0013-7227/90/1272-0891$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 2 Printed in U.S.A.
Differential Effects of in Vitro Glucocorticoids on Luteinizing Hormone and Follicle-Stimulating Hormone Secretion: Dependence on Sex of Pituitary Donor* JoBETH D'AGOSTINO, ROBERT J. VALADKA, AND NEENA B. SCHWARTZ Department of Neurology and Physiology, Northwestern University, Evanston, Illinois 60208
ABSTRACT. We have used a dynamic perifusion system to determine whether glucocorticoids exert a direct effect on the secretion of LH and FSH from rat anterior pituitaries. Anterior pituitary fragments from male, proestrous female, or metestrous female rats were perifused for 8 h in either the absence (basal secretion rate) or presence of pulsatile GnRH administration (50 ng/ml peak concentration). Perifusions used medium containing 0.05% ethanol (vehicle), 600 ng/ml corticosterone, or 600 ng/ml cortisol. GnRH-stimulated secretion of FSH was enhanced in pituitaries from both male and female rats after in vitro incubation with either corticosterone or cortisol. The basal secretion rate of FSH was also elevated in proestrous females after glucocorticoid treatment. The GnRH-stimulated secretion rate for LH was significantly decreased in pituitaries from male
T
rats treated with either glucocorticoid. In contrast, pituitaries from proestrous rats responded to either cortisol or corticosterone with an increase in LH secretion. Metestrous pituitaries showed divergent effects of the glucocorticoids on LH secretion; corticosterone enhanced secretion rates, and cortisol effected a decrease. Our data demonstrate that 1) glucocorticoids exert a direct effect on the secretion of LH and FSH from male and female rat pituitaries; 2) glucocorticoids elicit different effects on the secretion of LH and FSH, suggesting that they act at separate sites to regulate LH and FSH secretion; and 3) the effect of in vitro glucocorticoid treatment on gonadotropin secretion is dependent on sex and cycle stage of the pituitary donor and may be linked to prior in vivo concentrations of estrogen. {Endocrinology 127: 891-899, 1990)
glucocorticoids on basal and GnRH-stimulated secretion rates of both LH and FSH from the rat anterior pituitary. We have used male rats as well as female rats at two separate stages of the estrous cycle (proestrous and metestrous) to analyze both sex and cycle stage differences in the response.
HE EFFECTS of the stress hormones on the reproductive capacity of many mammalian species has been extensively studied. In animals (1-6) and humans (7-11), both stress and excess glucocorticoids have been shown to be effective disruptors of the reproductive cycle. The site(s) of action of the glucocorticoid hormones on the complex and precisely integrated reproductive system still remains elusive. In a number of in vivo studies a direct effect of the glucocorticoids on pituitary secretion of LH and FSH has been inferred, but not directly demonstrated (1, 3, 12-14). In vitro studies investigating whether the glucocorticoids exert their action at the level of the pituitary have reported discrepant results. Using static cell cultures, it has been demonstrated that glucocorticoid administration suppresses, enhances, or has no effect on pituitary gonadotropin secretion (15-20). In addition, there appear to be distinct LH and FSH responses to treatment (12-17). In the present study we have employed a dynamic perifusion system to study directly the effects of the
Materials and Methods Animals Male and female Sprague-Dawley CD rats (Charles River, Portage, MI) were obtained at 60 days of age and housed in temperature-controlled quarters on a 14-h light, 10-h dark cycle, with lights on at 0500 h. Food and water were provided ad libitum. For females, cycle stage was assessed by daily inspection of vaginal cytology. Only those females showing two consecutive 4-day cycles were used in this study. Males were allowed at least 1 week in the animal quarters before being used. Collection of tissue On the day of study (0900 h), animals were weighed and decapitated, and trunk blood was collected. Sera were obtained after centrifugation and stored at —20 C for subsequent LH and FSH RIAs. Pituitaries were immediately excised, and the posterior lobe was discarded. Anterior pituitaries were cut into
Received February 28, 1990. Address all correspondence and requests for reprints to: Dr. Neena B. Schwartz, Department of Neurobiology and Physiology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois, 60208. * This work was supported by NIH Grant POl-HD-07068. 891
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH
892
eighths and placed into warmed (37 C) oxygenated medium [medium 199 with 25 mM HEPES, Earle's salts, and L-glutamine (Gibco, Grand Island, NY) plus 0.5% BSA (Bovuminar Cohn fraction V powder, Armour Pharmaceutical, Kankakee, IL), 90 U/ml bacitracin (Sigma, St. Louis, MO), and 25 Mg/ml gentamicin (Garamycin, Schering Corp., Kenilworth, NJ)]. Pituitary fragments were rinsed twice in fresh medium before perifusion.
