0013-7227/90/1266-3022$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Secretion by Perifused Pituitary Cells from Normal, Gonadectomized, and Testicular Feminized Rats* RICHARD J. KRIEG, JR., JUDY M. BATSON, PAUL M. MARTHA, jR.f, DENNIS W. MATT, RONALD L. SALISBURY, AND WILLIAM S. EVANS Departments of Anatomy (R.J.K., J.M.B.) and Obstetrics and Gynecology (D.W.M.), Medical College of Virginia, Richmond, Virginia 23298; the Department of Biology, University of Akron (R.L.S.), Akron, Ohio 44325; and the Departments of Pediatrics (P.M.M.) and Medicine (W.S.E.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
ABSTRACT. To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King X Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P < 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P < 0.01) than that by cells from intact animals, but lower (P < 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-
I
T IS CLEAR that the pulsatile release of LH occurs in response to quanta of GnRH that are secreted by specialized neurons in the hypothalamus, are transported through the hypothalamic-hypophyseal portal circulation, and subsequently interact with specific receptors on gonadotropes within the anterior pituitary gland (13). Also well recognized is the fact that the secretion of LH is dependent on the gonadal hormone environment Received December 4,1989. Address all correspondence and requests for reprints to: Dr. Richard J. Krieg, Jr., Department of Anatomy, Medical College of Virginia, Box 709-MCV Station, Richmond, Virginia 23298. * This work was supported in part by NIH Grants HD-00711 and HD-19170 (to W.S.E.) and Diabetes and Hormone Action Training Grant DK-07320 and Clinical Investigator Award HD-00868 (to P.M.M.).f Current address: Department of Pediatrics, Baystate Medical Center, Springfield, Massachussets 01199.
dependent LH release. Pituitary cells from intact female rats showed an overall greater (P < 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P < 0.0001) or intact female (P < 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRHstimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P < 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact female rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens. {Endocrinology 126: 3022-3027,1990)
(4-9). Less well understood, however, are the mechanisms through which the gonadal hormones modulate the reproductive components of the hypothalamic-pituitary axis. A number of studies, primarily using sophisticated hormone pulse analysis algorithms, have suggested that the gonadal hormones may act directly on the hypothalamus to influence the activity of the GnRH pulse generator (10-17). In addition, significant investigative interest has been focussed on potential direct effects of testosterone and 17/?-estradiol on the anterior pituitary. In general, the latter studies have shown that GnRHstimulated LH release by cells from female animals is greater than that by cells from male animals (18, 19), that exposure of cells in vitro to testosterone diminishes GnRH-stimulated LH secretion (19-25), and that exposure to 17/3-estradiol may initially reduce (26, 27), but
3022
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE later increases (26-28), GnRH-stimulated LH secretion. In an effort to further our knowledge concerning gonadal hormone-modulated secretion of LH, we have undertaken studies using pituitary tissue from androgenresistant, testicular feminized (Tfm) rats. These animals represent a mutant strain in which the androgen receptors are functionally impaired (29), resulting in elevated circulating levels of LH (30, 31), presumably reflecting a lack of negative feedback by testosterone (32). Given that both the circulating concentrations of testosterone and 17/3-estradiol are elevated in the Tfm rat compared to those in normal male rats, but the pituitary is insensitive to the effects of the androgen, we designed the present series of experiments to characterize GnRHstimulated LH release by perifused cells from such animals and compared the results to those obtained using tissues from intact and gonadectomized rats of the same strain.
