaire pour limiter la buffke de LH au moment du proestms. Mots c&&: hormone lutkinisante, hormone de libkration des gonadotrophines. hormones stkroides, cellules hypophysaires cultiv6e.s. [Traduit par la rkdaction] 'Correspondence may be sent to the author at the following address: Chtedra de Fisiologia, Departamento de Famacologia, Facultad Ciencks Quimicas, Universidad Nacional de Cbrdoba, Suc. 16, Casilla de Correo 61, 58 16 C6rdoba, Argentina. Printed in Canada i Imprime au Canada
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Introduction Cyclic patterns of circulating gonadotrophins are a basic characteristic of the female rat estrous cycle. It is generally accepted that physiological levels of the gonadotrophins are mainly regulated by the hypothalamic gonadotroghin-releasing hormone (GnRH) and the ovarian hormones, although the mode of regulation and the interaction between these factors are not yet defined. Analysis of the sequence of hormonal events during the estrous cyck shows that rising titers s f plasma estrogen on the afternoon of diestrus and the morning of proestms act as a primaray stimulus for the preovulatory luteinizing hormone (LH) surge on the afternoon of proestms (Ferin et al. 1969; Legan et al. 1975). Once the preovulatory surge has been initiated, the ascending phase of LH secretion is accompanied by a rapid rise in progesterone and a decline in estrogen levels (Butcher et al. 1974; Smith et al. 1975). In addition, testosterone and androstenedione rise in the systemic circulation on proestms, in concert with the surge of gonadotrophin (Dupon and Kim 1973). While the role of estrogen in triggering the preovulatory surge of LH is well recognized, relatively less attention has been given to the role of progesterone in regulating LH secretion. For in vivo studies, a biphasic effect s f progesterone on LH release, with an acute stirnulatory and chronic inhibitory action, has been shown (Caligaris et al. I97 1; Mahesh and Muldson 1987). Much less is known on the functional significance of circulating androgens in a normal cycling rat. Some observations indicate that androgens can differentially affect the release of LH and folicle-stimulatinghormone (FSH) from the anterior pituitary, thereby favoring the proportional release s f FSH (Gay and Tomacari 1974). Undoubtedly, timing, magnitude, and duration of the sequence of changes in hormonal feedback are critical in achieving a normal reproductive cycle. Numerous studies have shown that ovarian steroids can either enhance or inhibit gonadotrophin secretion, depending upon the dosage, length of treatment, and hormonal environment at the time of administration. The steroid hormones modu%ate gonadotrophin secretion by acting at both the hypothalamic Bevel on GnRH secretion, mQ at the pituitary level on responsiveness of gonadotrophs to GnRH (Karsch 1987). The biochemical mechanisms and site of action by which the steroids influence GnRH-stimulated LH secretion are unhown. At the pituitary level, the feedback actions of steroids an gonadotrophs may involve both regulation of GnRH receptors (Clayton and Catt 1981; Menon et d. 198%)and modulation of post-receptor events. Several studies have demonstrated that steroids are able to act on different steps of the complex second messenger system involved in the GnRH transduction system (Emons et al. 1989; Liu and Jackson 1988). Nevertheless, the results do not provide direct evidence about their ~nechanismof action. In earlier studies with in vitro hemipituitary glands from ovariectomized rats (Apfelbaum 1983; Apfelbaum and Taleisnik 1977) we reported that ovarian steroids can exert specific effects on LH basal secretion by a direct action at the pituitary level. The purpose of the current experiments was to further characterize the short- and long-term effects of physiological doses of estradiol-170 (E2), progesterone (P) and the nonaromatizable androgen 5a-dihydrotestostersne (DHT), alone and in combination, on GwRH-induced LH secretion using an ovariectomized rat pituitary cells culture model as a preliminary study to explore subsequently the molecular events
mediating these actions around the critical preovulatory period in the rat.
Materids and methods Hornones Gonadotrophin-releasing hormone was obtained from Bachem Inc. (Torrance, CA, U. S.A .). Estradiol- 170, progesterone, and Sa-dihydrotestostersne were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), Stock solutions sf steroids were prepared in 0.9% NaCl - I % ethanol and were used at a I@3-fold dilution in the incubation medium, such that the final concentration of vehicle in the medium was 0,01%. Dispersion and culture of mt anterior pituitary cells Adult female rats (200 -250 g) from our colony of albino rats from Wistar strain were ovariectomized 30 clays before being used. Animals were housed in a controlled environment (21 f 1°C; 14-h light : 10-h darkness) with food and water available ad libitum. The animals were killed by decapitation. The anterior pituitary glands were rapidly excised and placed into a petri dish containing 5 mL of supplemented Spinner minimum essential medium (S-SMEM) calcium- and magnesium-free, which consisted of 0.25% BSA, 13 mM HEPES, 50 pg/mL streptomycin, and 50 U/mL penicillin. The pituitaries were cut with a razor blade in fine pieces and washed with 10 mE S-SMEM. The tissue blocks were then transferred to a 50-mL Erlenmeyer flask containing 0.25% trgipsin (1:258 porcine pancreatic trypsin; Bifco, Detroit, MI, U.S.A.) in S-SMEM (0.5 mL per pituitary) and incubated for 20 rnin at 37°C in a shaker bath. The medium was removed and replaced with 4 rnL S-SMBM containing I mg soybean trypsin inhibitor (type I-S; Sigma) and the incubation was continued for another 3 min. One hundred micrograms of deoxyribonuclease (DNase; bovine pancreatic deoxyribonuclease type DN-lQ0, Sigma) was present during all steps of incubation to avoid clumping. The tissue blocks were then transferred to a conical centrifuge tube and washed three times with 10 mL S-SMEM. The final dispersion of the pituitary cells was accomplished by m a n s of repeated gentle uptake and expulsion with a flame-polished Pasteur pipette. Aker complete dissociation, the cells were centrifuged (250 X g; 5 rnin), washed twice with S-SMEM, and resuspended in supplemented Medium 199 (S-Ml%9),which consisted of Medium 199 with Earle's salts plus 0.25 % BSA, 25 mM HEPES, 100 pg/rnL streptomycin, 100 U/mE penicillin, 50 pglmL Nystatin, 8% horse serum, and 2% fetal bovine serum. Both sera were pretreated with dextran-coated charcoal (Drouin and Labrie 19'96) to remove endogenous steroids. The number of cells obtained after dispersion was determined with a hemocytorneter; cell viability was established by the trypan blue exclusion technique. This procedure yields 3.54.5 x lo6 cells per pituitary, and more than 95% of cells were viable. Finally, the cells were plated in Falcon tissue culture dishes (3 x 106 cells per 35-mm dish) in 1.5 mL S-MI99 and cultured at 39°C under a humidified atmosphere of 95% air : 5 % CO,. An additional aliquot of 1 mL fresh culture medium was added to each dish 48 h later. After plating (72 h), the media were decanted and the cells were further cultured for 24 h in 1.5 mL S-MI99 in the presence or absence of the indicated steroid to be tested. Following the 96-h culture period, the culture medium was discarded and the cells were washed twice with 2 mL per dish of Dulbecco9s modified Eagle's medium (DEM) supplemented with 0.25 % BSA to remove serum and nonadhered cells, prior to use in short-term incubations. All media were filtered through 0.22-yrnMilipore filters (Millipore Corp., Bedford, MA) before use, the final pH being 7.4. The culture media, antibiotics, and chemicals were purchased from Sigma, and the sera from Grand Island Biochemical Co. (Grand Island, NY, U.S.A.). Short-term incubatioris of cultured cells After 96 h of culture, short-term incubations were carried out at 37 OC under a humidified atmosphere of 95% air : 5% CO,. Two experimental designs were used to establish the effects of ovarian steroid
TABLE1. Percentages of basal LH rdeased and contained in the total system (cells plus medium) by pituitary cells from ovariectomized rats cultured in suspension or in monolayer and incubated for different times
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Incubation time (h)
% LH in suspension
% LH in monolayer
Medium
Medium
Total
Total
NOTE:Control cultures were processed for hormone determinacells; monolayer: tion at 0 h (suspension: 4.04 k 0.30 kg13 x ceHHs). Amount of LH in cells incubated 5.26 k 0.45 pgIP x for 2,4, and 6 h was expressed as percentage of the respective control cultures (taken as 100%). Values are means f SEM of five cultures per group.
hormones on bas& and CwRH-stimulated release, cell content, and total (cells plus medium) LH by cultured pituitary cells from ovariectomized rats. In the first, the acute effects of gonadal steroids were studied. After 3 days in culture the medium was discarded and the cells were hrther cultured for 24 h in 1.5 mL S-MI99 with 0-017% ethanol (vehicle). Pituitary cell cultures were then washed twice with DEM and incubated for 5 h in DEM containing 1 nM E,, 100 nM P, 10 nM DHT, or 0.81% ethanol vehicle (control) with or without 1 nM G n W . In the second approach, the chronic effects of gonadal steroids were studied by pretreating the cells with steroids before the GnRH challenge. Pituitary cells were cultured for 3 days in S-M 199, followed by 24 h in fresh S-M189 containing I nM E,, 100 nM P, 10 nM DHT, or vehicle (control). The cells were then washed as above and subsequently incubated in DEM with vehicle in the presence or absence of 1 nM GanRH. Hn the third set of experiments the combined action of steroids on the GnRH response of pituitary cells was studied. Cells primed for 24 h with 1 nM E, or vehicle were incubated for 5 h with 1 nM G n M in the presence of 1 nM E2, 100 nM P, HO nM DHT, or vehicle. Incubations were terminated by adding 1 mL phosphate-buffered saline (PBS), pH 7, and chilling the dishes on ice. The media were removed, centrifuged at 2508 x g for 15 rnin at 4 "C, and kept frozen at -20°C until assayed. Cells were washed once with 2 mL PBS and lysed by incubation in 1.5 rnL of ice-cold 50 mM Na,C03 in 8.1 M NaOH for 10 min. The neutralized cell lysates were then frozen and thawed three times and treated with Triton X-100 (1 94 final concentration). The samples were then centrifuged at 35 000 x g for 30 min. Supernatants were decanted and stored at -20aC.
