Endocrinology 101: 1335, 1977 FSH AND LH STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY: STUDIES WITH PORCINE GRANULOSA CELLS D4 VITRO Juraj Osterman
and James M. Hammond
Division of Endocrinology, Department of Medicine, The Milton S. Hershey Medical Center of the Pennsylvania State University, Hershey, Penna. 17033 ABSTRACT: Under defined conditions in vitro FSH and LH caused a dosedependent stimulation of ornithine decarboxylase (ODC) activity in granulosa cells isolated from porcine ovarian follicles. In cells from small follicles, FSH was at least twice as potent an inducer of the enzyme activity as LH. The cells from medium-sized follicles were more responsive to both hormones than cells from small follicles. In addition, the cells from medium-sized follicles were approximately equally responsive to maximal FSH and LH stimulation. Incubation of cells from either small follicles or medium-sized follicles with maximum effective doses of LH plus FSH caused no additive effects.
Previous in_ vivo studies have implicated LH as the mediator of spontaneously occurring elevations of ovarian ODC activity in the rat (1). With exogenous administration, LH, but not FSH or various sex steroids, induced the enzyme activity (1-4). After LH stimulation iji vivo, the enzyme activity was significantly higher in Graafian follicles than in whole ovary (5). Here we report the first systematic study of the regulation of ovarian ODC activity under defined conditions ^n vitro. Using isolated porcine granulosa cells and in vitro techniques developed by Channing (6), we have found effects of FSH as well as LH and described the changes in hormonal responsiveness of these follicular cells as they mature. MATERIALS AND METHODS Ovine LH (NIH-LH-S19) was obtained from the Hormone Distribution Office, NIAMDD. The highly purified ovine FSH (FSH-HP,G4-150C) containing FSH activity approximately 50 x that of NIH-FSH-S1 was obtained from Dr. Harold Papkoff. D L - Q - ^ C ) ornithine monohydro-
Submitted May 20, 1977
chloride (specific activity 45 mCi/ mmole) was from New England Nuclear. Bovine albumin, fraction V, was from Miles Laboratories, Inc. All other reagents were of the best quality commercially available. Porcine ovaries were collected at a local slaughter house, and granulosa cells from small (1-2 mm) and medium-sized (3-5 mm) follicles were isolated as previously described (7). Approximately 10^ cells/flask were incubated at 37 C in room air for 1-6 h in 10 ml Medium 199 with Hanks' salts (Flow Laboratories) containing 25 mM HEPES buffer, 100 U/ml penicillin and 100 yg/ml streptomycin, pH 7.4. At the end of the incubation period, the cells were recovered by centrifugation and washed twice in 25 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 0.1 mM EDTA and 2.5 mM dithiothreitol. The cells were then homogenized in 2.0 ml of the same buffer without sucrose. The homogenates were centrifuged for 30 min at 20,000 x g and supernatants were used for the assay of ODC activity (8) and protein concentration (9). There was no appreciable loss of enzyme activity in frozen supernatants over a 72 h period. The assay mixture (total volume 0.25 ml) contained
1335
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 October 2015. at 18:05 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1977 Vol 101 • No 4
RAPID COMMUNICATIONS
Figure 1 illustrates the time course of gonadotropin induction of ODC activity of granulosa cells from small follicles. The enzyme activities are given in relative units since two pools of ovaries (50-100 ovaries/pool) were required to complete the experiment. FSH was a more potent stimulator than LH but the profile of activation was similar.
J _ -
*—* FSH o—o LH
1400
DECARBO (ILASE ACTIV )les/mg pe r 30 min)
>.
o o
To evaluate the precision of the culture technique and ODC assay, the responsiveness of granulosa cells to
o o
RESULTS
LH (200 ng/ml) was determined in 6 consecutive experiments with 3-5 replicate cultures per experiment. The coefficient of variation within experiment ranged from 1.4% to 13.0% (average 10.1%). However, substantial variation was encountered b e tween experiments involving different batches of cells. For example, in the 15 experiments incorporated into the estimate of LH responsivity of granulosa cells from small follicles depicted in Figure 4, the coefficient of variation between experiments was 47.1%.