E n d o • 1990 Vol 127* No 2
and S-ll antirat FSH antibody. Mean intra- and interassay coefficients of variation were 3% and 14% for serum LH and 7.2% and 8% for medium LH determinations, respectively. Mean intra- and interassay coefficients of variation were 4.4% and 6% for medium FSH determinations, respectively. The mean intraassay coefficient of variation for serum FSH was 2%. Data calculation and statistics
In vitro perifusion Anterior pituitary fragments from six animals were transferred to individual 200-^1 microchambers for perifusion using the ACUSYST (model 11501, Endotronics, Coon Rapids, MN). Tissue was perifused at a flow rate of 10 ml/h for a total of 8 h using medium 199 (described above). Five-minute fractions were collected and stored at —20 C for subsequent LH and FSH RIAs. At the conclusion of each perifusion (450 min), tissue was exposed to a 30-min KC1 challenge (60 mM) to verify tissue viability. Perifusions were conducted in either the absence (basal secretion rate determinations) or presence of pulsatile GnRH administration. GnRH pulses were administered 30 min after the start of the perifusion to allow LH and FSH secretion rates to achieve baseline values. Seven GnRH pulses (30-sec rising phase; 50 ng/ml peak concentration) (21) were then delivered hourly at 30, 90, 150, 210, 270, 330, and 390 min. Experimental design Basal and GnRH-stimulated secretion of LH and FSH were determined in vitro for anterior pituitaries derived from proestrous and metestrous female and male rats. Six anterior pituitaries were perifused during each perifusion run. Three anterior pituitaries received no pulsatile GnRH administration (basal secretion rate), and 3 anterior pituitaries received hourly GnRH pulses. Separate perifusion runs were conducted with medium containing 0.05% ethanol (vehicle), 600 ng/ml corticosterone (Sigma), or 600 ng/ml cortisol (Sigma). The entire study was repeated once for a total of 12 perifusions. This concentration of corticosterone is within the high physiological range for rats (22). Since the perifusion medium did not contain the corticosteroid-binding protein (CBG), the concentration of free glucocorticoid reaching the tissue would be somewhat higher than that seen in vivo. The total number of experimental animals was as follows. For determination of GnRH-stimulated secretion rates: proestrous vehicle-treated, n = 3; proestrous corticosterone-treated, n = 6; proestrous cortisol-treated, n = 6; and metestrous and males, all treatment groups, n = 4. For determination of basal secretion rates: proestrous vehicle-treated, n = 3; proestrous corticosterone-treated, n = 6; proestrous cortisol-treated, n = 6; and metestrous and males, all treatment groups, n = 3. Hormone RIAs Serum and medium fractions were assayed for LH using an ovine:rat RIA, employing NIH LH S25 as standard and S-9 antirat LH antibody. FSH concentrations were determined using the rat-rat NIDDK system, with FSH RP-1 as standard
All values are reported as the mean ± SE. Both basal secretion rates and GnRH-stimulated secretion rates for LH and FSH were calculated for each hour after the 30-min stabilization period and are expressed as nanograms per h. The ratio of FSH and LH secretion rates was also calculated for each hourly interval in pituitaries receiving GnRH pulses and is expressed as FSH/LH. Separate two-way analyses of variance were calculated for proestrous and metestrous females and for males. Data for basal secretion rates were analyzed in a two-way analysis of variance (ANOVA), with treatment (vehicle, corticosterone, and cortisol) as the between-subject factor and hour of perifusion (2-7 h) as the within-subject factor. Results from GnRHstimulated pituitaries and FSH/LH ratios were also analyzed by two-way ANOVA with treatment (vehicle, corticosterone, and cortisol) as the between-subject factor and GnRH pulse number (2-7) as the within-subject factor. A three-way ANOVA was conducted for pituitary response to the 60 mM KC1 challenge, with treatment (vehicle, corticosterone, and cortisol) and group (male, proestrous female, and metestrous female) as the between factors, and KC1 treatment (prepotassium baseline value and postpotassium peak value) as the within factor. Serum concentrations of LH and FSH were analyzed by oneway ANOVA with group (proestrous female, metestrous female, and male) as the between-subject factor. Scheffe's test was used for post-hoc comparisons to reveal significant differences between levels of the between- and within-subject factor. Statistics were computed using CRISP (Crunch Interactive Statistical Package, Crunch software, San Francisco, CA).