Preliminary experiments The purpose of the preliminary experiments was to establish our rat anterior pituitary cell culture system. Two systems of cultured cells either in suspension or in rnonolayer were tested. After dissociation, the pituitary cell suspension was aliquoted half into 5-mL glass tubes and the other half into culture dishes at 3 X 1Q6 cells per tube or dish, cells in tubes being cultured for 22 h while those in dishes for 96 h as described above. After the respective culture period, almost all of the cells placed in tubes are present in suspension, whereas those in dishes are attached to the surface as a monolayer, the viability of cells k i n g higher for cells in monolayers Q > 90%) than for those in suspension ( > 75 %). The time course of basal release, cell content, and total (cells plus medium) LM in both preparations was studied. The cultured cells, either in suspension or monolayer were washed twice with DEM and incubated for different periods (0 - 6 h) in 1.5 mL of the same medium. Control cultures were processed for homone determination at 0 h for the measurement of total LM content initially present in the incubation system. The response of cultured pituitary cells to GnRH was also analyzed in both preparations. Cultured cells, in suspension and in monolayer were incubated for 5 h
TABLE2. Effect of increasing concentrations of SnRH on LH released and contained in the total system (cells plus medium) by pituitary cells from ovariectomized rats cultured in suspension or in monolayer
GnRH (nM)
% LH in suspension
Medium
Total
% EH in monolayer
Medium
Total
NOTE:Pituitary cells were incubated for 5 k in the absence (control) or presence of different doses of GnRH. Responsiveness of pituitary cells to GnRH was expressed as percentage of the respective control (medium: 0.5 1 i 0.04 and 0.28 f 0.01 ygl3 X lo6 cells; total: 6.02 i 0.45 and 5.10 f_ 0.46 kg13 x lo6 cells far suspension and rnonolayer, respectively) cultures (taken as I%%). Values are means & §EM of five cultures per group.
in the absence (control) or presence of different doses of GnRH (0.1 - 100 nM).
LH mdioimrnunoassay lmmunoreactive LH was measured in the medium and the cell extract at two dose levels by a double-antibody radioimunsassay, with reagents supplied by the U.S. National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (NEDDK), National Institutes of Health. The amount of LH was calculated in terms of NIDDK rat standard rLH-RP-2. Hntraassay coefficient of variation (CV) for LH assay was 6.2%. Corresponding interassay CV was 10.0%.The results were expressed as micrograms of EH per 3 x 1Qh cells.
Statistics The data were analysed by analysis of variance and Duncan's multiple range test. A level sf p < 0.05 was considered statistically sigSEM. nificant. All results are expressed as the mean
+
Results Suspension versus rno~asla~~er cell culture system Two separate experiments were performed to evaluate the functional capacity of the cultured pituitary cells in both suspension and monolayer. The bas& release, cell content, and total (cells plus medium) LH as a function of incubation time as well as the ability of the cultured pituitary cells to respond to increasing doses of GnRH were studied in both systems. When the amount of basal LH in the medium is expressed as a percentage of total LH contained in the respective control cultures at 0 h (taken as loo%), LH is released in greater proportion from the cells in suspension than from the attached cells (Table I). Respnsiveness of pituitary cells to GnRH is expressed as a percentage of the respective untreated control cultures ts compare both preparations (Table 2). Addition of GnRH to the incubation medium greatly stimulated ( p < 0.01) the release sf LH in a dose-related manner in both systems, the LH net response being greater in cells in rnonolayer than in those in suspension. The EDSovalue sf GnRH action was calculated to be 0.69 anM for monolayers and 1.75 nM for suspensions. Hn addition, GnRH-stimulated total LH content was significantly increased (10 nM GnRH, p < 0.05; 100 nM GazRH, p < 0.01) in the case of rnonolayers, while that of cells in suspensisn was unchanged. On the basis of these data. cultured cells in monolayer were selected as the experimental model for the study of steroid hsrmane action on LH secretion.
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FIG. I. Effects of ovarian steroids on basal LH secretion after short- and long-term treatment. Amount of LH released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by pituitary cells from ovariectotniaed rats incubated for 5 h is shown, as described in Materials and Methods. Cultured cells were (a) acutely (5 h) or (b) chronically (24 h) exposed to 1 nM estradiol-By@(B), 100 nM progesterone (El), and 10 nM Sa-dihydrotestosterone (B). Control were incubated in absence of steroid hormones. The groups (8) height of the bar gives the mean value of five cultures and the dot represents the %EM.
Eflects of steroids on basal LH secretion a@er short- and longterm trealneent Figure I summarizes the effects of actue (5 h) and chronic (24 19) treatment with 1 IM E2, 106) nM B, and BO nM DHT om basal LH secretion by pituitary cells from ovariectomized rats. Ethanol (vehicle), added into the medium at the final concentration of 0.0 1% (vlv), did not modify basal release, cell content, or t s t d (cells plus medium) LM by pituitary cell cultures irrespectively of the time of treatment (Table 3). When pituitary cells were pretreated for 24 h with vehicle and subsequently incubated for 5 h in medium containing steroids (Fig. IQ), basal release of LH was not affected by E2 and DHT, whereas a small though not significant increase (1 16.3% of control, p > 0.05) in LH release was observed in cells acutely exposed to P. Priming the cells for 24 h with steroids also failed to affect basd release of LH from cultures incubated for 5 h with vehicle (Fig. 1b). At the end of incubations the amount of LH remaining in the cells was higher than that in control in all the groups studied (Fig. la, and Ib). Comparatively, the effect of E2 after both acute (117.I%, p < 0.05) and chronic (123.6%, p < 0.81) treatment was greater than that of B (acute: 115.1%, p > 0.05; and chronic: 111.9%, g > 0.05) or DHT (114.7%, p > 0.05, and 118.9%,p < 0.81). Treatment s f pituitary cells with steroids resulted in an enhanced total (cells plus medium) LH in the system (Fig. l a and Ib). This stimulating effect was more pronounced for E2
PIG. 2. Effects of ovarian steroids on GnRH-stimulated LH secretion after short- and long-term treatment. Amount of LH released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by pituitary cells from ovariectomized rats incubated for 5 h with 1 wM GnRH is shown, as described in Materials and Methods. Cultured cells were (a) acutely (5 h) or (b) chronically (24 h) exposed to 1 nM estradiol-178 (B), 180 nM progesterone (B), and BO wM Sa-dihydrotestosterone (P). Control groups (a)were incubated in absence of steroid hormones. Groups of cultures incubated in medium alone (we GnRH or steroids) (B)were also included to check the response to GnRH in this experiment. The height of the bar gives the mean value of five cultures -and the dot represents the SEG.
(acute: 116.1%,pa > 0.05; and chronic: 1%3.3%,p < 0.81) than for P (115.276, p > 0.05; and 111.3%,p > 0.05) or DHT (1%4.0%,p > 0.05; and 117.8%, p < 0.05). Eflects of steroids on GnRH-stkmalated EH secretion afier short- and long-term treatment Figure 2 shows the LH secretory response to 1 nM GnRH by cells acutely or chronically exposed to 1 nM E2, 100 nM P, or 10 HIMDHT. Vehicle treatment s f the cells did not induce any changes in the LH response to GnRH administration (Table 3). When cells were pretreated with vehicle for 24 h and then incubated for 5 h in medium containing GnRH and vehicle, GwRH stimulated LH release (567.5 % of control, g < 0.01) and total LH (1 16-676,p < 0.05) but was without effect on LH cell content (94.4%, p > 8.05). GnRH-stimulated LH release was modified by the treatment with steroids. Addition s f E2 or DHT during the 5-h incubation period decreased to 63.9% ( p < 0.0%)and 82.4 % ( p < 8-05), respectively, the LH release having been induced by GnRH (Fig. 2u). On the contrary, pretreatment s f cultures with E2 or DHT before challenging the cells with GnRH increased (I58 %, g < 0.01; and 115.4 9% ,p > 0.85, respectively) the response to GnRH (Fig. 2b). Conversely, acute administration of P enhanced (1 18.5% , p < 8.01) (Fig. 2a), and chronic administration sf P slightly decreased (90.7%, p > 0.05) (Fig. 2b), the LH released in response to GnRHH.
P ~ R E ZAND APFELBAUM
TABLE3. Effect of ethanol (vehicle) on basal and GnRH-induced LM intracellular, released, and contained in the total system (cells plus medium) by cultured pituitary cells from ovariectomized rats
LH &g/3 --
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Time of treatment
-
X
lo6 cells)
-
Basal Medium
Cells
GnRH Total
Medium
Cells
Total
NOTE: Pituitary cell cultures incubated for 5 h in the absence (Basal) or presence of 1 wM CnRH were either 5 h) with co-incubated (5 h), pretreated (24 h) before incubation, or exposed ta the combined treatment (24 h 0.81 % ethanol. Values are means % SEM of five cultures per group.
