0013-7227/91/1282-0710$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
Interrelationship between the Actions of Testosterone and Primate Sertoli Cell Inhibin in the Control of Gonadotropin Secretion by Cultured Pituitary Cells* SATOSHI KITAHARA, FUMIKAZU KOTSUJI, HUGH S. KEEPING, HIROYUKI OSHIMA, PHILIP TROEN, AND STEPHEN J. WINTERS Department of Medicine, Montefiore Hospital, and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15213
ABSTRACT. There is accumulating evidence that the differential regulation of LH and FSH secretion in the male is partly accomplished by the direct actions of testosterone (T) and inhibin on the pituitary. The present study was designed to examine the interaction between T and inhibin, in the presence and absence of GnRH, using dispersed pituitary cells in monolayer culture and cells perifused with pulses of GnRH from intact, 2-week castrated, and castrated T-replaced young adult male rats. The effect of partially purified inhibin from primate Sertoli cell culture medium (pSCI) to suppress basal FSH secretion was similar with pituitary cells from intact and castrated rats. T increased basal FSH secretion in the presence or absence of pSCI but did not alter the dose-dependent suppression of
M
FSH by pSCI with cells from either intact or castrate rats. Castration increased basal FSH and LH secretion, whereas only basal FSH release was increased with cells from T-replaced castrates. T pretreatment increased the action of pSCI to suppress GnRH-stimulated FSH and LH release from perifused pituitary cells. These data indicate that T and inhibin exert opposite but independent effects on basal FSH release. The action of inhibin to suppress basal FSH secretion is not impaired by the absence of T and inhibin subsequent to castration. By contrast, the actions of T and inhibin to suppress GnRHstimulated gonadotropin secretion are coordinated and interrelated. (Endocrinology 128: 710-716, 1991)
release. For T, the effect on LH is greater than on FSH; for inhibin the effect on FSH exceeds the effect on LH. These different responses to GnRH may relate to differing amounts of FSH available for release because of altered FSH synthesis (11). There is one study using pituitary cells in monolayer culture that indicates that androgens enhance the action of inhibin to suppress GnRH-stimulated LH and FSH release (12). In light of the evidence for an interaction between T and inhibin in regulating FSH and LH secretion, we examined the influence of T on the control of gonadotropin secretion by inhibin using pituitary cells perifused with pulses of GnRH and cells in monolayer culture from intact, castrated, and T-replaced orchidectomized rats.
ORE than 75% of gonadotrophs contain both FSH and LH, and this number increases to 90% after orchidectomy (1). Further, the secretion (2), glycosylation (3), and almost certainly the synthesis (4) of both gonadotropins is stimulated by GnRH. Yet, under certain circumstances, FSH and LH are produced disproportionately. In the male, there is accumulating evidence that both testosterone (T) and inhibin play a role in the differential regulation of FSH and LH secretion. In vitro, inhibin decreases interpulse basal FSH secretion by pituitary cells perifused with pulses of GnRH (5), whereas T has the opposite action (6). The effect of inhibin on basal FSH is partly explained by a rapid and profound reduction of FSH/? messenger RNA (mRNA) levels (7), whereas under certain conditions T increases FSH/3 mRNA (8). On the other hand, both T (6, 9) and inhibin (5, 10, 11) suppress GnRH-stimulated FSH and LH
Materials and Methods Animals
Received September 27, 1990. Address all correspondence and requests for reprints to: Dr. Stephen J. Winters, Department of Medicine, Montefiore University Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213. * This work was supported in part by NIH Grants R01-HD-19546 (to S.J.W.) and R01-HD-25272 (to H.S.K.) Portions of this work were published in abstract form in J Androl 10(l):23, 1989, and in the Program of the Eighth International Congress of Endocrinology, Kyoto, 1988, Abstract 03-19-067.
