Estradiol-Induced Increase of the LH Responsiveness to LH Releasing Hormone (LHRH) in Rat Anterior Pituitary Cells in Culture JACQUES DROUIN,1 LISETTE LAGAC£, AND FERNAND LABRIE2 Medical Research Council Group in Molecular Endocrinology, he Centre Hospitaller de I'Universite Laval, Quebec, GlV 4G2, Canada ABSTRACT. The effect of 17/3-estradiol (E2) on LH secretion was studied using rat adenohypophyseal cells in primary culture. Preincubation of cells with 1 X 10~9M E2 for 40 h decreased the concentration of LHRH required for half-maximal stimulation (ED50) of LH release from 3.0 ± 0.3 to 1.6 ± 0.2 x 10~10M (P < 0.01). Basal LH release was increased from 84 ± 4 to 182 ± 8 ng LH-RP-l/ml/4h (P < 0.01) by E2 pretreatment. Time-course experiments showed that the stimulatory effect of 10~8M E2 on the LH response to LHRH can be first measured after 10 h of incubation in the presence of E2 and that this effect is
E
STRADIOL (E2) is known to exert both negative (1) and positive (2-4) feedback effects on gonadotropin secretion. The sensitivity of the LH and FSH responses to LHRH is increased following estrogen administration in man (4-6) and rat (3,7). The increased pituitary sensitivity to LHRH found at midcycle in women (8,9) and at proestrus in the rat (10,11) is also thought to follow increased estrogen secretion. Although these data strongly suggest a pituitary site of action of estrogens on LH release, the recent observation that pretreatment with LHRH can potentiate the LH responses to subsequent injection of the neurohormone (7,12,13) adds further complication to the interpretation of in vivo experiments using LHRH. It was thus felt of interest to study the effect of E2 on both basal and LHRH-induced LH release using rat anterior pituitary cells in primary culture. Received May 3, 1976. 1 Fellow of the Medical Research Council of Canada (MRC). 2 MRC Associate.
maximal after 24 h of incubation with the steroid. While E2 increases the LH responsiveness to LHRH, androgens decrease the sensitivity of LHsecreting cells to the neurohormone. The LHRH ED50 value of testosterone-treated cells is of 7.2 ± 0.4 vs. 3.7 ± 0.3 x 10-'°M for control cells (P < 0.01). E2 can only partially reverse this inhibitory effect of androgens on the LH response to LHRH. These data show clearly that E2 can have a direct stimulatory effect on LH-secreting cells to increase the sensitivity of their response to LHRH. (Endocrinology 99: 1477, 1976)
Materials and Methods Materials Synthetic LHRH (AY-24, 031-4) was kindly supplied by Drs. M. Gotz and R. Deghenghi, Ayerst Research Laboratories, Montreal. Stock solutions of steroids obtained from Steraloids were prepared in 0.9% NaCl-1% ethanol and were used at a hundredfold dilution in the incubation medium. Such a concentration of ethanol (0.01%) in the incubation medium did not affect basal or LHRH-stimulated LH release. Media and sera for tissue culture were obtained from Grand Island Biologicals Co. Dextrancoated charcoal (DCC)-adsorbed sera were prepared by overnight incubation of the sera at 4 C under constant agitation with 1% charcoal (Norit A) and 0.1% Dextran T 70 obtained from Fisher and Pharmacia, respectively. This treatment was found to remove more than 99% of added tracer amounts of [;!H]-E2 and to lower E2 concentrations as measured by radioimmunoassay to undetectable levels. Purified goat anti-rabbit y-globulins were a product of Endocrinolab Ltd, Quebec. Preparation of dispersed anterior pituitary cells Adult female Sprague-Dawley rats (150-300 g) at random stages of the estrous cycle (obtained
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Endo • 1976 Vol 99 • No 6
DROUIN, LAGACE AND LABRIE
These incubations were followed by a short period (4 to 6 h) of incubation with increasing concentrations of LHRH or a fixed concentration of tlie neurohormone corresponding to the ED5() (50% effective dose) of LHRH stimulation ofLH release. These short-term incubations were performed in DMEM without sera in the presence or absence of the indicated concentrations of steroids. At the end of incubations, media were centrifuged at 100 x g for 5 min at 4 C and the supernatant frozen at - 2 0 C until assayed. Hormone assays LH was measured by double-antibody radioimmunoassay (15,16) using rat hormones (LH-I-1 and LH-RP-1) and rabbit antiserum to ovine LH (anti-LH-Sl) kindly supplied by Dr. A. F. Parlow for the National Institute of Arthritis, Metabolism, and Digestive Diseases, Rat Pituitary Hormone Program. Calculations -7
FIG. 1. Effect of increasing concentrations of LHRH on LH release by anterior pituitary cells in primary culture preincubated for about 40 h in the presence (•) or absence (O) of 1 x 10~9M 17/3-estradiol. The results are presented as means ± SEM of triplicate determinations.