Materials and Methods Animals Male, female, and Tfm King x Holtzman rats (Stanley Gumbreck Tfm, Introgene, Oklahoma City, OK) were raised in an environmentally controlled animal facility (14 h of light, 10 h of darkness), with the temperature maintained at 21-23 C. The gonadectomized animals underwent orchiectomy/ovariectomy 3 weeks before use. All animals were age matched for each replication of the experiment, with the mean ± SD age being 112 ± 43 days. The mean ± SD body weights (grams) were 337.9 ± 68.3 for intact males, 231.3 ± 44.8 for intact females, 305.0 ± 33.4 for Tfm, 332.8 ± 45.1 for gonadectomized (GNX) males, and 244.3 ± 33.1 for GNX females. Standard laboratory chow and water were available ad libitum. Experimental design GnRH-induced LH release by cells from intact male, intact female, Tfm, GNX male, and GNX female rats was documented using a perifusion system previously described by Evans and colleagues (33). Three or four rats provided pituitaries for each experimental group, and for each group five to eight repetitions (on separate days) were performed. After rapid decapitation, the posterior lobe of the pituitary was removed, and the anterior pituitary was diced in Earle's Balanced Salt Solution (EBSS; Gibco, Grand Island, NY). Fragments were incubated for 20 min in 10 ml EBSS with 0.2% (wt/vol) trypsin and 0.15 mg DNAse (deoxyribonuclease-I, Sigma Chemical Co., St. Louis, MO) at 37 C. Fragments were washed with calcium- and magnesium-free EBSS, and cells were dispersed by gentle trituration with a 1.0-ml pipette (Pipetman, Rainin Instrument Co., Woburn, MA). Stranded DNA was removed, and an aliquot of cells was taken for determination of the number of viable cells by the trypan blue exclusion test, using a standard hemocytometer. Cells were gently mixed with Bio-Gel P2 (200400 mesh; Bio-Rad Laboratories, Richmond, CA), which had been preswollen overnight in normal saline. The pituitary cell-
3023
Bio-Gel mixture was packed into 2.0-ml plastic syringes (Sabre International Products Ltd., Reading, Berkshire, England), which served as the perifusion chambers. Cells were maintained at 37 C by submersion of the chambers in a water bath, and perifusion was performed with Medium 199 (Gibco), containing 0.25% BSA, 10 U/ml penicillin, 0.5 Mg/ml streptomycin, 187.5 ng/ml amphotericin-B, and 5 fig/m\ gentamicin at a mean flow rate of approximately 0.43 ml/min. Cells were allowed to equilibrate to perifusion conditions for 4 h, at which time GnRH was administered as 2.5-min pulses at 30-min intervals. The concentrations of GnRH (0.03, 0.1, 0.3,1.0, 3.0,10, 30, and 100 nM) were applied in a random order. Eluate was collected as 5min fractions and stored at —20 C until measurement of LH by R1A. RIA Concentrations of LH in the eluate fractions were measured by RIA, using reagents kindly supplied by Dr. A. F. Parlow and the National Pituitary Hormone Program of the NIDDK. Antirat LH serum (S-8) and rLH reference preparation RP-2 were used. The assay standards were set up in triplicate. Intraassay coefficients of variation were 4.3% and 5.7% at 0.2 and 1.0 ng/ tube, respectively, and the interassay coefficient of variation was 7.9% at 0.125 ng/tube. Data analysis The immunoactive LH concentrations (nanograms per ml) were converted to a secretory rate for each eluate fraction (nanograms per min) and expressed as secretion per 10 million cells (nanograms per min/107 cells). The secretory response to each concentration of GnRH was expressed as the mean secretory rate above baseline during the 30-min period immediately following administration of the appropriate concentration. The slope of the baseline was calculated using the first preinfusion value, the trough levels between GnRH-stimulated surges, and the last postinfusion LH value. The baseline value for each fraction was determined using the initial preinfusion value, the slope, and the sample number, and the amount of LH secreted above baseline was calculated by subtracting the individual baseline value from the total value of LH for that fraction. The resulting values were summed over the 30-min period following the initiation of the GnRH pulse, and the sum was divided by the number of fractions (six 5-min fractions per 30-min period) to produce a mean value for the response, expressed again as the secretory rate (nanograms per min/10 million cells). The sequence of concentrations was then rearranged from random to ascending order, and the overall means (±SEM) of these data were calculated for each experiment. Concentration-response curves were determined for each treatment group using univariate linear regression. All other statistical comparisons among group means were accomplished using analysis of variance with Duncan's multiple range test. Statistical significance was accepted at P < 0.05.
Results Number of pituitary cells and viability after dispersion The mean (±SEM) number (in millions) of cells recovered after trypsin dispersion per pituitary obtained
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE
3024
Endo • 1990 Voll26«No6
GnRH
from intact male rats (2.58 ± 0.30) was similar to that obtained from GNX male (2.19 ± 0.37) and GNX female (2.35 ± 0.21) rats, but significantly less than that obtained from intact female (4.03 ± 0.55; P = 0.016) rats. The number of cells recovered from the pituitaries of Tfm rats (3.81 ± 0.41) was intermediate between but not significantly different from the numbers obtained from intact male and intact female animals. Between 8-12 million cells were introduced into each perifusion chamber. Cell viability, as demonstrated by the ability to exclude trypan blue, was 90% in every case.
3
1
(nM) 30
100
10
0.1
O.3
I I
I I I
5.0)
I I
100
30
10
1.0
0.3
I
I
I
I
I
100 30 I , ,
10 I
0.1 I
0.3 I
Basal LH release
Unstimulated LH secretory rates, determined just before administration of the initial GnRH concentration, are shown in Table 1. As can be seen, basal LH secretory rates documented for cells from intact male and female rats were similar. Basal secretory rates for intact animals were significantly lower than those for cells from GNX male and female rats. Basal LH secretion by cells obtained from Tfm rats was higher than that by cells from intact rats, but lower than that by cells from GNX rats.