o
L-ornithine 0.4 mM, DLthine 0.5 yCi, pyridoxal phosphate 0.08 mM, dithiothreitol 2.5 mM, TrisHC1 buffer 50 mM, pH 7.2 and 75-100 yg of supernatant protein. The l^C02 released was trapped in hyamine. The assay was linear for at least 1 h when the reaction mixture contained between 0.04 and 0.2 mg of protein. All assays were carried out in duplicate for 30 min. The enzyme activity was expressed as pmoles 1^002 released/mg protein per 30 min.
o
1336
2 E
J °
S
400 200
f
3
12
3 4 TIME (hr)
Figure 1. Time course of ODC activation by FSH (50 ng/ml) and LH (200 ng/ml). Granulosa cells were from small ovarian follicles. Each point represents the mean and range of duplicate assays from a single culture. For several points the range falls within the width of the symbol.
i
i
5
20
1
50 100 DOSE (ng/ml)
200
Figure 2. The dose-response curve of FSH and LH at 4 h of incubation. Granulosa cells were from a single batch of small ovarian follicles. (Results are the mean and range of duplicate assays.) The dose-response curve for LH and FSH at 4 h of incubation is shown in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 October 2015. at 18:05 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Fig. 2. The minimum effective dose for both hormones was 5 ng/ml. The saturating dose of LH was 20-50 ng/ ml, whereas with FSH the saturation was not complete even at 200 ng/ml. At. any hormonal dose employed, the FSH was a more potent stimulator of ODC activity than LH, and the maximal response to FSH was at least twice that to LH. The response of granulosa cells from small vs. medium-sized follicles to increasing doses of LH is shown in Fig. 3. The cells from medium-sized follicles showed a greater response to each dose of LH.
1337
treated with maximally effective doses of LH plus FSH exhibited a response no greater than that to FSH alone (p >.4, paired _t test).
SMALL FOLLICLES
3000
MEDIUM FOLLICLES
2600 2200 1800 1400 1000
SMALL FOLLICLES MEDIUM FOLLICLES
!=iioo
600
I
900
200
15 CONTROL
15 CONTROL
700 500
II
300
s 100 "
2
I
5
I
10 20 LH (ng/ml)
I
50
Figure 3. The effect of LH on ODC activity of granulosa cells from small and medium-sized follicles at 4 h of incubation. Results are mean and range of duplicate assays. Figure 4 provides data from multiple experiments to support several points previously illustrated with data from single experiments: Granulosa cells from small follicles are more responsive to maximal doses of FSH than LH (p
and James M. Hammond
Division of Endocrinology, Department of Medicine, The Milton S. Hershey Medical Center of the Pennsylvania State University, Hershey, Penna. 17033 ABSTRACT: Under defined conditions in vitro FSH and LH caused a dosedependent stimulation of ornithine decarboxylase (ODC) activity in granulosa cells isolated from porcine ovarian follicles. In cells from small follicles, FSH was at least twice as potent an inducer of the enzyme activity as LH. The cells from medium-sized follicles were more responsive to both hormones than cells from small follicles. In addition, the cells from medium-sized follicles were approximately equally responsive to maximal FSH and LH stimulation. Incubation of cells from either small follicles or medium-sized follicles with maximum effective doses of LH plus FSH caused no additive effects.
Previous in_ vivo studies have implicated LH as the mediator of spontaneously occurring elevations of ovarian ODC activity in the rat (1). With exogenous administration, LH, but not FSH or various sex steroids, induced the enzyme activity (1-4). After LH stimulation iji vivo, the enzyme activity was significantly higher in Graafian follicles than in whole ovary (5). Here we report the first systematic study of the regulation of ovarian ODC activity under defined conditions ^n vitro. Using isolated porcine granulosa cells and in vitro techniques developed by Channing (6), we have found effects of FSH as well as LH and described the changes in hormonal responsiveness of these follicular cells as they mature. MATERIALS AND METHODS Ovine LH (NIH-LH-S19) was obtained from the Hormone Distribution Office, NIAMDD. The highly purified ovine FSH (FSH-HP,G4-150C) containing FSH activity approximately 50 x that of NIH-FSH-S1 was obtained from Dr. Harold Papkoff. D L - Q - ^ C ) ornithine monohydro-
Submitted May 20, 1977
chloride (specific activity 45 mCi/ mmole) was from New England Nuclear. Bovine albumin, fraction V, was from Miles Laboratories, Inc. All other reagents were of the best quality commercially available. Porcine ovaries were collected at a local slaughter house, and granulosa cells from small (1-2 mm) and medium-sized (3-5 mm) follicles were isolated as previously described (7). Approximately 10^ cells/flask were incubated at 37 C in room air for 1-6 h in 10 ml Medium 199 with Hanks' salts (Flow Laboratories) containing 25 mM HEPES buffer, 100 U/ml penicillin and 100 yg/ml streptomycin, pH 7.4. At the end of the incubation period, the cells were recovered by centrifugation and washed twice in 25 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 0.1 mM EDTA and 2.5 mM dithiothreitol. The cells were then homogenized in 2.0 ml of the same buffer without sucrose. The homogenates were centrifuged for 30 min at 20,000 x g and supernatants were used for the assay of ODC activity (8) and protein concentration (9). There was no appreciable loss of enzyme activity in frozen supernatants over a 72 h period. The assay mixture (total volume 0.25 ml) contained
1335
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 October 2015. at 18:05 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1977 Vol 101 • No 4
RAPID COMMUNICATIONS
Figure 1 illustrates the time course of gonadotropin induction of ODC activity of granulosa cells from small follicles. The enzyme activities are given in relative units since two pools of ovaries (50-100 ovaries/pool) were required to complete the experiment. FSH was a more potent stimulator than LH but the profile of activation was similar.