Five-week-old male Sprague Dawley rats (Zivic Miller Laboratories, Inc, Allison Park, PA) were divided into two groups. Group 1 was castrated under methoxyfluorane anesthesia (Pitman-Moore, Inc., Washington Crossing, NJ); group 2 was an intact control. In selected experiments, a third group of rats was implanted with T (Sigma Chemical Co., St. Louis, MO)filled Silastic capsules (25 mm X 1.6 mm id, Dow Corning
710
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T AND INHIBIN EFFECTS ON THE PITUITARY Corp., Midland, MI) at the time of orchidectomy. Two weeks later, at age 7 weeks, animals were killed by decapitation, and serum was saved for LH, FSH, and T RIAs. Pituitary cell dispersion The methods for the preparation of pituitary cell cultures were similar to those described previously (5, 6, 13). In brief, anterior pituitaries from 7-week-old control and experimental rats were minced and enzymatically digested for 60 min at 37 C with 0.4% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.003% DNase (Sigma Chemical Co.) followed by treatment with 0.25% pancreatin (Sigma Chemical Co.) for 8 min. Cells were washed with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum. Dispersed cells were harvested with 0.1% Triton-X 100 (BioRad Laboratories, Richmond, CA) for determination of intracellular LH and FSH content by RIA. Inhibin preparation Inhibin was partially purified from cynomolgus monkey Sertoli cell culture medium by filtration on Bio-Gel P-6DG columns (Bio-Rad Laboratories), affinity chromatography on Concanavalin A-Sepharose (Pharmacia, Piscataway, NJ), and reverse phase HPLC, as described previously (5). Two batches of inhibin from primate Sertoli cell culture medium (pSCI) were used for this study. Batch 1 with an ED50 of 20 ng protein/ ml (based on suppression of basal FSH secretion over 72 h), was used for the monolayer culture studies. Batch 2, used for the perifusion experiments, had an ED50 of 250 ng protein/ml. Cell culture and perifusion Dispersed pituitary cells were plated at a density of 1.2-2.0 X 105 cells/ml in DMEM containing 10% fetal calf serum in 1.5-cm diameter multi-well culture dishes (Nunc, Roskilde, Denmark) at 37 C in a 5% CO2-95% air atmosphere. For perifusion experiments, dispersed cells were cultured with preswollen Cytodex beads type 3 (Pharmacia) at a ratio of 1.0 X 107 cells : 100 mg beads : 30 ml DMEM in 10 ml siliconized Petri dishes for 48 h. Then the cell-bead mixtures were transferred to spinner flasks and stirred with magnetic rods for 24 h. The mixtures, containing approximately 1 x 107 cells per culture vessel, were packed into perifusion chambers and perifused at 0.3 ml/min with DMEM-F12 containing 14.8 mM NaHCO3 and 0.2% glucose (pH 7.3) gassed with 10% CO290% O2. Chambers were stimulated with 10 nM GnRH for 2 min every 60 min. Fractions of the column effluents were collected at 10-min intervals for determination of LH and FSH by RIA. Experimental design Exp 1: effects of castration on inhibin action. Pituitary cells from intact or castrated rats were precultured for 2 days. Then the medium was changed to include pSCI at concentrations of 0-200 ng protein/ml, and the incubation was continued for 3 days. The medium from each well was collected and stored at -20 C for LH and FSH RIA.
711
Exp 2: effects of T on inhibin-regulated basal and GnRH-stimulated gonadotropin secretion. Pituitary cells from intact or castrated rats were precultured for 2 days, and 10 nM T was added to designated culture wells beginning on day 3. On day 4, the medium was changed to include pSCI (0-200 ng/ml) as well as T. After 72 h the medium was collected, and fresh medium was added, which also contained 10 nM GnRH. The incubation was continued for an additional 6 h. Exp 3: effects of castration and T replacement on inhibin action. Inhibin deficiency was produced by T replacement of orchidectomized rats. At age 7 weeks, 2 weeks after surgery, rats were decapitated, and blood samples were obtained from intact, bilaterally orchidectomized, and T-implanted bilaterally orchidectomized rats. Dispersed pituitary cells were cultured and treated with pSCI as described in Exp 1. Exp 4: effects of T on inhibin-regulated gonadotropin secretion by perifused pituitary cells from intact rats. The pituitary cellbead mixtures were precultured for 48 h. Ten nanomolar T or control medium was added for 24 h before beginning the perifusion. Cells were exposed to 3 hourly pulses of GnRH (10 nM); then pSCI (225 ng protein/ml) was added to the perifusion medium of one chamber for an additional 4 h, during which time the hourly stimulation with GnRH was continued. In all experiments a second control chamber containing cells from the same dispersion not treated with pSCI was perifused simultaneously with pulses of GnRH. Data analysis RIA potency estimates were performed using the computer program of Rodbard (14). Dose-response curves were analyzed using the Allfit program of DeLean et al. (15). The effects of T and inhibin on LH and FSH secretion were analyzed using a two-way analysis of variance. Because basal secretion differed between replicate experiments, the data were transformed for this analysis. In each study, basal secretion in the absence of T and inhibin was set at 100%. Hormone concentrations in medium from treated cultures were expressed as percentages of concentrations of control cultures. All values are reported as mean ± SEM. In the perifusion experiments, LH and FSH pulse amplitudes were calculated by subtracting the basal concentration before adding GnRH from the hormone concentration in each of the following three 10-min fractions, which were then summed. Comparisons were performed between the experimental and control columns in the same study, because the absolute gonadotropin levels differed between replicate experiments.