+
As illustrated in Fig. 2, & and DHT increased the LH cell content in the presence of GnRH after both acute (133.0%, p < 0.01; and 118.7%,p < 0.01, respectively) and chronic (1 18.5%,p < 0.05; and 118.0%,p < 0.05) administration. Total (cells plus medium) LH in cultures incubated with GnRH was also enhanced by E2 (acute: B 17.2% , p < 0.05; and chronic: 127.5%, p < 0.01) and DHT (acute: 110.4%, p > 0.05; and chronic: 117.4% ,p < 0.05) treatment. Neither cell content nor total LEI was significantly modified by P. Acute eflects of steroids on GRRH-stimahted LH secretion by estradiol-primed cells The acute effects of E2 (1 nM), P (100 nM), and DHT (10 nM) on the GnRH (1 nM) response in E2-primed (I nM) cells during a short-term incubation were tested. It can be seen in Fig. 3 that again, pretreatment of the pituitary cell cultures for 24 h with E2 leads to a significant increase of the response to GnRH. This augmentated LH response remained unchanged when E2-primed cells were hrther incubated for 5 h with the same dose of &. On the other hand, the addition of P to pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of E2, greatly increasing the releasing effect of GnRH as compared with nonprirned (205.2%, p < 0.8%)as well as &-primed (133.2 % p < 0.01) control groups. On the contrary, DHT decreased (63.1 % , p < 0.81) the LH release by E2-primed cells incubated with GnRH, reaching values similar to those found in & nonprimed cells. None of the steroids acutely administered was capable of modifying the amount of LH remaining in the E2-primed cells in response to GnRH. GnRH-stimulated total LH by b-primed cells was not affected by incubating the cells with E2 or DHT, whereas in the presence of B it increased ( p < 0.05) to 111 8.8 % of control values.
1
Discussion This study analyses the short- and long-term effects of physiological doses of E2, B, and DHT, alone and in combination, on the GnRH responsiveness of cultured anterior pituitary cells from ovariectomized rats. Since we had not previously established in our laboratory the pituitary cell culture model used in the present study, two systems of cultured cells either in suspension or in monolayer were tested. More satisfactory results were obtained from the monolayer than from the suspension cell cultures when comparing the bas& secretory function and the response to GnRH of cells in both systems. On
FIG. 3. Acute effects of ovarian steroids on GnRH-stimulated LH secretion by estradiol-primed cells. Annsunt of LM released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by cells from ovariectomized rats incubated for 5 h with 1 nM GnRH is shown, as described in Materials and Methods. Cultured cells pretreated for 24 h with 1 nM estradisl-17D were incubated f ~ 5r h with 1 nM estradiol 17-fi (R), 108 nM progesterone (Ed), and 10 nM 5a-dihydrotestosterone (a).The control group (63) was incubated in absence of steroid hormones. The group of nonprimed cultures incubated in medium contained G n W (m) was also included to check the response to estradiol priming in this experiment. The height of the bar gives the mean value of five cultures and the dot represents SEM.
the basis of these preliminary experiments, cultured cells in monolayer were selected as the experimental model. The addition of GnRH to the incubation medium caused increases of LH in the medium as well as in total combined LH content of cells plus medium. These results are in agreement with our previous observations (Apfelbaum 1981 ; Apfelbaum and Taleisnik 1976) and those of other investigators (Labrie et al. 1973; Ramey et al. 1987; Starzec et al. 19861, showing that GnRH stimulates not only the release but dso the synthe-
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sis of LH. The pituitary responsiveness to GnRH was modified by the steroid kormones and the effect varied according to the treatment. The present data show that the steroid hormones exert o p p site effects on the release of LH induced by GnRH, which seem to be dependent upon the length of time the pituitary cells have been exposed to the steroid. Like others (Apfelbaum and Tdeisnik 1976; Frawley and Neil 1984; Ernons et al. 1986) we found that short-term (5 h) action of E2 resulted in a partial inhibition whereas long-term (24 h) exposure enhanced the release of LH induced by GmRH. Similar results were obtained with BHT, although the magnitude of the effects was lower than with Q. Even though androgens are the characteristic male hormones, previous studies have shown that pituitary cells from female rats exhibit physiological responses to androgens (Drouin and Eabrie 1976; Kamel et al. 1887). Furthermore, androgen receptors are found in female pituitaries, which have similar affinity and specificity to those in male tissues (Handa et d . 1986). Conversely, P caused an acute stimulatory action on the LH released in response to GnRH and a slightly inhibitory effect after chronic treatment. It is recognized that P facilitates gonadotrophin release and that a previous estrogenic action is critical in determining the feedback effect of P on the release of LH (Caligaris et al. 1971; Kalra et al. 1973; Easley et d. 1975). In contrast, the observations made in the present study regarding the stimulatory effect of P on EH release in response to GnRH merit consideration. Although our results correspond qualitatively to those obtained in cultured cells from intact female rats (Turgeon and Waring 1981) or ovariectomized rats primed with E2 (Ortrnann et al. 1989), we found expressed effects of P using pituitary cells from ovariectomized rats in the absence of estrogenic priming. In an attempt to assess whether or not GnRH-induced LH biosynthesis is also influenced by steroid treatments, we measured the total (intracellular plus released) LH in the system. Other investigators have reported that GnRH-stimulated incorporation of radiolabeled precursors into kH was enhanced by E2 pretreatment either in vivo (Liu and Jackson 1977) or in vitrs (Rarney et al. 1987). This increase of hormone synthesis, dso suggested by our previous data (Apfelbaum and Taleisnik 1976), is in agreement with the results sf the present study. Significant increases in total EH were evident in pituitary cells treated with EL or DHT, E2 being more effective in this respect. Differences in the response were observed when comparing the steroid-induced increases in total LEH. While the stimulatory effect of DHT appears to be exerted mainly after long-term pretreatment, that of E2 was evident after both chronic and acute treatment. Fu~hermore,it should be noted that GnRH-induced total LH from cells incubated for 5 h with E2 was higher than that from nontreated cells in spite of the fact that the release of the hormone was reduced, indicating hat the effects on release are not simply the consequence of changes in hormone content. Therefore, these results suggest that 6 modulates LH secretion by acting at both synthesis and release level. On the other hand, the pstentiating effect of P on GnRH-induced LH release seems to be due to a stimulation at the Bevel of the release mechanism rather than to an enhanced gonadotrophin synthesis, since acute stimulation with P did not affect the total hormone content. Regarding the physiological significance of the steroid modulatory effects on GnRH action, consideration was taken
VOE. 70,
1992
of the in vivo hormonal environment found around the time of the preovulatory gonadotrophin surge. Since the secretion of E2 starting 24 h before proestrus acts as a trigger for the preovulatory LH release (Ferin et al. 1969; Legan et al. f 975), we intended to examine the estrogen dependence of the steroid actions on the final response to GnRH by priming the cells with E2 prior to short-term incubations. The net effect produced by the combined treatment with E2 plus P shows an increase in the overdl pituitary responsiveness to GnRH. In fact, while P done had a relative smaller effect, its addition to pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of estrogen on GnRH-induced EH release and total content. In essence, this increased response indicates that the LH release and synthesis from E2-treated cells in the presence of P was higher than the sum s f effects that can result from each treatment independently. Conversely, the augmentative E2 effect on pituitary responsiveness to GnRH was abolished by DHT. Our data also show that the effects of E2 and DHT influenced each other in such a way that the expected negative consequences for gonadotrophs GmRH responsiveness of treatment with DHT (in terms of LH release) and potentiation by E2 of LH stimulation by GnRH were not observed. In other words, androgens, in the presence of estrogens, do not suppress the GnRH responsiveness of the pituitary gland, but they do prevent the expression of the positive effect of E2 pretreatment. The current results indicate that the pituitary cells respond to P and DHT more efficiently when E2 is present, which can be correlated with the wellknown regulatory effect of estrogen on progestin and androgen receptor population. Recent studies have shown that the anterior pituitary progestin and androgen receptors are particularly sensitive to the action of circulating estrogen (Attardi 1984; Handa et al. I986), increasing on proestrus and during periods sf high circulating estrogen, and decreasing in the absence of estrogen. Physiologically, the effect of P may play an important role at proestrus by dictating the find magnitude of the secretory response, at the time when high pituitary responsiveness is required. The hnctional significance of the inhibitory effect of DHT could then be to confine the LH surge to that day of proestrus. -