J _ -
*—* FSH o—o LH
1400
DECARBO (ILASE ACTIV )les/mg pe r 30 min)
>.
o o
To evaluate the precision of the culture technique and ODC assay, the responsiveness of granulosa cells to
o o
RESULTS
LH (200 ng/ml) was determined in 6 consecutive experiments with 3-5 replicate cultures per experiment. The coefficient of variation within experiment ranged from 1.4% to 13.0% (average 10.1%). However, substantial variation was encountered b e tween experiments involving different batches of cells. For example, in the 15 experiments incorporated into the estimate of LH responsivity of granulosa cells from small follicles depicted in Figure 4, the coefficient of variation between experiments was 47.1%.
o
L-ornithine 0.4 mM, DLthine 0.5 yCi, pyridoxal phosphate 0.08 mM, dithiothreitol 2.5 mM, TrisHC1 buffer 50 mM, pH 7.2 and 75-100 yg of supernatant protein. The l^C02 released was trapped in hyamine. The assay was linear for at least 1 h when the reaction mixture contained between 0.04 and 0.2 mg of protein. All assays were carried out in duplicate for 30 min. The enzyme activity was expressed as pmoles 1^002 released/mg protein per 30 min.
o
1336
2 E
J °
S
400 200
f
3
12
3 4 TIME (hr)
Figure 1. Time course of ODC activation by FSH (50 ng/ml) and LH (200 ng/ml). Granulosa cells were from small ovarian follicles. Each point represents the mean and range of duplicate assays from a single culture. For several points the range falls within the width of the symbol.
i
i
5
20
1
50 100 DOSE (ng/ml)
200
Figure 2. The dose-response curve of FSH and LH at 4 h of incubation. Granulosa cells were from a single batch of small ovarian follicles. (Results are the mean and range of duplicate assays.) The dose-response curve for LH and FSH at 4 h of incubation is shown in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 October 2015. at 18:05 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Fig. 2. The minimum effective dose for both hormones was 5 ng/ml. The saturating dose of LH was 20-50 ng/ ml, whereas with FSH the saturation was not complete even at 200 ng/ml. At. any hormonal dose employed, the FSH was a more potent stimulator of ODC activity than LH, and the maximal response to FSH was at least twice that to LH. The response of granulosa cells from small vs. medium-sized follicles to increasing doses of LH is shown in Fig. 3. The cells from medium-sized follicles showed a greater response to each dose of LH.
1337
treated with maximally effective doses of LH plus FSH exhibited a response no greater than that to FSH alone (p >.4, paired _t test).
SMALL FOLLICLES
3000
MEDIUM FOLLICLES
2600 2200 1800 1400 1000
SMALL FOLLICLES MEDIUM FOLLICLES
!=iioo
600
I
900
200
15 CONTROL
15 CONTROL
700 500
II
300
s 100 "
2
I
5
I
10 20 LH (ng/ml)
I
50
Figure 3. The effect of LH on ODC activity of granulosa cells from small and medium-sized follicles at 4 h of incubation. Results are mean and range of duplicate assays. Figure 4 provides data from multiple experiments to support several points previously illustrated with data from single experiments: Granulosa cells from small follicles are more responsive to maximal doses of FSH than LH (p