Results Expl pSCI suppressed basal FSH secretion by pituitary cells from both intact and castrated rats in a dose-dependent fashion (Fig. 1). Castration increased (P < 0.01) maximum and minimum FSH secretion but did not influence the percentage suppression from maximum to minimum levels or the ED50 for pSCI (Table 1). Castration also
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 07:28 For personal use only. No other uses without permission. . All rights reserved.
T AND INHIBIN EFFECTS ON THE PITUITARY
712
Endo«1991 Voll28-No2
CASTRATE INTACT
•O— CONTROL
CASTRATE
- - Q- -
TESTOSTERONE
CONTROL TESTOSTERONE
S 40
0 2.5
15
2.5
50 100 200
5
2 . 5S
1 5
5 0 1 0 0 2 0 0
pSC Inhibin (ng/ml)
pSC Inhlbln (ng/ml)
0
2 . 5 5
1 5 5 0 1 0 0 2 0 0
15 50 100 200 30 CASTRATE
FIG.
1. Suppression of basal FSH and LH secretion by pituitary cell
cultures from intact and castrated rats with increasing concentrations of inhibin from primate Sertoli cell culture medium. The results are from a representative experiment and are the mean ± SEM of triplicate or quadruplicate wells.
• --••--
——O - - O- -
CONTROL
CONTROL TESTOSTERONE
TESTOSTERONE
= 20-
TABLE 1. Effects of Inhibin on FSH release by cultured pituitary cells from intact and castrated rats
Maximum FSH release ° (ng/
Intact
Castrated
24.0 ± 3.5
40.6 ± 6.76
4.4 ± 0.4
6.3 ± 0.76
2 x 10 5 c e l l s -72 h )
Minimum FSH release0 (ng/2 x 106 cells • 72 h) Percent suppression ED60 (ng/ml)
2.5
79.0 ± 4.0 20.1 ± 1.4
78.0 ± 6.8 24.5 ± 3.2
Cells from intact or castrated rats were cultured with pSCI (2.5-200 ng/ml) for 72 h. The results are the mean ± SEM of 6 independent experiments. Values were determined after curve fitting using the Allfit program of DeLean et al. (15). 0 FSH secretion in the absence of inhibin. 6 P < 0.01, castrate us. intact. c Minimal FSH response in the presence of excess pSCI.
increased basal LH secretion. There was no significant effect of pSCI on basal LH secretion (Fig. 1).
Exp2 Figure 2 illustrates that pretreatment of pituitary cells for 24 h with T-increased (P < 0.01) basal FSH secretion in the presence or absence of inhibin. A comparable effect was observed with cells from intact and castrated rats. T pretreatment did not significantly change the ED50 for suppression of basal FSH secretion by pSCI by cells from either intact (25.0 ± 3.6 vs. 19.6 ± 0.5 ng/ml; T vs. control) or castrated rats (24.1 ± 4.0 vs. 18.6 ± 4.5 ng/ml). There was no effect of either T or pSCI together with T on basal LH secretion in either group (Fig. 2, C and D). GnRH (10 nM) was then added to all culture wells, and the incubation was continued for 6 h. The results in Fig. 3 show that T pretreatment suppressed GnRH-
5
1 5
5 0
1 0 0 2 0 0
2.5
5
1 5 5 0 1 0 0 2 0 0
pSC INHIBIN (ng/ml) FIG. 2. Effects of T pretreatment on the suppression of basal FSH (A, B) and LH (C, D) secretion by pituitary cells from intact and castrated rats. Cells were pretreated with 10 nM T for 24 h and then incubated for 72 h with pSCI (0-200 ng/ml) as well as T. The results are the mean ± SEM of triplicate wells from one of three experiments, each with similar results. T significantly increased basal FSH secretion by cells from intact (F = 33.2, P < 0.01) and castrated rats (F = 31.5, P < 0.01).