Results Serum concentrations of LH and FSH for proestrous and metestrous female and male rats autopsied at 0900 h are shown in Table 1. Serum LH concentrations were uniformly low, as expected, in all three groups. Serum FSH concentrations were significantly higher in males than in females at either stage of the estrous cycle, which is consistent with previous observations (21). TABLE 1. Serum concentrations of LH and FSH at 0900 h Group
LH (ng/ml)
FSH (ng/ml)
Proestrous females Metestrous females Males
1.1 ± 0.2 0.9 ±0.1 0.8 ± 0.1
146.2 ± 6.5 176.9 ± 7.0 355.6 ± 17.9
Values are given as the mean ± SE. One-way ANOVA revealed a significant difference among groups for serum FSH concentrations (P < 0.000).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH
893
The secretion of LH and FSH from perifused anterior pituitaries derived from male, metestrous female, and proestrous female rats was determined under both basal and GnRH-stimulated conditions. The overall results of this study are summarized in Table 2, and the data appear in Figs. 1-4.
and metestrus-derived pituitaries increased over time regardless of treatment. This effect was not present in male rats.
Basal secretion of LH and FSH in proestrous and metestrous female and male rats
The in vitro secretion profiles of LH and FSH from three representative rat pituitaries receiving vehicle treatment only are shown in Fig. 2. GnRH-stimulated secretion of both LH and FSH varied significantly as a function of sex and cycle stage. LH secretion in response to exogenous GnRH was dramatically higher in pituitaries from proestrous rats than in those from metestrous or male rats (Fig. 2). FSH secretion rates after GnRH stimulation were highest in pituitaries obtained from male and proestrous rats (Fig. 2). Pituitaries derived from female rats exhibited an upward trend in the secretion rate of FSH as a function of time, an effect that was absent in male rat pituitaries. The effect of in vitro glucocorticoid treatment on the secretion of both LH and FSH from anterior pituitaries receiving pulsatile GnRH administration is shown in Fig. 3. In proestrous females a priming effect of GnRH on LH release was evident in the vehicle-treated as well as glucocorticoid-treated pituitaries (Fig. 3). The interaction between treatment and pulse number was significant due to dynamic changes in the effect of the glucocorticoids on LH secretion. Specifically, corticosterone treatment elevated LH secretion relative to control values during the first half of the perifusion only. Cortisol, in contrast, had a minimal effect on LH secretion during the early hours of the perifusion, but a stimulatory effect toward the end of the run. In metestrous females (Fig. 3) GnRH priming of LH secretion was evident in the vehicle-treated pituitaries, although it was not as dramatic as that in proestrous females. Treatment in vitro with either corticosterone or cortisol abolished this priming effect. The statistical interaction between treatment and pulse number was significant in the metestrous rats due to distinct temporal effects of cortisol and corticosterone on LH release. Increased secretion rates were detected in corticosteronetreated pituitaries during the early hours of the perifusion. In contrast, cortisol treatment resulted in LH secretion rates that were similar to control values at the beginning of the perifusion, but significantly lower toward the end of the run. In both glucocorticoid groups LH was secreted at a relatively constant rate throughout the perifusion. The significant interaction between treatment and time was a function of changes in the secretion rate in the vehicle-treated pituitaries.