We are gratefugll to the NIDDK Rat Pituitary Hormone Distribution Program for providing the LH RIA kit. This work was supported by the research grant 446/88 from Consejo Nacisnal Be Investigaciones Cientificas y Tecnicas of Argentina and grant 1@2/90 from Consejo de Investigaciones Cientificas y Teenol6gicas de la Provincia de Cdrdoba, Argentina. Apfelbaurm, M. E. 1981. Basal and GwRH-induced release and synthesis of LH and FSH from incubated pituitary glands throughout the 4-day estrous cycle of the rat. Horm. Res. (Basel), 15: 109121. Apfelbaum, M. E. 1983. Influence of gonadal steroids on the timecourse of release and synthesis of luteinizing hormone, folliclestimulating hormone and prolactin from rat pituitary glands in vitro. J. Endocrinol. 96: 171- 179. Apfelbaum, M. E., and Taleisnik, S. 1976. Interaction between oestrogen anad gonadotrophin-releasing hormone on the release and synthesis of luteinizing hormone and follicle-stimulating hormone from incubated pituitaries. J. Endocrinol. 68: 127 - 136. Apfelbaum, M. E., and Taleisnik, S. 1977. Influence s f oestrogen administration in vivo and in vitro on the release and synthesis of
P ~ E Z AND APFELBALJM
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LH and FSH from incubated pituitaries. Acta Endocrinol. $6: 704-713. Attardi, B. 1984. Progesterone mdulation of the luteinizing hormone surge: regulation of hypothalamic and pituitary progestin receptors. Endocrinology (Baltimore), 115: 2 113-2 122. Butcher, R. L., Collins, W. E., and Fugo, N. W. 1974. Plasma concentration of LH, FSH, prolactin. progesterone and estradiol-17P throughout the 4-day estrous cycle of the rat. Endocrinology (Baltimore), 94: 1704- 1708. Caligaris, L., Astrada, J. J., and Taleisnik, S. 1971. Biphasic effect of progesterone on the release of gonadotropins in rats. Endocrinology (Baltimore), 89: 33 1- 337. Clayton, R. N . , and Catt, K. J. 1981. Regulation of pituitary gonadotropin-releasing hormone receptors by gonadal hormones. Endocrinology (Baltimore), 108: 887 - 894. Drouin, J., and Labrie, F. 1976. Selective effect of androgen on LH and FSH release in anterior pituitary cells in culture. Endocrinology (Baltimore), 98: 1528- 1534. Dupon, C., and Kim, M. H. 1973. Peripheral plasma level of testosterone, androstenedione, and oetradiol during the rat oestrous cycle. J. Endmrinol. 59: 653 -654. Emons. G . , Ortmann, O., Fingscheidt, U.,et a!. 1986. Short-term effects of oestradiol and 4-hydroxyoestradiol on gonadotropinreleasing hormone induced luteinizing hormone secretion by rat pituitary cells in culture. Acta Endocrinol. 1111 312-320. Emons, G., Hrevert, E. U.,Ortrnann, O., eb a!. 1989. Studies on the subeellular mechanisms mediating the negative estradiol effect on GnRH-induced LM-release by rat pituitary cells in culture. Acta Endocrinol. 121: 350 - 360. Ferin, M., Tempone, A., Zimmering, P., and Vande WBle, R. L. 1969. Effects of antibodies to 170-estradiol and progesterone on the estrous cycle of the rat. Endocrinology (Baltimore), 85: 10701878. Frawley, L. S o ,and Neil, 5 . D. 1984. Bighasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer culthares of rat and monkey pituitary cells. Endocrinology (Baltimore), 614: 459 -663. Gay, V. L., and Tomacari, R. L. 1974. Follicle-stimulating hormone secretion in the female rat: cyclic release is dependent on circulating androgen. Science (Washington, DC), 84: 75-77. Handa, R. J., Reid, D. L., and Resko, J. A. 1986. Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male. BioH. Reprod. 34: 293-303. Kalra, P. S., Fawcett, C. P., Kmlich, L., and McCann, S. M. 1953. The effect of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrinology (Baltimore), 92: 1256- 1268. Kamel, F., Balz, S. A., Kubajak, C. L., and Schneider, V. A. 1987. Gonadal steroids modulate pulsatile luteinizing hormone secretion by p e r k e d rat anterior pituitary cells. Endocrinology (Baltimore), 120: 1451- 1654. Karseh, F. J. 1987. Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing homone. Annu. Rev. Physiol. 49: 345-382. Kmmmen, L. A., and Baldwin, D. M. 1988. Regulation of luteinizing hormone subunit biosynthesis in cultured male anterior
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pituitary cells: effects of gonadotropin-releasing hormone and testosterone. Endocrinology (Baltimore), 123: H 868 - 1878. Labrie, F., Pelletier, G., Lemay, A., eb a!. 1973. Control of protein synthesis in anterior pituitary gland. Ach Endocrinol. Suppl. 180: 301 -334. Lasley, B. L., Wang, C. F., and Ven, S. S. C. 1975. The effects of oestrogen and progesterone on the functional capacity of the gonadotrophs. J. Clin. Endocrinol. Metab. 41: 828 - 826. Legan, S. J., Coon, G . A., and Karsch, F. 5 . 1975. Role of estrogen as initiator of daily LH surges in ovariectomized rat. Endocrinology (Baltimore), 96: 58-56. Liu, T. C., and Jackson, G . L. 1977. Effect of in vivo treatment with estrogen on luteinizing hormone synthesis and release by rat pituitaries in vitro. Endocrinology (Baltimore). 108: 1294- 1382. Liu, T. C., and Jackson, G . L. 1988. Actions of 176-estradiol on gonadotropin release induced by drugs that activate intracellular signal transduction mechanisms in rat anterior pituitary cells. Biol. Reprod. 39: 787-796. Mahesh, V., and Muldoon, T. G 1987. Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretions. 9. Steroid Biochem. 27: 665 -675. Menon, M. M. D., Peegel, H. M. S., and Katta, V. B. S . 1985. Estradiol potentiation of gonadotropin-releasing hormone responsiveness in the anterior pituitary mediated by an increase in gonadotropin-releasing hormone receptors. Am. J. Bbstet. Gynecol. 851: 534-540. Qrtmann, O . , Wiese, H., Knuppen, W.,and Emons, G. 1989. Acute facilitory action of progesterone on gonadotropin secretion of perifused rat pituitary cells. Aeta Endocrinol. 12%:426-434. Ramey, J. W., Highsmith, R. F.,Wilfinger, W. W., and Baldwin, D. M. 1987. The effects of gonadotropin-releasing homone and estradiol on luteinizing homone biosynthesis in cultured rat anterior pituitary cells. Endocrinology (Baltimore), 120: 15031513. Schally, A. V., Redding, T. W., and Arimura, A. 1943. Effect of sex steroids to LH and FSW-releasing homone in vitro. Endocrinology (Baltimore), 93: 893- 902. Smith, M., Freeman, M., and Neill, J. 1975. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteurn of pxudopregnancy. Endocrinology (Baltimore), 96: 2 19-226. Starzec, A., Counis, R., and Sutisz, M. 1986. Gonadotropinreleasing hormone stimulates the synthesis of the polypeptide chains of luteinizing hormone. Endocrinology (Baltimore), 119: 561 -566. Turgeon, J. L., and Waring, D. W. 1981. Acute progesterone and 176-estradiol modulation of luteinizing hormone secretion by pituitaries of cycling rats perfused in vitro. Endocrinology (Baltimore), 188: 413-419. Turgeon, J. L., and Waring, D. W. 1990, Rapid augmentation by progesterone of agonist-stimulated luteinizing homone secretion by cultured pituitary cells. Endocrinology (Baltimore), 627: 773 780.
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Received September 24, 1991 PEREZ,G. T., and APPELBAWM, M, E. 1992. Modulatory effect of steroid hormones on CnRH-induced LH secretion by cultured rat pituitary cells. Can. J . Physiol. Pharmacol. 70: 363 -969. The purpose of the present experiments was to examine the short- and long-term effects of estradiol-119B (E,), progesterone (P), and 5a-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 Ea in steroid-free medium, pituitary cells were further cultklred for 24 h in medium with or without E2 (1 nM), P (100 nM), s r DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of l nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show ghat the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E, resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E,. Conversely, B caused an acute stimulatsry action (118%) on the LH released in response to GnIZH and a slightly inhibitory effect (90%)after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) kH were observed in pituitary cells treated with E2 or BHT. While the stimulatory effect of E, was evident after both acute (133 %) and chronic (1 19%) treatment, that of DHT appears to be exerted mainly after long-term priming (1 18 %). These results suggest that the steroids modulate GnRH-induced LH secretion by acting on both synthesis and release sf LH. On the other hand, total hormone content was not affected by P. The acute (5 h) effects of E2, P, and BHT on the GnRH response in E,-primed (24 h) cells during a short-term incubation, were also tested. Addition of P to the pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of E2 on GnRH-induced LH release and total content. Conversely, the augmentative E2 effect on pituitary responsiveness to GnRH was abolished by DHT. Taken together, these findings suggest that the physiological significance of the stimulatory action of progesterone could be to define the final magnitude of the LH preovulatory surge, while the inhibition by DHT could be required to limit the LH surge to that day of proestms. Key words: luteinizing hormone, gonadotrophin-releasing hormone, steroid hormones, cultured pituitary cells.