stimulated LH secretion by cells from intact (F = 143, P < 0.01) and castrated (F = 67, P < 0.01) rats in the presence or absence of pSCI, whereas no significant effect of T pretreatment was observed on GnRH-stimulated FSH release. pSCI suppressed GnRH-stimulated FSH (F = 2.63, P < 0.05, and F = 34.6, P < 0.01) and LH (F = 18.1, P < 0.01 and F = 6.0, P < 0.01) secretion by cells from both intact and castrated rats, respectively.
Exp3 Table 2 summarizes the effects of orchidectomy and orchidectomy together with T replacement on serum hormone concentrations and gonadotropin cell content of dispersed pituitary cells. T-Filled Silastic capsules that produced serum T levels of 5.4 ± 0.5 nM suppressed serum LH and FSH concentrations to levels indistinguishable from those of intact rats. Pituitary cell LH
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T AND INHIBIN EFFECTS ON THE PITUITARY ^
•— --•--
0
CASTRATE
INTACT
2.5
5
CONTROL
— O —
CONTROL
- - Q- -
TESTOSTERONE
15
50
0
1 0 0 2 0 0
2.5
5
15
50
—
CONTROL
- -
TESTOSTERONE
5
15
o
5
15 50 100 200
pSC INHIBIN (ng/ml)
INTACT
2.5
•
CASTRATE
50
O—
1 0 0 2 0 0
0
2.5
TESTOSTERONE
5
15
50
CASTRATE CASTRATE . TESTOSTERONE
2.5
5
15
50 100 200
pSC INHIBIN (ng/ml)
FIG. 4. Effects of pSCI on basal FSH and LH secretion by pituitary cells from intact, castrated, and T-replaced castrated rats. The results are the mean ± SEM of triplicate wells from one of four experiments, each with similar results.
CONTROL
--•Q--
--•ft--
INTACT
1 0 0 2 0 0
2.5
0
INTACT
CASTRATE . TESTOSTERONE
TESTOSTERONE
713
1 0 0 2 0 0
cells from intact rats. T treatment of castrate rats selectively suppressed basal LH release to the level observed with cells from intact rats; FSH secretion, by contrast, remained at the levels observed with cells from castrates. Increasing doses of inhibin suppressed (P < 0.01) basal FSH secretion similarly in all three groups but was without effect on basal LH secretion.
pSC INHIBIN (ng/ml) FIG. 3. Suppression of GnRH-stimulated FSH and LH secretion by increasing concentrations of pSCI in the presence or absence of T. After collection of the media used to calculate the results in Fig. 2, cells were treated for 6 h with 10 nM GnRH. The experiment was performed three times with similar results. TABLE 2. Effects of castration and testosterone replacement on serum hormone levels and gonadotropin cell content Intact Serum (nM) Serum LH (ng/ml) Serum FSH (ng/ml) LH content (ng/2 x 105 cells) FSH content (ng/2 X 106 cells)
Castrated
Castrated + T replaced
5.4 ± O.50'* (10) 7.9 ± 0.7 0.9 ± 0.1° 6 0.50 ±0.05 7.13 ±0.80° 0.95 ± 0.31 (10) 13.9 ± 0.7 32.4 ± 1.7° 18.6 ± 3.4" (10) 135.6 ± 5.2 303.0 ± 8.6° 136.3 ± 15.5" (3) 100.3 ± 8.2
84.7 ± 10.0 164.9 ± 27.0a-i> (3)
The number of samples is in parentheses. 0 P < 0.05 vs. intact. b P < 0.05 castrated + T replacement us. castrated.
content increased 2-fold 2 weeks after orchidectomy, and T replacement maintained LH content at the level found in pituitary cells from intact rats. By contrast, T treatment of castrated rats, but not castration alone, increased the FSH content of dispersed pituitary cells by 65% (P
Vol. 128, No. 2 Printed in U.S.A.