from Canadian Breeding Farms, St. Constant) were used for the preparation of primary cultures of anterior pituitary cells. The cells were prepared as described (14), except that the cell suspension was washed by centrifugation through a layer of 4% bovine serum albumin after the Viokase treatment. Cells (5-10 x 105) in 1.5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% DCC-adsorbed horse serum and 2.5% DCC-adsorbed fetal calf serum were plated in 35 x 10 mm Falcon petri dishes. Cells were usually used 3 days after plating. Incubation procedure Cells were washed 4 times with sterile DMEM without sera before further incubation for about 48 h in 1.5 ml of DMEM containing DCC-adsorbed sera in the presence or absence of the indicated concentrations of steroids to be tested. In time-course experiments, steroids were added at the indicated times in 10 fA volumes.
Radioimmunoassay data were analyzed with a Hewlett-Packard desk-top calculator using a program based on model II of Rodbard and Lewald (17). Statistical significance was analyzed using Student's t test. Dose-response curves and 50% effective doses (ED50) were calculated using an iterative non-linear least squares regression according to die method described (18). Each treatment was applied to 3 separate culture dishes and the data are expressed as mean ± SEM (when the SEM was greater than the symbol used). Results As shown in Fig. 1, preincubation of anterior pituitary cells in primary culture for 40 h in the presence of 1 x 10~9M E2 increased the LH responsiveness to LHRH. The LHRH concentration required to produce an half-maximal stimulation (ED50) of LH release was decreased from 3.0 ± 0.3 to 1.6 ± 0.2 x 10~10M by E2-pretreatment (P
E
STRADIOL (E2) is known to exert both negative (1) and positive (2-4) feedback effects on gonadotropin secretion. The sensitivity of the LH and FSH responses to LHRH is increased following estrogen administration in man (4-6) and rat (3,7). The increased pituitary sensitivity to LHRH found at midcycle in women (8,9) and at proestrus in the rat (10,11) is also thought to follow increased estrogen secretion. Although these data strongly suggest a pituitary site of action of estrogens on LH release, the recent observation that pretreatment with LHRH can potentiate the LH responses to subsequent injection of the neurohormone (7,12,13) adds further complication to the interpretation of in vivo experiments using LHRH. It was thus felt of interest to study the effect of E2 on both basal and LHRH-induced LH release using rat anterior pituitary cells in primary culture. Received May 3, 1976. 1 Fellow of the Medical Research Council of Canada (MRC). 2 MRC Associate.