P ~ R E ZG. , T., et APFELBAUM, M. E. 1992. Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells. Can. J . Phy siol. Pharmacol . 78: 963 - 969. Le but des pr6sentes expkriences a 6tC d'examiner les effets B court et 2 long terme de 190estradiol-1'7b(E,), de la progestCrone (P) et de la 5a-dihydroxytestosthne (BHT), seuls ou csmbinCs, sur la sCcr6tion de I'hormone 1utCinisante (LH) induite par l'homone de liberation des gonadotrophines (GnRH), en utilisant un modkle de culture de cellules hypophysaires de rats ovariectomises. Aprbs une incubation de 72 h dans un milieu d 6 p o u m d'hormones stCroYdes, les cellules hypophysaires ont CtC cultivCes pedant une autre pCriode de 24 h avec ou sans E, (1 nM), P (1W nM) ou DHT (10 nM). Ensuite, les cellules cultivCes ont CtC incub6es pendant 5 h en l'absence ou en prCsence de 1 nM de GnRH, avec saa sans homones stCroides. h i s , la LH a CtC CvaluCe dans le milieu de culture et les extraits des cellules par radio-immunodosage. Les rksultats rnontrent que les homones stkroides exercent des effets opposks sur la 1ibCration de LH irnduite par la GnRH, libkration qui semble &re dCpendante de la longueur de la phiode d'exposition des cellules hypophysaires auw hormones stCroYdes. En effet, l'action A court terms (5 h) de I'E, a provoquk une inhibition partielle (64% des valeurs tCmoins) de la libkration de LH en rCponse 2 la GnRH, alors que I'exposition de longue dur6e (24 h) a stimulk sa libCration (158%). Bes 5Csultats similaires ont CtC obtenus avec la DHT, bien que l'amplitude de l'effet ait 6t6 infkrieure 2i celle obtenue avec l'E2. A l'oppos6, la P a eu un effet stimulateur aigu (1 18%) sur la libkration de LH en rCponse B la GnRM et un effet l6gkement inhibiteur (90%)aprks un traitement chronique. La biosynthbse de la LH stimulCe par la GnRH a aussi CtC influencke par le traitement a m hormones stCroTdes. Des augmentations signifieatives de la teneur totale en LH (cellules plus milieu) ont Ctk observkes dans les cellules hypophysaires traitees avec l'E2 ou la BHT. Alors que l'effet stimulateur de l'E, a dtC manifeste tant aprks le traitement chronique (1 19 %) qu'aprks le traitement aigu (133 %), celui de la DWT semble s7Ctreprincipalement exerck aprks une sensibilisation 8 long terme (118%). Ces rksultats suggtrent que les hormones stkroi'des modulent la skcretion de LH induite par la GnRH en agissant autant sur la synthkse que sur la libkration de LM. Par ailleurs, le teneur hornonale totale n'a pas kt6 influencke par la B. Les effets aigus (5 h) de 17E2,de la P et de la DHT sur la rCponse 8 la GnRH dans les cellules sensibiliskes (24 h) B 19E,, durant une incubation de couae duke, ont aussi kt6 examinks. L'addition de P aux celldes hyp'~physairessensibiliskes avec 1'E2 a\ entrain6 une potentialisation aigue Be l'effet stimulateur de l'E, sur la teneur totale en LH et aa libkration par la GnRH. A I'opposC, 19effetstimulant de 19E2sur la rCactivitC pituitaire B la GnRH a Ctd supprim6 par la BHT. Ensemble, ces rCsultats suggkrent qu'en termes physiologiques, le sens de l'action stimulatrice de la progestkrone poumait etre de dCfinir l'amplitude finale de la bouff6e prkovulatoire de LH, alors que I'inhibition par la DHT pourrait &re nkcessaire pour limiter la buffke de LH au moment du proestms. Mots c&&: hormone lutkinisante, hormone de libkration des gonadotrophines. hormones stkroides, cellules hypophysaires cultiv6e.s. [Traduit par la rkdaction] 'Correspondence may be sent to the author at the following address: Chtedra de Fisiologia, Departamento de Famacologia, Facultad Ciencks Quimicas, Universidad Nacional de Cbrdoba, Suc. 16, Casilla de Correo 61, 58 16 C6rdoba, Argentina. Printed in Canada i Imprime au Canada
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Introduction Cyclic patterns of circulating gonadotrophins are a basic characteristic of the female rat estrous cycle. It is generally accepted that physiological levels of the gonadotrophins are mainly regulated by the hypothalamic gonadotroghin-releasing hormone (GnRH) and the ovarian hormones, although the mode of regulation and the interaction between these factors are not yet defined. Analysis of the sequence of hormonal events during the estrous cyck shows that rising titers s f plasma estrogen on the afternoon of diestrus and the morning of proestms act as a primaray stimulus for the preovulatory luteinizing hormone (LH) surge on the afternoon of proestms (Ferin et al. 1969; Legan et al. 1975). Once the preovulatory surge has been initiated, the ascending phase of LH secretion is accompanied by a rapid rise in progesterone and a decline in estrogen levels (Butcher et al. 1974; Smith et al. 1975). In addition, testosterone and androstenedione rise in the systemic circulation on proestms, in concert with the surge of gonadotrophin (Dupon and Kim 1973). While the role of estrogen in triggering the preovulatory surge of LH is well recognized, relatively less attention has been given to the role of progesterone in regulating LH secretion. For in vivo studies, a biphasic effect s f progesterone on LH release, with an acute stirnulatory and chronic inhibitory action, has been shown (Caligaris et al. I97 1; Mahesh and Muldson 1987). Much less is known on the functional significance of circulating androgens in a normal cycling rat. Some observations indicate that androgens can differentially affect the release of LH and folicle-stimulatinghormone (FSH) from the anterior pituitary, thereby favoring the proportional release s f FSH (Gay and Tomacari 1974). Undoubtedly, timing, magnitude, and duration of the sequence of changes in hormonal feedback are critical in achieving a normal reproductive cycle. Numerous studies have shown that ovarian steroids can either enhance or inhibit gonadotrophin secretion, depending upon the dosage, length of treatment, and hormonal environment at the time of administration. The steroid hormones modu%ate gonadotrophin secretion by acting at both the hypothalamic Bevel on GnRH secretion, mQ at the pituitary level on responsiveness of gonadotrophs to GnRH (Karsch 1987). The biochemical mechanisms and site of action by which the steroids influence GnRH-stimulated LH secretion are unhown. At the pituitary level, the feedback actions of steroids an gonadotrophs may involve both regulation of GnRH receptors (Clayton and Catt 1981; Menon et d. 198%)and modulation of post-receptor events. Several studies have demonstrated that steroids are able to act on different steps of the complex second messenger system involved in the GnRH transduction system (Emons et al. 1989; Liu and Jackson 1988). Nevertheless, the results do not provide direct evidence about their ~nechanismof action. In earlier studies with in vitro hemipituitary glands from ovariectomized rats (Apfelbaum 1983; Apfelbaum and Taleisnik 1977) we reported that ovarian steroids can exert specific effects on LH basal secretion by a direct action at the pituitary level. The purpose of the current experiments was to further characterize the short- and long-term effects of physiological doses of estradiol-170 (E2), progesterone (P) and the nonaromatizable androgen 5a-dihydrotestostersne (DHT), alone and in combination, on GwRH-induced LH secretion using an ovariectomized rat pituitary cells culture model as a preliminary study to explore subsequently the molecular events
mediating these actions around the critical preovulatory period in the rat.
Materids and methods Hornones Gonadotrophin-releasing hormone was obtained from Bachem Inc. (Torrance, CA, U. S.A .). Estradiol- 170, progesterone, and Sa-dihydrotestostersne were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), Stock solutions sf steroids were prepared in 0.9% NaCl - I % ethanol and were used at a I@3-fold dilution in the incubation medium, such that the final concentration of vehicle in the medium was 0,01%. Dispersion and culture of mt anterior pituitary cells Adult female rats (200 -250 g) from our colony of albino rats from Wistar strain were ovariectomized 30 clays before being used. Animals were housed in a controlled environment (21 f 1°C; 14-h light : 10-h darkness) with food and water available ad libitum. The animals were killed by decapitation. The anterior pituitary glands were rapidly excised and placed into a petri dish containing 5 mL of supplemented Spinner minimum essential medium (S-SMEM) calcium- and magnesium-free, which consisted of 0.25% BSA, 13 mM HEPES, 50 pg/mL streptomycin, and 50 U/mL penicillin. The pituitaries were cut with a razor blade in fine pieces and washed with 10 mE S-SMEM. The tissue blocks were then transferred to a 50-mL Erlenmeyer flask containing 0.25% trgipsin (1:258 porcine pancreatic trypsin; Bifco, Detroit, MI, U.S.A.) in S-SMEM (0.5 mL per pituitary) and incubated for 20 rnin at 37°C in a shaker bath. The medium was removed and replaced with 4 rnL S-SMBM containing I mg soybean trypsin inhibitor (type I-S; Sigma) and the incubation was continued for another 3 min. One hundred micrograms of deoxyribonuclease (DNase; bovine pancreatic deoxyribonuclease type DN-lQ0, Sigma) was present during all steps of incubation to avoid clumping. The tissue blocks were then transferred to a conical centrifuge tube and washed three times with 10 mL S-SMEM. The final dispersion of the pituitary cells was accomplished by m a n s of repeated gentle uptake and expulsion with a flame-polished Pasteur pipette. Aker complete dissociation, the cells were centrifuged (250 X g; 5 rnin), washed twice with S-SMEM, and resuspended in supplemented Medium 199 (S-Ml%9),which consisted of Medium 199 with Earle's salts plus 0.25 % BSA, 25 mM HEPES, 100 pg/rnL streptomycin, 100 U/mE penicillin, 50 pglmL Nystatin, 8% horse serum, and 2% fetal bovine serum. Both sera were pretreated with dextran-coated charcoal (Drouin and Labrie 19'96) to remove endogenous steroids. The number of cells obtained after dispersion was determined with a hemocytorneter; cell viability was established by the trypan blue exclusion technique. This procedure yields 3.54.5 x lo6 cells per pituitary, and more than 95% of cells were viable. Finally, the cells were plated in Falcon tissue culture dishes (3 x 106 cells per 35-mm dish) in 1.5 mL S-MI99 and cultured at 39°C under a humidified atmosphere of 95% air : 5 % CO,. An additional aliquot of 1 mL fresh culture medium was added to each dish 48 h later. After plating (72 h), the media were decanted and the cells were further cultured for 24 h in 1.5 mL S-MI99 in the presence or absence of the indicated steroid to be tested. Following the 96-h culture period, the culture medium was discarded and the cells were washed twice with 2 mL per dish of Dulbecco9s modified Eagle's medium (DEM) supplemented with 0.25 % BSA to remove serum and nonadhered cells, prior to use in short-term incubations. All media were filtered through 0.22-yrnMilipore filters (Millipore Corp., Bedford, MA) before use, the final pH being 7.4. The culture media, antibiotics, and chemicals were purchased from Sigma, and the sera from Grand Island Biochemical Co. (Grand Island, NY, U.S.A.). Short-term incubatioris of cultured cells After 96 h of culture, short-term incubations were carried out at 37 OC under a humidified atmosphere of 95% air : 5% CO,. Two experimental designs were used to establish the effects of ovarian steroid
TABLE1. Percentages of basal LH rdeased and contained in the total system (cells plus medium) by pituitary cells from ovariectomized rats cultured in suspension or in monolayer and incubated for different times
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Incubation time (h)
% LH in suspension
% LH in monolayer
Medium
Medium
Total
Total
NOTE:Control cultures were processed for hormone determinacells; monolayer: tion at 0 h (suspension: 4.04 k 0.30 kg13 x ceHHs). Amount of LH in cells incubated 5.26 k 0.45 pgIP x for 2,4, and 6 h was expressed as percentage of the respective control cultures (taken as 100%). Values are means f SEM of five cultures per group.