Interrelationship between the Actions of Testosterone and Primate Sertoli Cell Inhibin in the Control of Gonadotropin Secretion by Cultured Pituitary Cells* SATOSHI KITAHARA, FUMIKAZU KOTSUJI, HUGH S. KEEPING, HIROYUKI OSHIMA, PHILIP TROEN, AND STEPHEN J. WINTERS Department of Medicine, Montefiore Hospital, and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15213
ABSTRACT. There is accumulating evidence that the differential regulation of LH and FSH secretion in the male is partly accomplished by the direct actions of testosterone (T) and inhibin on the pituitary. The present study was designed to examine the interaction between T and inhibin, in the presence and absence of GnRH, using dispersed pituitary cells in monolayer culture and cells perifused with pulses of GnRH from intact, 2-week castrated, and castrated T-replaced young adult male rats. The effect of partially purified inhibin from primate Sertoli cell culture medium (pSCI) to suppress basal FSH secretion was similar with pituitary cells from intact and castrated rats. T increased basal FSH secretion in the presence or absence of pSCI but did not alter the dose-dependent suppression of
M
FSH by pSCI with cells from either intact or castrate rats. Castration increased basal FSH and LH secretion, whereas only basal FSH release was increased with cells from T-replaced castrates. T pretreatment increased the action of pSCI to suppress GnRH-stimulated FSH and LH release from perifused pituitary cells. These data indicate that T and inhibin exert opposite but independent effects on basal FSH release. The action of inhibin to suppress basal FSH secretion is not impaired by the absence of T and inhibin subsequent to castration. By contrast, the actions of T and inhibin to suppress GnRHstimulated gonadotropin secretion are coordinated and interrelated. (Endocrinology 128: 710-716, 1991)
release. For T, the effect on LH is greater than on FSH; for inhibin the effect on FSH exceeds the effect on LH. These different responses to GnRH may relate to differing amounts of FSH available for release because of altered FSH synthesis (11). There is one study using pituitary cells in monolayer culture that indicates that androgens enhance the action of inhibin to suppress GnRH-stimulated LH and FSH release (12). In light of the evidence for an interaction between T and inhibin in regulating FSH and LH secretion, we examined the influence of T on the control of gonadotropin secretion by inhibin using pituitary cells perifused with pulses of GnRH and cells in monolayer culture from intact, castrated, and T-replaced orchidectomized rats.
ORE than 75% of gonadotrophs contain both FSH and LH, and this number increases to 90% after orchidectomy (1). Further, the secretion (2), glycosylation (3), and almost certainly the synthesis (4) of both gonadotropins is stimulated by GnRH. Yet, under certain circumstances, FSH and LH are produced disproportionately. In the male, there is accumulating evidence that both testosterone (T) and inhibin play a role in the differential regulation of FSH and LH secretion. In vitro, inhibin decreases interpulse basal FSH secretion by pituitary cells perifused with pulses of GnRH (5), whereas T has the opposite action (6). The effect of inhibin on basal FSH is partly explained by a rapid and profound reduction of FSH/? messenger RNA (mRNA) levels (7), whereas under certain conditions T increases FSH/3 mRNA (8). On the other hand, both T (6, 9) and inhibin (5, 10, 11) suppress GnRH-stimulated FSH and LH
Materials and Methods Animals
Received September 27, 1990. Address all correspondence and requests for reprints to: Dr. Stephen J. Winters, Department of Medicine, Montefiore University Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213. * This work was supported in part by NIH Grants R01-HD-19546 (to S.J.W.) and R01-HD-25272 (to H.S.K.) Portions of this work were published in abstract form in J Androl 10(l):23, 1989, and in the Program of the Eighth International Congress of Endocrinology, Kyoto, 1988, Abstract 03-19-067.