maximal after 24 h of incubation with the steroid. While E2 increases the LH responsiveness to LHRH, androgens decrease the sensitivity of LHsecreting cells to the neurohormone. The LHRH ED50 value of testosterone-treated cells is of 7.2 ± 0.4 vs. 3.7 ± 0.3 x 10-'°M for control cells (P < 0.01). E2 can only partially reverse this inhibitory effect of androgens on the LH response to LHRH. These data show clearly that E2 can have a direct stimulatory effect on LH-secreting cells to increase the sensitivity of their response to LHRH. (Endocrinology 99: 1477, 1976)
Materials and Methods Materials Synthetic LHRH (AY-24, 031-4) was kindly supplied by Drs. M. Gotz and R. Deghenghi, Ayerst Research Laboratories, Montreal. Stock solutions of steroids obtained from Steraloids were prepared in 0.9% NaCl-1% ethanol and were used at a hundredfold dilution in the incubation medium. Such a concentration of ethanol (0.01%) in the incubation medium did not affect basal or LHRH-stimulated LH release. Media and sera for tissue culture were obtained from Grand Island Biologicals Co. Dextrancoated charcoal (DCC)-adsorbed sera were prepared by overnight incubation of the sera at 4 C under constant agitation with 1% charcoal (Norit A) and 0.1% Dextran T 70 obtained from Fisher and Pharmacia, respectively. This treatment was found to remove more than 99% of added tracer amounts of [;!H]-E2 and to lower E2 concentrations as measured by radioimmunoassay to undetectable levels. Purified goat anti-rabbit y-globulins were a product of Endocrinolab Ltd, Quebec. Preparation of dispersed anterior pituitary cells Adult female Sprague-Dawley rats (150-300 g) at random stages of the estrous cycle (obtained
1477
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
1478
Endo • 1976 Vol 99 • No 6
DROUIN, LAGACE AND LABRIE
These incubations were followed by a short period (4 to 6 h) of incubation with increasing concentrations of LHRH or a fixed concentration of tlie neurohormone corresponding to the ED5() (50% effective dose) of LHRH stimulation ofLH release. These short-term incubations were performed in DMEM without sera in the presence or absence of the indicated concentrations of steroids. At the end of incubations, media were centrifuged at 100 x g for 5 min at 4 C and the supernatant frozen at - 2 0 C until assayed. Hormone assays LH was measured by double-antibody radioimmunoassay (15,16) using rat hormones (LH-I-1 and LH-RP-1) and rabbit antiserum to ovine LH (anti-LH-Sl) kindly supplied by Dr. A. F. Parlow for the National Institute of Arthritis, Metabolism, and Digestive Diseases, Rat Pituitary Hormone Program. Calculations -7
FIG. 1. Effect of increasing concentrations of LHRH on LH release by anterior pituitary cells in primary culture preincubated for about 40 h in the presence (•) or absence (O) of 1 x 10~9M 17/3-estradiol. The results are presented as means ± SEM of triplicate determinations.
from Canadian Breeding Farms, St. Constant) were used for the preparation of primary cultures of anterior pituitary cells. The cells were prepared as described (14), except that the cell suspension was washed by centrifugation through a layer of 4% bovine serum albumin after the Viokase treatment. Cells (5-10 x 105) in 1.5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% DCC-adsorbed horse serum and 2.5% DCC-adsorbed fetal calf serum were plated in 35 x 10 mm Falcon petri dishes. Cells were usually used 3 days after plating. Incubation procedure Cells were washed 4 times with sterile DMEM without sera before further incubation for about 48 h in 1.5 ml of DMEM containing DCC-adsorbed sera in the presence or absence of the indicated concentrations of steroids to be tested. In time-course experiments, steroids were added at the indicated times in 10 fA volumes.
Radioimmunoassay data were analyzed with a Hewlett-Packard desk-top calculator using a program based on model II of Rodbard and Lewald (17). Statistical significance was analyzed using Student's t test. Dose-response curves and 50% effective doses (ED50) were calculated using an iterative non-linear least squares regression according to die method described (18). Each treatment was applied to 3 separate culture dishes and the data are expressed as mean ± SEM (when the SEM was greater than the symbol used). Results As shown in Fig. 1, preincubation of anterior pituitary cells in primary culture for 40 h in the presence of 1 x 10~9M E2 increased the LH responsiveness to LHRH. The LHRH concentration required to produce an half-maximal stimulation (ED50) of LH release was decreased from 3.0 ± 0.3 to 1.6 ± 0.2 x 10~10M by E2-pretreatment (P