hormones on bas& and CwRH-stimulated release, cell content, and total (cells plus medium) LH by cultured pituitary cells from ovariectomized rats. In the first, the acute effects of gonadal steroids were studied. After 3 days in culture the medium was discarded and the cells were hrther cultured for 24 h in 1.5 mL S-MI99 with 0-017% ethanol (vehicle). Pituitary cell cultures were then washed twice with DEM and incubated for 5 h in DEM containing 1 nM E,, 100 nM P, 10 nM DHT, or 0.81% ethanol vehicle (control) with or without 1 nM G n W . In the second approach, the chronic effects of gonadal steroids were studied by pretreating the cells with steroids before the GnRH challenge. Pituitary cells were cultured for 3 days in S-M 199, followed by 24 h in fresh S-M189 containing I nM E,, 100 nM P, 10 nM DHT, or vehicle (control). The cells were then washed as above and subsequently incubated in DEM with vehicle in the presence or absence of 1 nM GanRH. Hn the third set of experiments the combined action of steroids on the GnRH response of pituitary cells was studied. Cells primed for 24 h with 1 nM E, or vehicle were incubated for 5 h with 1 nM G n M in the presence of 1 nM E2, 100 nM P, HO nM DHT, or vehicle. Incubations were terminated by adding 1 mL phosphate-buffered saline (PBS), pH 7, and chilling the dishes on ice. The media were removed, centrifuged at 2508 x g for 15 rnin at 4 "C, and kept frozen at -20°C until assayed. Cells were washed once with 2 mL PBS and lysed by incubation in 1.5 rnL of ice-cold 50 mM Na,C03 in 8.1 M NaOH for 10 min. The neutralized cell lysates were then frozen and thawed three times and treated with Triton X-100 (1 94 final concentration). The samples were then centrifuged at 35 000 x g for 30 min. Supernatants were decanted and stored at -20aC.
Preliminary experiments The purpose of the preliminary experiments was to establish our rat anterior pituitary cell culture system. Two systems of cultured cells either in suspension or in rnonolayer were tested. After dissociation, the pituitary cell suspension was aliquoted half into 5-mL glass tubes and the other half into culture dishes at 3 X 1Q6 cells per tube or dish, cells in tubes being cultured for 22 h while those in dishes for 96 h as described above. After the respective culture period, almost all of the cells placed in tubes are present in suspension, whereas those in dishes are attached to the surface as a monolayer, the viability of cells k i n g higher for cells in monolayers Q > 90%) than for those in suspension ( > 75 %). The time course of basal release, cell content, and total (cells plus medium) LM in both preparations was studied. The cultured cells, either in suspension or monolayer were washed twice with DEM and incubated for different periods (0 - 6 h) in 1.5 mL of the same medium. Control cultures were processed for homone determination at 0 h for the measurement of total LM content initially present in the incubation system. The response of cultured pituitary cells to GnRH was also analyzed in both preparations. Cultured cells, in suspension and in monolayer were incubated for 5 h
TABLE2. Effect of increasing concentrations of SnRH on LH released and contained in the total system (cells plus medium) by pituitary cells from ovariectomized rats cultured in suspension or in monolayer
GnRH (nM)
% LH in suspension
Medium
Total
% EH in monolayer
Medium
Total
NOTE:Pituitary cells were incubated for 5 k in the absence (control) or presence of different doses of GnRH. Responsiveness of pituitary cells to GnRH was expressed as percentage of the respective control (medium: 0.5 1 i 0.04 and 0.28 f 0.01 ygl3 X lo6 cells; total: 6.02 i 0.45 and 5.10 f_ 0.46 kg13 x lo6 cells far suspension and rnonolayer, respectively) cultures (taken as I%%). Values are means & §EM of five cultures per group.
in the absence (control) or presence of different doses of GnRH (0.1 - 100 nM).
LH mdioimrnunoassay lmmunoreactive LH was measured in the medium and the cell extract at two dose levels by a double-antibody radioimunsassay, with reagents supplied by the U.S. National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (NEDDK), National Institutes of Health. The amount of LH was calculated in terms of NIDDK rat standard rLH-RP-2. Hntraassay coefficient of variation (CV) for LH assay was 6.2%. Corresponding interassay CV was 10.0%.The results were expressed as micrograms of EH per 3 x 1Qh cells.
Statistics The data were analysed by analysis of variance and Duncan's multiple range test. A level sf p < 0.05 was considered statistically sigSEM. nificant. All results are expressed as the mean
+
Results Suspension versus rno~asla~~er cell culture system Two separate experiments were performed to evaluate the functional capacity of the cultured pituitary cells in both suspension and monolayer. The bas& release, cell content, and total (cells plus medium) LH as a function of incubation time as well as the ability of the cultured pituitary cells to respond to increasing doses of GnRH were studied in both systems. When the amount of basal LH in the medium is expressed as a percentage of total LH contained in the respective control cultures at 0 h (taken as loo%), LH is released in greater proportion from the cells in suspension than from the attached cells (Table I). Respnsiveness of pituitary cells to GnRH is expressed as a percentage of the respective untreated control cultures ts compare both preparations (Table 2). Addition of GnRH to the incubation medium greatly stimulated ( p < 0.01) the release sf LH in a dose-related manner in both systems, the LH net response being greater in cells in rnonolayer than in those in suspension. The EDSovalue sf GnRH action was calculated to be 0.69 anM for monolayers and 1.75 nM for suspensions. Hn addition, GnRH-stimulated total LH content was significantly increased (10 nM GnRH, p < 0.05; 100 nM GazRH, p < 0.01) in the case of rnonolayers, while that of cells in suspensisn was unchanged. On the basis of these data. cultured cells in monolayer were selected as the experimental model for the study of steroid hsrmane action on LH secretion.
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FIG. I. Effects of ovarian steroids on basal LH secretion after short- and long-term treatment. Amount of LH released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by pituitary cells from ovariectotniaed rats incubated for 5 h is shown, as described in Materials and Methods. Cultured cells were (a) acutely (5 h) or (b) chronically (24 h) exposed to 1 nM estradiol-By@(B), 100 nM progesterone (El), and 10 nM Sa-dihydrotestosterone (B). Control were incubated in absence of steroid hormones. The groups (8) height of the bar gives the mean value of five cultures and the dot represents the %EM.
Eflects of steroids on basal LH secretion a@er short- and longterm trealneent Figure I summarizes the effects of actue (5 h) and chronic (24 19) treatment with 1 IM E2, 106) nM B, and BO nM DHT om basal LH secretion by pituitary cells from ovariectomized rats. Ethanol (vehicle), added into the medium at the final concentration of 0.0 1% (vlv), did not modify basal release, cell content, or t s t d (cells plus medium) LM by pituitary cell cultures irrespectively of the time of treatment (Table 3). When pituitary cells were pretreated for 24 h with vehicle and subsequently incubated for 5 h in medium containing steroids (Fig. IQ), basal release of LH was not affected by E2 and DHT, whereas a small though not significant increase (1 16.3% of control, p > 0.05) in LH release was observed in cells acutely exposed to P. Priming the cells for 24 h with steroids also failed to affect basd release of LH from cultures incubated for 5 h with vehicle (Fig. 1b). At the end of incubations the amount of LH remaining in the cells was higher than that in control in all the groups studied (Fig. la, and Ib). Comparatively, the effect of E2 after both acute (117.I%, p < 0.05) and chronic (123.6%, p < 0.81) treatment was greater than that of B (acute: 115.1%, p > 0.05; and chronic: 111.9%, g > 0.05) or DHT (114.7%, p > 0.05, and 118.9%,p < 0.81). Treatment s f pituitary cells with steroids resulted in an enhanced total (cells plus medium) LH in the system (Fig. l a and Ib). This stimulating effect was more pronounced for E2
PIG. 2. Effects of ovarian steroids on GnRH-stimulated LH secretion after short- and long-term treatment. Amount of LH released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by pituitary cells from ovariectomized rats incubated for 5 h with 1 wM GnRH is shown, as described in Materials and Methods. Cultured cells were (a) acutely (5 h) or (b) chronically (24 h) exposed to 1 nM estradiol-178 (B), 180 nM progesterone (B), and BO wM Sa-dihydrotestosterone (P). Control groups (a)were incubated in absence of steroid hormones. Groups of cultures incubated in medium alone (we GnRH or steroids) (B)were also included to check the response to GnRH in this experiment. The height of the bar gives the mean value of five cultures -and the dot represents the SEG.