Five-week-old male Sprague Dawley rats (Zivic Miller Laboratories, Inc, Allison Park, PA) were divided into two groups. Group 1 was castrated under methoxyfluorane anesthesia (Pitman-Moore, Inc., Washington Crossing, NJ); group 2 was an intact control. In selected experiments, a third group of rats was implanted with T (Sigma Chemical Co., St. Louis, MO)filled Silastic capsules (25 mm X 1.6 mm id, Dow Corning
710
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T AND INHIBIN EFFECTS ON THE PITUITARY Corp., Midland, MI) at the time of orchidectomy. Two weeks later, at age 7 weeks, animals were killed by decapitation, and serum was saved for LH, FSH, and T RIAs. Pituitary cell dispersion The methods for the preparation of pituitary cell cultures were similar to those described previously (5, 6, 13). In brief, anterior pituitaries from 7-week-old control and experimental rats were minced and enzymatically digested for 60 min at 37 C with 0.4% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.003% DNase (Sigma Chemical Co.) followed by treatment with 0.25% pancreatin (Sigma Chemical Co.) for 8 min. Cells were washed with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum. Dispersed cells were harvested with 0.1% Triton-X 100 (BioRad Laboratories, Richmond, CA) for determination of intracellular LH and FSH content by RIA. Inhibin preparation Inhibin was partially purified from cynomolgus monkey Sertoli cell culture medium by filtration on Bio-Gel P-6DG columns (Bio-Rad Laboratories), affinity chromatography on Concanavalin A-Sepharose (Pharmacia, Piscataway, NJ), and reverse phase HPLC, as described previously (5). Two batches of inhibin from primate Sertoli cell culture medium (pSCI) were used for this study. Batch 1 with an ED50 of 20 ng protein/ ml (based on suppression of basal FSH secretion over 72 h), was used for the monolayer culture studies. Batch 2, used for the perifusion experiments, had an ED50 of 250 ng protein/ml. Cell culture and perifusion Dispersed pituitary cells were plated at a density of 1.2-2.0 X 105 cells/ml in DMEM containing 10% fetal calf serum in 1.5-cm diameter multi-well culture dishes (Nunc, Roskilde, Denmark) at 37 C in a 5% CO2-95% air atmosphere. For perifusion experiments, dispersed cells were cultured with preswollen Cytodex beads type 3 (Pharmacia) at a ratio of 1.0 X 107 cells : 100 mg beads : 30 ml DMEM in 10 ml siliconized Petri dishes for 48 h. Then the cell-bead mixtures were transferred to spinner flasks and stirred with magnetic rods for 24 h. The mixtures, containing approximately 1 x 107 cells per culture vessel, were packed into perifusion chambers and perifused at 0.3 ml/min with DMEM-F12 containing 14.8 mM NaHCO3 and 0.2% glucose (pH 7.3) gassed with 10% CO290% O2. Chambers were stimulated with 10 nM GnRH for 2 min every 60 min. Fractions of the column effluents were collected at 10-min intervals for determination of LH and FSH by RIA. Experimental design Exp 1: effects of castration on inhibin action. Pituitary cells from intact or castrated rats were precultured for 2 days. Then the medium was changed to include pSCI at concentrations of 0-200 ng protein/ml, and the incubation was continued for 3 days. The medium from each well was collected and stored at -20 C for LH and FSH RIA.
711
Exp 2: effects of T on inhibin-regulated basal and GnRH-stimulated gonadotropin secretion. Pituitary cells from intact or castrated rats were precultured for 2 days, and 10 nM T was added to designated culture wells beginning on day 3. On day 4, the medium was changed to include pSCI (0-200 ng/ml) as well as T. After 72 h the medium was collected, and fresh medium was added, which also contained 10 nM GnRH. The incubation was continued for an additional 6 h. Exp 3: effects of castration and T replacement on inhibin action. Inhibin deficiency was produced by T replacement of orchidectomized rats. At age 7 weeks, 2 weeks after surgery, rats were decapitated, and blood samples were obtained from intact, bilaterally orchidectomized, and T-implanted bilaterally orchidectomized rats. Dispersed pituitary cells were cultured and treated with pSCI as described in Exp 1. Exp 4: effects of T on inhibin-regulated gonadotropin secretion by perifused pituitary cells from intact rats. The pituitary cellbead mixtures were precultured for 48 h. Ten nanomolar T or control medium was added for 24 h before beginning the perifusion. Cells were exposed to 3 hourly pulses of GnRH (10 nM); then pSCI (225 ng protein/ml) was added to the perifusion medium of one chamber for an additional 4 h, during which time the hourly stimulation with GnRH was continued. In all experiments a second control chamber containing cells from the same dispersion not treated with pSCI was perifused simultaneously with pulses of GnRH. Data analysis RIA potency estimates were performed using the computer program of Rodbard (14). Dose-response curves were analyzed using the Allfit program of DeLean et al. (15). The effects of T and inhibin on LH and FSH secretion were analyzed using a two-way analysis of variance. Because basal secretion differed between replicate experiments, the data were transformed for this analysis. In each study, basal secretion in the absence of T and inhibin was set at 100%. Hormone concentrations in medium from treated cultures were expressed as percentages of concentrations of control cultures. All values are reported as mean ± SEM. In the perifusion experiments, LH and FSH pulse amplitudes were calculated by subtracting the basal concentration before adding GnRH from the hormone concentration in each of the following three 10-min fractions, which were then summed. Comparisons were performed between the experimental and control columns in the same study, because the absolute gonadotropin levels differed between replicate experiments.