The basal secretion rates of LH and FSH from anterior pituitaries in the presence or absence of glucocorticoids was assessed in male and female rats. There was a marginal stimulatory effect of treatment on the secretion rate for LH in proestrous females (Fig. 1). Post-hoc analyses revealed that while corticosterone treatment did not significantly alter the release of LH, cortisol significantly elevated LH secretion above control values (P < 0.002). There was also a significant interaction between treatment and hour of perifusion, since the effect of cortisol diminished as the perifusion proceeded. There was no significant effect of glucocorticoid treatment on the release of LH in either metestrous female or male rats (Fig. 1). Both males and females exhibited a downward trend in the secretion rate of LH as the perifusion progressed, regardless of treatment (Fig. 1). Pituitaries derived from proestrous females exhibited a significant increase in the basal secretion of FSH with glucocorticoid treatment (Fig. 1). Post-hoc analysis revealed that both cortisol and corticosterone significantly increased the secretion rate of FSH (P < 0.001). The effect of corticosterone on the basal secretion of FSH was more dramatic than that of cortisol and increased with time of perifusion. The basal release of FSH from either metestrous female or male rat pituitaries (Fig. 1) was not significantly affected by in vitro glucocorticoid treatment. Basal FSH secretion from both proestrusTABLE 2. Summary of glucocorticoid effects on GnRH-stimulated and basal secretion of LH and FSH Basal secretion
GnRH-stimulated secretion
LH
FSH
LH
FSH
Proestrus
B NE Ft(sust)
Bf(sust) Ff(early)
Bf(early) Ff(late)
Bf(early) Ff(late)
Metestrus
NE
NE
Bf(early) Fj(late)
Bt(sust) FNE
Male
NE
NE
BJ(sust) FJ(sust)
Bt(early) F|(early)
Changes in secretion rate relative to controls: f, increase; j , decrease; and NE, no effect on hourly secretion rates relative to controls. The temporal nature of the response is indicated in parentheses: early, first half of perifusion only; late, second half of perifusion only; sust, sustained through majority of perifusion.
GnRH-stimulated secretion of LH and FSH in proestrous and metestrous female and male rats
In pituitaries obtained from male rats (Fig. 3) both cortisol and corticosterone significantly depressed the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
894
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH 150
Proestrus
en
Vehicle
Proestrus
4000
Endo •1990 Vol 127 •No 2 Vehicle Corticosterone Cortisol
CD
Corticosterone
ES
Cortisol
3000
100
I< <
Vol. 127, No. 2 Printed in U.S.A.
Differential Effects of in Vitro Glucocorticoids on Luteinizing Hormone and Follicle-Stimulating Hormone Secretion: Dependence on Sex of Pituitary Donor* JoBETH D'AGOSTINO, ROBERT J. VALADKA, AND NEENA B. SCHWARTZ Department of Neurology and Physiology, Northwestern University, Evanston, Illinois 60208
ABSTRACT. We have used a dynamic perifusion system to determine whether glucocorticoids exert a direct effect on the secretion of LH and FSH from rat anterior pituitaries. Anterior pituitary fragments from male, proestrous female, or metestrous female rats were perifused for 8 h in either the absence (basal secretion rate) or presence of pulsatile GnRH administration (50 ng/ml peak concentration). Perifusions used medium containing 0.05% ethanol (vehicle), 600 ng/ml corticosterone, or 600 ng/ml cortisol. GnRH-stimulated secretion of FSH was enhanced in pituitaries from both male and female rats after in vitro incubation with either corticosterone or cortisol. The basal secretion rate of FSH was also elevated in proestrous females after glucocorticoid treatment. The GnRH-stimulated secretion rate for LH was significantly decreased in pituitaries from male
T
rats treated with either glucocorticoid. In contrast, pituitaries from proestrous rats responded to either cortisol or corticosterone with an increase in LH secretion. Metestrous pituitaries showed divergent effects of the glucocorticoids on LH secretion; corticosterone enhanced secretion rates, and cortisol effected a decrease. Our data demonstrate that 1) glucocorticoids exert a direct effect on the secretion of LH and FSH from male and female rat pituitaries; 2) glucocorticoids elicit different effects on the secretion of LH and FSH, suggesting that they act at separate sites to regulate LH and FSH secretion; and 3) the effect of in vitro glucocorticoid treatment on gonadotropin secretion is dependent on sex and cycle stage of the pituitary donor and may be linked to prior in vivo concentrations of estrogen. {Endocrinology 127: 891-899, 1990)
glucocorticoids on basal and GnRH-stimulated secretion rates of both LH and FSH from the rat anterior pituitary. We have used male rats as well as female rats at two separate stages of the estrous cycle (proestrous and metestrous) to analyze both sex and cycle stage differences in the response.