(acute: 116.1%,pa > 0.05; and chronic: 1%3.3%,p < 0.81) than for P (115.276, p > 0.05; and 111.3%,p > 0.05) or DHT (1%4.0%,p > 0.05; and 117.8%, p < 0.05). Eflects of steroids on GnRH-stkmalated EH secretion afier short- and long-term treatment Figure 2 shows the LH secretory response to 1 nM GnRH by cells acutely or chronically exposed to 1 nM E2, 100 nM P, or 10 HIMDHT. Vehicle treatment s f the cells did not induce any changes in the LH response to GnRH administration (Table 3). When cells were pretreated with vehicle for 24 h and then incubated for 5 h in medium containing GnRH and vehicle, GwRH stimulated LH release (567.5 % of control, g < 0.01) and total LH (1 16-676,p < 0.05) but was without effect on LH cell content (94.4%, p > 8.05). GnRH-stimulated LH release was modified by the treatment with steroids. Addition s f E2 or DHT during the 5-h incubation period decreased to 63.9% ( p < 0.0%)and 82.4 % ( p < 8-05), respectively, the LH release having been induced by GnRH (Fig. 2u). On the contrary, pretreatment s f cultures with E2 or DHT before challenging the cells with GnRH increased (I58 %, g < 0.01; and 115.4 9% ,p > 0.85, respectively) the response to GnRH (Fig. 2b). Conversely, acute administration of P enhanced (1 18.5% , p < 8.01) (Fig. 2a), and chronic administration sf P slightly decreased (90.7%, p > 0.05) (Fig. 2b), the LH released in response to GnRHH.
P ~ R E ZAND APFELBAUM
TABLE3. Effect of ethanol (vehicle) on basal and GnRH-induced LM intracellular, released, and contained in the total system (cells plus medium) by cultured pituitary cells from ovariectomized rats
LH &g/3 --
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Time of treatment
-
X
lo6 cells)
-
Basal Medium
Cells
GnRH Total
Medium
Cells
Total
NOTE: Pituitary cell cultures incubated for 5 h in the absence (Basal) or presence of 1 wM CnRH were either 5 h) with co-incubated (5 h), pretreated (24 h) before incubation, or exposed ta the combined treatment (24 h 0.81 % ethanol. Values are means % SEM of five cultures per group.
+
As illustrated in Fig. 2, & and DHT increased the LH cell content in the presence of GnRH after both acute (133.0%, p < 0.01; and 118.7%,p < 0.01, respectively) and chronic (1 18.5%,p < 0.05; and 118.0%,p < 0.05) administration. Total (cells plus medium) LH in cultures incubated with GnRH was also enhanced by E2 (acute: B 17.2% , p < 0.05; and chronic: 127.5%, p < 0.01) and DHT (acute: 110.4%, p > 0.05; and chronic: 117.4% ,p < 0.05) treatment. Neither cell content nor total LEI was significantly modified by P. Acute eflects of steroids on GRRH-stimahted LH secretion by estradiol-primed cells The acute effects of E2 (1 nM), P (100 nM), and DHT (10 nM) on the GnRH (1 nM) response in E2-primed (I nM) cells during a short-term incubation were tested. It can be seen in Fig. 3 that again, pretreatment of the pituitary cell cultures for 24 h with E2 leads to a significant increase of the response to GnRH. This augmentated LH response remained unchanged when E2-primed cells were hrther incubated for 5 h with the same dose of &. On the other hand, the addition of P to pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of E2, greatly increasing the releasing effect of GnRH as compared with nonprirned (205.2%, p < 0.8%)as well as &-primed (133.2 % p < 0.01) control groups. On the contrary, DHT decreased (63.1 % , p < 0.81) the LH release by E2-primed cells incubated with GnRH, reaching values similar to those found in & nonprimed cells. None of the steroids acutely administered was capable of modifying the amount of LH remaining in the E2-primed cells in response to GnRH. GnRH-stimulated total LH by b-primed cells was not affected by incubating the cells with E2 or DHT, whereas in the presence of B it increased ( p < 0.05) to 111 8.8 % of control values.
1
Discussion This study analyses the short- and long-term effects of physiological doses of E2, B, and DHT, alone and in combination, on the GnRH responsiveness of cultured anterior pituitary cells from ovariectomized rats. Since we had not previously established in our laboratory the pituitary cell culture model used in the present study, two systems of cultured cells either in suspension or in monolayer were tested. More satisfactory results were obtained from the monolayer than from the suspension cell cultures when comparing the bas& secretory function and the response to GnRH of cells in both systems. On
FIG. 3. Acute effects of ovarian steroids on GnRH-stimulated LH secretion by estradiol-primed cells. Annsunt of LM released (medium, left), remaining in the cells (cells, middle), and contained in the total system (cells plus medium: total, right) by cells from ovariectomized rats incubated for 5 h with 1 nM GnRH is shown, as described in Materials and Methods. Cultured cells pretreated for 24 h with 1 nM estradisl-17D were incubated f ~ 5r h with 1 nM estradiol 17-fi (R), 108 nM progesterone (Ed), and 10 nM 5a-dihydrotestosterone (a).The control group (63) was incubated in absence of steroid hormones. The group of nonprimed cultures incubated in medium contained G n W (m) was also included to check the response to estradiol priming in this experiment. The height of the bar gives the mean value of five cultures and the dot represents SEM.
the basis of these preliminary experiments, cultured cells in monolayer were selected as the experimental model. The addition of GnRH to the incubation medium caused increases of LH in the medium as well as in total combined LH content of cells plus medium. These results are in agreement with our previous observations (Apfelbaum 1981 ; Apfelbaum and Taleisnik 1976) and those of other investigators (Labrie et al. 1973; Ramey et al. 1987; Starzec et al. 19861, showing that GnRH stimulates not only the release but dso the synthe-
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CAN. J. PHYSIBL. PHARMACOL.
sis of LH. The pituitary responsiveness to GnRH was modified by the steroid kormones and the effect varied according to the treatment. The present data show that the steroid hormones exert o p p site effects on the release of LH induced by GnRH, which seem to be dependent upon the length of time the pituitary cells have been exposed to the steroid. Like others (Apfelbaum and Tdeisnik 1976; Frawley and Neil 1984; Ernons et al. 1986) we found that short-term (5 h) action of E2 resulted in a partial inhibition whereas long-term (24 h) exposure enhanced the release of LH induced by GmRH. Similar results were obtained with BHT, although the magnitude of the effects was lower than with Q. Even though androgens are the characteristic male hormones, previous studies have shown that pituitary cells from female rats exhibit physiological responses to androgens (Drouin and Eabrie 1976; Kamel et al. 1887). Furthermore, androgen receptors are found in female pituitaries, which have similar affinity and specificity to those in male tissues (Handa et d . 1986). Conversely, P caused an acute stimulatory action on the LH released in response to GnRH and a slightly inhibitory effect after chronic treatment. It is recognized that P facilitates gonadotrophin release and that a previous estrogenic action is critical in determining the feedback effect of P on the release of LH (Caligaris et al. 1971; Kalra et al. 1973; Easley et d. 1975). In contrast, the observations made in the present study regarding the stimulatory effect of P on EH release in response to GnRH merit consideration. Although our results correspond qualitatively to those obtained in cultured cells from intact female rats (Turgeon and Waring 1981) or ovariectomized rats primed with E2 (Ortrnann et al. 1989), we found expressed effects of P using pituitary cells from ovariectomized rats in the absence of estrogenic priming. In an attempt to assess whether or not GnRH-induced LH biosynthesis is also influenced by steroid treatments, we measured the total (intracellular plus released) LH in the system. Other investigators have reported that GnRH-stimulated incorporation of radiolabeled precursors into kH was enhanced by E2 pretreatment either in vivo (Liu and Jackson 1977) or in vitrs (Rarney et al. 1987). This increase of hormone synthesis, dso suggested by our previous data (Apfelbaum and Taleisnik 1976), is in agreement with the results sf the present study. Significant increases in total EH were evident in pituitary cells treated with EL or DHT, E2 being more effective in this respect. Differences in the response were observed when comparing the steroid-induced increases in total LEH. While the stimulatory effect of DHT appears to be exerted mainly after long-term pretreatment, that of E2 was evident after both chronic and acute treatment. Fu~hermore,it should be noted that GnRH-induced total LH from cells incubated for 5 h with E2 was higher than that from nontreated cells in spite of the fact that the release of the hormone was reduced, indicating hat the effects on release are not simply the consequence of changes in hormone content. Therefore, these results suggest that 6 modulates LH secretion by acting at both synthesis and release level. On the other hand, the pstentiating effect of P on GnRH-induced LH release seems to be due to a stimulation at the Bevel of the release mechanism rather than to an enhanced gonadotrophin synthesis, since acute stimulation with P did not affect the total hormone content. Regarding the physiological significance of the steroid modulatory effects on GnRH action, consideration was taken