Results Expl pSCI suppressed basal FSH secretion by pituitary cells from both intact and castrated rats in a dose-dependent fashion (Fig. 1). Castration increased (P < 0.01) maximum and minimum FSH secretion but did not influence the percentage suppression from maximum to minimum levels or the ED50 for pSCI (Table 1). Castration also
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 07:28 For personal use only. No other uses without permission. . All rights reserved.
T AND INHIBIN EFFECTS ON THE PITUITARY
712
Endo«1991 Voll28-No2
CASTRATE INTACT
•O— CONTROL
CASTRATE
- - Q- -
TESTOSTERONE
CONTROL TESTOSTERONE
S 40
0 2.5
15
2.5
50 100 200
5
2 . 5S
1 5
5 0 1 0 0 2 0 0
pSC Inhibin (ng/ml)
pSC Inhlbln (ng/ml)
0
2 . 5 5
1 5 5 0 1 0 0 2 0 0
15 50 100 200 30 CASTRATE
FIG.
1. Suppression of basal FSH and LH secretion by pituitary cell
cultures from intact and castrated rats with increasing concentrations of inhibin from primate Sertoli cell culture medium. The results are from a representative experiment and are the mean ± SEM of triplicate or quadruplicate wells.
• --••--
——O - - O- -
CONTROL
CONTROL TESTOSTERONE
TESTOSTERONE
= 20-
TABLE 1. Effects of Inhibin on FSH release by cultured pituitary cells from intact and castrated rats
Maximum FSH release ° (ng/
Intact
Castrated
24.0 ± 3.5
40.6 ± 6.76
4.4 ± 0.4
6.3 ± 0.76
2 x 10 5 c e l l s -72 h )
Minimum FSH release0 (ng/2 x 106 cells • 72 h) Percent suppression ED60 (ng/ml)
2.5
79.0 ± 4.0 20.1 ± 1.4
78.0 ± 6.8 24.5 ± 3.2
Cells from intact or castrated rats were cultured with pSCI (2.5-200 ng/ml) for 72 h. The results are the mean ± SEM of 6 independent experiments. Values were determined after curve fitting using the Allfit program of DeLean et al. (15). 0 FSH secretion in the absence of inhibin. 6 P < 0.01, castrate us. intact. c Minimal FSH response in the presence of excess pSCI.
increased basal LH secretion. There was no significant effect of pSCI on basal LH secretion (Fig. 1).
Exp2 Figure 2 illustrates that pretreatment of pituitary cells for 24 h with T-increased (P < 0.01) basal FSH secretion in the presence or absence of inhibin. A comparable effect was observed with cells from intact and castrated rats. T pretreatment did not significantly change the ED50 for suppression of basal FSH secretion by pSCI by cells from either intact (25.0 ± 3.6 vs. 19.6 ± 0.5 ng/ml; T vs. control) or castrated rats (24.1 ± 4.0 vs. 18.6 ± 4.5 ng/ml). There was no effect of either T or pSCI together with T on basal LH secretion in either group (Fig. 2, C and D). GnRH (10 nM) was then added to all culture wells, and the incubation was continued for 6 h. The results in Fig. 3 show that T pretreatment suppressed GnRH-
5
1 5
5 0
1 0 0 2 0 0
2.5
5
1 5 5 0 1 0 0 2 0 0
pSC INHIBIN (ng/ml) FIG. 2. Effects of T pretreatment on the suppression of basal FSH (A, B) and LH (C, D) secretion by pituitary cells from intact and castrated rats. Cells were pretreated with 10 nM T for 24 h and then incubated for 72 h with pSCI (0-200 ng/ml) as well as T. The results are the mean ± SEM of triplicate wells from one of three experiments, each with similar results. T significantly increased basal FSH secretion by cells from intact (F = 33.2, P < 0.01) and castrated rats (F = 31.5, P < 0.01).