HE EFFECTS of the stress hormones on the reproductive capacity of many mammalian species has been extensively studied. In animals (1-6) and humans (7-11), both stress and excess glucocorticoids have been shown to be effective disruptors of the reproductive cycle. The site(s) of action of the glucocorticoid hormones on the complex and precisely integrated reproductive system still remains elusive. In a number of in vivo studies a direct effect of the glucocorticoids on pituitary secretion of LH and FSH has been inferred, but not directly demonstrated (1, 3, 12-14). In vitro studies investigating whether the glucocorticoids exert their action at the level of the pituitary have reported discrepant results. Using static cell cultures, it has been demonstrated that glucocorticoid administration suppresses, enhances, or has no effect on pituitary gonadotropin secretion (15-20). In addition, there appear to be distinct LH and FSH responses to treatment (12-17). In the present study we have employed a dynamic perifusion system to study directly the effects of the
Materials and Methods Animals Male and female Sprague-Dawley CD rats (Charles River, Portage, MI) were obtained at 60 days of age and housed in temperature-controlled quarters on a 14-h light, 10-h dark cycle, with lights on at 0500 h. Food and water were provided ad libitum. For females, cycle stage was assessed by daily inspection of vaginal cytology. Only those females showing two consecutive 4-day cycles were used in this study. Males were allowed at least 1 week in the animal quarters before being used. Collection of tissue On the day of study (0900 h), animals were weighed and decapitated, and trunk blood was collected. Sera were obtained after centrifugation and stored at —20 C for subsequent LH and FSH RIAs. Pituitaries were immediately excised, and the posterior lobe was discarded. Anterior pituitaries were cut into
Received February 28, 1990. Address all correspondence and requests for reprints to: Dr. Neena B. Schwartz, Department of Neurobiology and Physiology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois, 60208. * This work was supported by NIH Grant POl-HD-07068. 891
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH
892
eighths and placed into warmed (37 C) oxygenated medium [medium 199 with 25 mM HEPES, Earle's salts, and L-glutamine (Gibco, Grand Island, NY) plus 0.5% BSA (Bovuminar Cohn fraction V powder, Armour Pharmaceutical, Kankakee, IL), 90 U/ml bacitracin (Sigma, St. Louis, MO), and 25 Mg/ml gentamicin (Garamycin, Schering Corp., Kenilworth, NJ)]. Pituitary fragments were rinsed twice in fresh medium before perifusion.
E n d o • 1990 Vol 127* No 2
and S-ll antirat FSH antibody. Mean intra- and interassay coefficients of variation were 3% and 14% for serum LH and 7.2% and 8% for medium LH determinations, respectively. Mean intra- and interassay coefficients of variation were 4.4% and 6% for medium FSH determinations, respectively. The mean intraassay coefficient of variation for serum FSH was 2%. Data calculation and statistics
In vitro perifusion Anterior pituitary fragments from six animals were transferred to individual 200-^1 microchambers for perifusion using the ACUSYST (model 11501, Endotronics, Coon Rapids, MN). Tissue was perifused at a flow rate of 10 ml/h for a total of 8 h using medium 199 (described above). Five-minute fractions were collected and stored at —20 C for subsequent LH and FSH RIAs. At the conclusion of each perifusion (450 min), tissue was exposed to a 30-min KC1 challenge (60 mM) to verify tissue viability. Perifusions were conducted in either the absence (basal secretion rate determinations) or presence of pulsatile GnRH administration. GnRH pulses were administered 30 min after the start of the perifusion to allow LH and FSH secretion rates to achieve baseline values. Seven GnRH pulses (30-sec rising phase; 50 ng/ml peak concentration) (21) were then delivered hourly at 30, 90, 150, 210, 270, 330, and 390 min. Experimental design Basal and GnRH-stimulated secretion of LH and FSH were determined in vitro for anterior pituitaries derived from proestrous and metestrous female and male rats. Six anterior pituitaries were perifused during each perifusion