VOE. 70,
1992
of the in vivo hormonal environment found around the time of the preovulatory gonadotrophin surge. Since the secretion of E2 starting 24 h before proestrus acts as a trigger for the preovulatory LH release (Ferin et al. 1969; Legan et al. f 975), we intended to examine the estrogen dependence of the steroid actions on the final response to GnRH by priming the cells with E2 prior to short-term incubations. The net effect produced by the combined treatment with E2 plus P shows an increase in the overdl pituitary responsiveness to GnRH. In fact, while P done had a relative smaller effect, its addition to pituitary cells primed with E2 led to an acute potentiation of the stimulatory effect of estrogen on GnRH-induced EH release and total content. In essence, this increased response indicates that the LH release and synthesis from E2-treated cells in the presence of P was higher than the sum s f effects that can result from each treatment independently. Conversely, the augmentative E2 effect on pituitary responsiveness to GnRH was abolished by DHT. Our data also show that the effects of E2 and DHT influenced each other in such a way that the expected negative consequences for gonadotrophs GmRH responsiveness of treatment with DHT (in terms of LH release) and potentiation by E2 of LH stimulation by GnRH were not observed. In other words, androgens, in the presence of estrogens, do not suppress the GnRH responsiveness of the pituitary gland, but they do prevent the expression of the positive effect of E2 pretreatment. The current results indicate that the pituitary cells respond to P and DHT more efficiently when E2 is present, which can be correlated with the wellknown regulatory effect of estrogen on progestin and androgen receptor population. Recent studies have shown that the anterior pituitary progestin and androgen receptors are particularly sensitive to the action of circulating estrogen (Attardi 1984; Handa et al. I986), increasing on proestrus and during periods sf high circulating estrogen, and decreasing in the absence of estrogen. Physiologically, the effect of P may play an important role at proestrus by dictating the find magnitude of the secretory response, at the time when high pituitary responsiveness is required. The hnctional significance of the inhibitory effect of DHT could then be to confine the LH surge to that day of proestrus. -
We are gratefugll to the NIDDK Rat Pituitary Hormone Distribution Program for providing the LH RIA kit. This work was supported by the research grant 446/88 from Consejo Nacisnal Be Investigaciones Cientificas y Tecnicas of Argentina and grant 1@2/90 from Consejo de Investigaciones Cientificas y Teenol6gicas de la Provincia de Cdrdoba, Argentina. Apfelbaurm, M. E. 1981. Basal and GwRH-induced release and synthesis of LH and FSH from incubated pituitary glands throughout the 4-day estrous cycle of the rat. Horm. Res. (Basel), 15: 109121. Apfelbaum, M. E. 1983. Influence of gonadal steroids on the timecourse of release and synthesis of luteinizing hormone, folliclestimulating hormone and prolactin from rat pituitary glands in vitro. J. Endocrinol. 96: 171- 179. Apfelbaum, M. E., and Taleisnik, S. 1976. Interaction between oestrogen anad gonadotrophin-releasing hormone on the release and synthesis of luteinizing hormone and follicle-stimulating hormone from incubated pituitaries. J. Endocrinol. 68: 127 - 136. Apfelbaum, M. E., and Taleisnik, S. 1977. Influence s f oestrogen administration in vivo and in vitro on the release and synthesis of
P ~ E Z AND APFELBALJM
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LH and FSH from incubated pituitaries. Acta Endocrinol. $6: 704-713. Attardi, B. 1984. Progesterone mdulation of the luteinizing hormone surge: regulation of hypothalamic and pituitary progestin receptors. Endocrinology (Baltimore), 115: 2 113-2 122. Butcher, R. L., Collins, W. E., and Fugo, N. W. 1974. Plasma concentration of LH, FSH, prolactin. progesterone and estradiol-17P throughout the 4-day estrous cycle of the rat. Endocrinology (Baltimore), 94: 1704- 1708. Caligaris, L., Astrada, J. J., and Taleisnik, S. 1971. Biphasic effect of progesterone on the release of gonadotropins in rats. Endocrinology (Baltimore), 89: 33 1- 337. Clayton, R. N . , and Catt, K. J. 1981. Regulation of pituitary gonadotropin-releasing hormone receptors by gonadal hormones. Endocrinology (Baltimore), 108: 887 - 894. Drouin, J., and Labrie, F. 1976. Selective effect of androgen on LH and FSH release in anterior pituitary cells in culture. Endocrinology (Baltimore), 98: 1528- 1534. Dupon, C., and Kim, M. H. 1973. Peripheral plasma level of testosterone, androstenedione, and oetradiol during the rat oestrous cycle. J. Endmrinol. 59: 653 -654. Emons. G . , Ortmann, O., Fingscheidt, U.,et a!. 1986. Short-term effects of oestradiol and 4-hydroxyoestradiol on gonadotropinreleasing hormone induced luteinizing hormone secretion by rat pituitary cells in culture. Acta Endocrinol. 1111 312-320. Emons, G., Hrevert, E. U.,Ortrnann, O., eb a!. 1989. Studies on the subeellular mechanisms mediating the negative estradiol effect on GnRH-induced LM-release by rat pituitary cells in culture. Acta Endocrinol. 121: 350 - 360. Ferin, M., Tempone, A., Zimmering, P., and Vande WBle, R. L. 1969. Effects of antibodies to 170-estradiol and progesterone on the estrous cycle of the rat. Endocrinology (Baltimore), 85: 10701878. Frawley, L. S o ,and Neil, 5 . D. 1984. Bighasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer culthares of rat and monkey pituitary cells. Endocrinology (Baltimore), 614: 459 -663. Gay, V. L., and Tomacari, R. L. 1974. Follicle-stimulating hormone secretion in the female rat: cyclic release is dependent on circulating androgen. Science (Washington, DC), 84: 75-77. Handa, R. J., Reid, D. L., and Resko, J. A. 1986. Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male. BioH. Reprod. 34: 293-303. Kalra, P. S., Fawcett, C. P., Kmlich, L., and McCann, S. M. 1953. The effect of gonadal steroids on plasma gonadotropins and prolactin in the rat. Endocrinology (Baltimore), 92: 1256- 1268. Kamel, F., Balz, S. A., Kubajak, C. L., and Schneider, V. A. 1987. Gonadal steroids modulate pulsatile luteinizing hormone secretion by p e r k e d rat anterior pituitary cells. Endocrinology (Baltimore), 120: 1451- 1654. Karseh, F. J. 1987. Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing homone. Annu. Rev. Physiol. 49: 345-382. Kmmmen, L. A., and Baldwin, D. M. 1988. Regulation of luteinizing hormone subunit biosynthesis in cultured male anterior
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pituitary cells: effects of gonadotropin-releasing hormone and testosterone. Endocrinology (Baltimore), 123: H 868 - 1878. Labrie, F., Pelletier, G., Lemay, A., eb a!. 1973. Control of protein synthesis in anterior pituitary gland. Ach Endocrinol. Suppl. 180: 301 -334. Lasley, B. L., Wang, C. F., and Ven, S. S. C. 1975. The effects of oestrogen and progesterone on the functional capacity of the gonadotrophs. J. Clin. Endocrinol. Metab. 41: 828 - 826. Legan, S. J., Coon, G . A., and Karsch, F. 5 . 1975. Role of estrogen as initiator of daily LH surges in ovariectomized rat. Endocrinology (Baltimore), 96: 58-56. Liu, T. C., and Jackson, G . L. 1977. Effect of in vivo treatment with estrogen on luteinizing hormone synthesis and release by rat pituitaries in vitro. Endocrinology (Baltimore). 108: 1294- 1382. Liu, T. C., and Jackson, G . L. 1988. Actions of 176-estradiol on gonadotropin release induced by drugs that activate intracellular signal transduction mechanisms in rat anterior pituitary cells. Biol. Reprod. 39: 787-796. Mahesh, V., and Muldoon, T. G 1987. Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretions. 9. Steroid Biochem. 27: 665 -675. Menon, M. M. D., Peegel, H. M. S., and Katta, V. B. S . 1985. Estradiol potentiation of gonadotropin-releasing hormone responsiveness in the anterior pituitary mediated by an increase in gonadotropin-releasing hormone receptors. Am. J. Bbstet. Gynecol. 851: 534-540. Qrtmann, O . , Wiese, H., Knuppen, W.,and Emons, G. 1989. Acute facilitory action of progesterone on gonadotropin secretion of perifused rat pituitary cells. Aeta Endocrinol. 12%:426-434. Ramey, J. W., Highsmith, R. F.,Wilfinger, W. W., and Baldwin, D. M. 1987. The effects of gonadotropin-releasing homone and estradiol on luteinizing homone biosynthesis in cultured rat anterior pituitary cells. Endocrinology (Baltimore), 120: 15031513. Schally, A. V., Redding, T. W., and Arimura, A. 1943. Effect of sex steroids to LH and FSW-releasing homone in vitro. Endocrinology (Baltimore), 93: 893- 902. Smith, M., Freeman, M., and Neill, J. 1975. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteurn of pxudopregnancy. Endocrinology (Baltimore), 96: 2 19-226. Starzec, A., Counis, R., and Sutisz, M. 1986. Gonadotropinreleasing hormone stimulates the synthesis of the polypeptide chains of luteinizing hormone. Endocrinology (Baltimore), 119: 561 -566. Turgeon, J. L., and Waring, D. W. 1981. Acute progesterone and 176-estradiol modulation of luteinizing hormone secretion by pituitaries of cycling rats perfused in vitro. Endocrinology (Baltimore), 188: 413-419. Turgeon, J. L., and Waring, D. W. 1990, Rapid augmentation by progesterone of agonist-stimulated luteinizing homone secretion by cultured pituitary cells. Endocrinology (Baltimore), 627: 773 780.