stimulated LH secretion by cells from intact (F = 143, P < 0.01) and castrated (F = 67, P < 0.01) rats in the presence or absence of pSCI, whereas no significant effect of T pretreatment was observed on GnRH-stimulated FSH release. pSCI suppressed GnRH-stimulated FSH (F = 2.63, P < 0.05, and F = 34.6, P < 0.01) and LH (F = 18.1, P < 0.01 and F = 6.0, P < 0.01) secretion by cells from both intact and castrated rats, respectively.
Exp3 Table 2 summarizes the effects of orchidectomy and orchidectomy together with T replacement on serum hormone concentrations and gonadotropin cell content of dispersed pituitary cells. T-Filled Silastic capsules that produced serum T levels of 5.4 ± 0.5 nM suppressed serum LH and FSH concentrations to levels indistinguishable from those of intact rats. Pituitary cell LH
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T AND INHIBIN EFFECTS ON THE PITUITARY ^
•— --•--
0
CASTRATE
INTACT
2.5
5
CONTROL
— O —
CONTROL
- - Q- -
TESTOSTERONE
15
50
0
1 0 0 2 0 0
2.5
5
15
50
—
CONTROL
- -
TESTOSTERONE
5
15
o
5
15 50 100 200
pSC INHIBIN (ng/ml)
INTACT
2.5
•
CASTRATE
50
O—
1 0 0 2 0 0
0
2.5
TESTOSTERONE
5
15
50
CASTRATE CASTRATE . TESTOSTERONE
2.5
5
15
50 100 200
pSC INHIBIN (ng/ml)
FIG. 4. Effects of pSCI on basal FSH and LH secretion by pituitary cells from intact, castrated, and T-replaced castrated rats. The results are the mean ± SEM of triplicate wells from one of four experiments, each with similar results.
CONTROL
--•Q--
--•ft--
INTACT
1 0 0 2 0 0
2.5
0
INTACT
CASTRATE . TESTOSTERONE
TESTOSTERONE
713
1 0 0 2 0 0
cells from intact rats. T treatment of castrate rats selectively suppressed basal LH release to the level observed with cells from intact rats; FSH secretion, by contrast, remained at the levels observed with cells from castrates. Increasing doses of inhibin suppressed (P < 0.01) basal FSH secretion similarly in all three groups but was without effect on basal LH secretion.
pSC INHIBIN (ng/ml) FIG. 3. Suppression of GnRH-stimulated FSH and LH secretion by increasing concentrations of pSCI in the presence or absence of T. After collection of the media used to calculate the results in Fig. 2, cells were treated for 6 h with 10 nM GnRH. The experiment was performed three times with similar results. TABLE 2. Effects of castration and testosterone replacement on serum hormone levels and gonadotropin cell content Intact Serum (nM) Serum LH (ng/ml) Serum FSH (ng/ml) LH content (ng/2 x 105 cells) FSH content (ng/2 X 106 cells)
Castrated
Castrated + T replaced
5.4 ± O.50'* (10) 7.9 ± 0.7 0.9 ± 0.1° 6 0.50 ±0.05 7.13 ±0.80° 0.95 ± 0.31 (10) 13.9 ± 0.7 32.4 ± 1.7° 18.6 ± 3.4" (10) 135.6 ± 5.2 303.0 ± 8.6° 136.3 ± 15.5" (3) 100.3 ± 8.2
84.7 ± 10.0 164.9 ± 27.0a-i> (3)
The number of samples is in parentheses. 0 P < 0.05 vs. intact. b P < 0.05 castrated + T replacement us. castrated.
content increased 2-fold 2 weeks after orchidectomy, and T replacement maintained LH content at the level found in pituitary cells from intact rats. By contrast, T treatment of castrated rats, but not castration alone, increased the FSH content of dispersed pituitary cells by 65% (P