run. Three anterior pituitaries received no pulsatile GnRH administration (basal secretion rate), and 3 anterior pituitaries received hourly GnRH pulses. Separate perifusion runs were conducted with medium containing 0.05% ethanol (vehicle), 600 ng/ml corticosterone (Sigma), or 600 ng/ml cortisol (Sigma). The entire study was repeated once for a total of 12 perifusions. This concentration of corticosterone is within the high physiological range for rats (22). Since the perifusion medium did not contain the corticosteroid-binding protein (CBG), the concentration of free glucocorticoid reaching the tissue would be somewhat higher than that seen in vivo. The total number of experimental animals was as follows. For determination of GnRH-stimulated secretion rates: proestrous vehicle-treated, n = 3; proestrous corticosterone-treated, n = 6; proestrous cortisol-treated, n = 6; and metestrous and males, all treatment groups, n = 4. For determination of basal secretion rates: proestrous vehicle-treated, n = 3; proestrous corticosterone-treated, n = 6; proestrous cortisol-treated, n = 6; and metestrous and males, all treatment groups, n = 3. Hormone RIAs Serum and medium fractions were assayed for LH using an ovine:rat RIA, employing NIH LH S25 as standard and S-9 antirat LH antibody. FSH concentrations were determined using the rat-rat NIDDK system, with FSH RP-1 as standard
All values are reported as the mean ± SE. Both basal secretion rates and GnRH-stimulated secretion rates for LH and FSH were calculated for each hour after the 30-min stabilization period and are expressed as nanograms per h. The ratio of FSH and LH secretion rates was also calculated for each hourly interval in pituitaries receiving GnRH pulses and is expressed as FSH/LH. Separate two-way analyses of variance were calculated for proestrous and metestrous females and for males. Data for basal secretion rates were analyzed in a two-way analysis of variance (ANOVA), with treatment (vehicle, corticosterone, and cortisol) as the between-subject factor and hour of perifusion (2-7 h) as the within-subject factor. Results from GnRHstimulated pituitaries and FSH/LH ratios were also analyzed by two-way ANOVA with treatment (vehicle, corticosterone, and cortisol) as the between-subject factor and GnRH pulse number (2-7) as the within-subject factor. A three-way ANOVA was conducted for pituitary response to the 60 mM KC1 challenge, with treatment (vehicle, corticosterone, and cortisol) and group (male, proestrous female, and metestrous female) as the between factors, and KC1 treatment (prepotassium baseline value and postpotassium peak value) as the within factor. Serum concentrations of LH and FSH were analyzed by oneway ANOVA with group (proestrous female, metestrous female, and male) as the between-subject factor. Scheffe's test was used for post-hoc comparisons to reveal significant differences between levels of the between- and within-subject factor. Statistics were computed using CRISP (Crunch Interactive Statistical Package, Crunch software, San Francisco, CA).
Results Serum concentrations of LH and FSH for proestrous and metestrous female and male rats autopsied at 0900 h are shown in Table 1. Serum LH concentrations were uniformly low, as expected, in all three groups. Serum FSH concentrations were significantly higher in males than in females at either stage of the estrous cycle, which is consistent with previous observations (21). TABLE 1. Serum concentrations of LH and FSH at 0900 h Group
LH (ng/ml)
FSH (ng/ml)
Proestrous females Metestrous females Males
1.1 ± 0.2 0.9 ±0.1 0.8 ± 0.1
146.2 ± 6.5 176.9 ± 7.0 355.6 ± 17.9
Values are given as the mean ± SE. One-way ANOVA revealed a significant difference among groups for serum FSH concentrations (P < 0.000).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH
893
The secretion of LH and FSH from perifused anterior pituitaries derived from male, metestrous female, and proestrous female rats was determined under both basal and GnRH-stimulated conditions. The overall results of this study are summarized in Table 2, and the data appear in Figs. 1-4.
and metestrus-derived pituitaries increased over time regardless of treatment. This effect was not present in male rats.
Basal secretion of LH and FSH in proestrous and metestrous female and male rats
The in vitro secretion profiles of LH and FSH from three representative rat pituitaries receiving vehicle treatment only are shown in Fig. 2. GnRH-stimulated secretion of both LH and FSH varied significantly as a function of sex and cycle stage. LH secretion in response to exogenous GnRH was dramatically higher in pituitaries from proestrous rats than in those from metestrous or male rats (Fig. 2). FSH secretion rates after GnRH stimulation were highest in pituitaries obtained from male and proestrous rats (Fig. 2). Pituitaries derived from female rats exhibited an upward trend in the secretion rate of FSH as a function of time, an effect that was absent in male rat pituitaries. The effect of in vitro glucocorticoid treatment on the secretion of both LH and FSH from anterior pituitaries receiving pulsatile GnRH administration is shown in Fig. 3. In proestrous females a priming effect of GnRH on LH release was evident in the vehicle-treated as well as glucocorticoid-treated pituitaries (Fig. 3). The interaction between treatment and pulse number was significant due to dynamic changes in the effect of the glucocorticoids on LH secretion. Specifically, corticosterone treatment elevated LH secretion relative to control values during the first half of the perifusion only. Cortisol, in contrast, had a minimal effect on LH secretion during the early hours of the perifusion, but a stimulatory effect toward the end of the run. In metestrous females (Fig. 3) GnRH priming of LH secretion was evident in the vehicle-treated pituitaries, although it was not as dramatic as that in proestrous females. Treatment in vitro with either corticosterone or cortisol abolished this priming effect. The statistical interaction between treatment and pulse number was significant in the metestrous rats due to distinct temporal effects of cortisol and corticosterone on LH release. Increased secretion rates were detected in corticosteronetreated pituitaries during the early hours of the perifusion. In contrast, cortisol treatment resulted in LH secretion rates that were similar to control values at the beginning of the perifusion, but significantly lower toward the end of the run. In both glucocorticoid groups LH was secreted at a relatively constant rate throughout the perifusion. The significant interaction between treatment and time was a function of changes in the secretion rate in the vehicle-treated pituitaries.
The basal secretion rates of LH and FSH from anterior pituitaries in the presence or absence of glucocorticoids was assessed in male and female rats. There was a marginal stimulatory effect of treatment on the secretion rate for LH in proestrous females (Fig. 1). Post-hoc analyses revealed that while corticosterone treatment did not significantly alter the release of LH, cortisol significantly elevated LH secretion above control values (P < 0.002). There was also a significant interaction between treatment and hour of perifusion, since the effect of cortisol diminished as the perifusion proceeded. There was no significant effect of glucocorticoid treatment on the release of LH in either metestrous female or male rats (Fig. 1). Both males and females exhibited a downward trend in the secretion rate of LH as the perifusion progressed, regardless of treatment (Fig. 1). Pituitaries derived from proestrous females exhibited a significant increase in the basal secretion of FSH with glucocorticoid treatment (Fig. 1). Post-hoc analysis revealed that both cortisol and corticosterone significantly increased the secretion rate of FSH (P < 0.001). The effect of corticosterone on the basal secretion of FSH was more dramatic than that of cortisol and increased with time of perifusion. The basal release of FSH from either metestrous female or male rat pituitaries (Fig. 1) was not significantly affected by in vitro glucocorticoid treatment. Basal FSH secretion from both proestrusTABLE 2. Summary of glucocorticoid effects on GnRH-stimulated and basal secretion of LH and FSH Basal secretion
GnRH-stimulated secretion
LH
FSH
LH
FSH
Proestrus
B NE Ft(sust)
Bf(sust) Ff(early)
Bf(early) Ff(late)
Bf(early) Ff(late)
Metestrus
NE
NE
Bf(early) Fj(late)
Bt(sust) FNE
Male
NE
NE
BJ(sust) FJ(sust)
Bt(early) F|(early)
Changes in secretion rate relative to controls: f, increase; j , decrease; and NE, no effect on hourly secretion rates relative to controls. The temporal nature of the response is indicated in parentheses: early, first half of perifusion only; late, second half of perifusion only; sust, sustained through majority of perifusion.
GnRH-stimulated secretion of LH and FSH in proestrous and metestrous female and male rats
In pituitaries obtained from male rats (Fig. 3) both cortisol and corticosterone significantly depressed the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:21 For personal use only. No other uses without permission. . All rights reserved.
894
EFFECT OF IN VITRO GLUCOCORTICOIDS ON LH AND FSH 150
Proestrus
en
Vehicle
Proestrus
4000
Endo •1990 Vol 127 •No 2 Vehicle Corticosterone Cortisol
CD
Corticosterone
ES
Cortisol
3000
100
I< <