3 I
i DC
5
O
i
1 I
2.0
.-J\.
o.o L.
9.0]
GnRH-stimulated LH release Representative profiles of the LH secretory responses to several concentrations of GnRH administered in a random order are shown in Fig. 1. Visual inspection suggested that the responses occurred in a concentrationdependent manner and differed according to experimental group. Figure 2 illustrates the mean LH secretory responses, which have been rearranged into order of ascending GnRH concentration. As shown in Table 2, application of univariate linear regression analysis to these data demonstrated that the responses within each study group were strongly concentration dependent. Overall mean GnRH-stimulated LH secretory rates (picograms per min/10 million cells) were calculated using all responses to all GnRH concentrations (Table 1). As can be seen, overall mean GnRH-stimulated LH secretion by cells from intact male rats was significantly lower than that by all other expermental groups. Overall mean TABLE 1. Basal and overall mean GnRH-stimulated LH secretory rates (above basal) by perifused dispersed antior pituitary cells obtained from intact male and female, Tfm, and GNX male and female rats Group
Basal
GnRH-stimulated
Intact male Intact female Tfm GNX male GNX female
232 ± 11.6° 211 ± 9.1° 376 ± 11.66 569 ± 18.0c 608 ± 19.5C
37.8 ± 4.3° 167 ± 266 566 ± 63C 281 ± 23.2M 317 ± 58d
All values represent the mean ± SEM secretory rates (picograms per min/10 million cells). Secretory rates within columns identified by different superscripts differ significantly (P < 0.05).
100
30
1
0.3
0.1
10
I
I
I
I
|
|
JJ TIME
T (HOURS)
FIG. 1. LH secretion by perifused anterior pituitary cells obtained from intact male and female, Tfm, and GNX male and female rats. Individual data points are expressed as secretory rate (nanograms per min/10 million cells) for each 5-min fraction. The concentrations of GnRH were administered in random order.
secretion by cells from Tfm animals was higher than that in all other groups. Cells from GNX male and female rats displayed indistinguishable overall mean LH secretory rates. Although statistically intermediate between the intact male and Tfm groups, overall mean secretion by cells from intact female rats did not differ significantly (P = 0.079) from the rate for GNX male rats.
Discussion To our knowledge, the current in vitro study is the first in which both basal and GnRH-stimulated LH secretory rates have been compared using pituitary cells obtained from Tfm rats and their intact and gonadectomized littermates. Our results in the intact animals con-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE
GnRH RESPONSE 1.4 1.0
• Male • Female gS Gx Male 0 Gx Female • Tfm
.8
1
.6
c
.4
I
.2 0
0.1
0.3 1.0 3.0 10 30 100 GnRH Concentrations (nM) FIG. 2. Mean (±SEM) GnRH-stimulated LH release by pituitary cells from intact male (8 repetitions; n = 30), intact female (8 repetitions; n = 24), Tfm (10 repetitions; n = 35), GNX male (6 repetitions; n = 24), and GNX female (5 repetitions; n = 23) rats in response to several concentrations of GnRH. Although the GnRH challenges were given in random order, they have, for purposes of illustration, been rearranged into ascending order of concentration. The responses were concentration dependent and linear for all groups. TABLE 2. Concentration-response relationships between GnRH and LH release by cells from intact male and female, Tfm, and GNX male and female rats Slope (pg/min • 10 million cells/nM GnRH)a Intact male Intact female Tfm
GNX male GNX female 0 6
6.8 ± 45.5 ± 179.0 ± 83.3 ± 93.8 ±
0.8 8.5 20.2 8.7 19.9
r2
P"
0.45 0.45 0.62 0.76 0.59
Vol. 126, No. 6 Printed in U.S.A.
Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Secretion by Perifused Pituitary Cells from Normal, Gonadectomized, and Testicular Feminized Rats* RICHARD J. KRIEG, JR., JUDY M. BATSON, PAUL M. MARTHA, jR.f, DENNIS W. MATT, RONALD L. SALISBURY, AND WILLIAM S. EVANS Departments of Anatomy (R.J.K., J.M.B.) and Obstetrics and Gynecology (D.W.M.), Medical College of Virginia, Richmond, Virginia 23298; the Department of Biology, University of Akron (R.L.S.), Akron, Ohio 44325; and the Departments of Pediatrics (P.M.M.) and Medicine (W.S.E.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
ABSTRACT. To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King X Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P < 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P < 0.01) than that by cells from intact animals, but lower (P < 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-
I
T IS CLEAR that the pulsatile release of LH occurs in response to quanta of GnRH that are secreted by specialized neurons in the hypothalamus, are transported through the hypothalamic-hypophyseal portal circulation, and subsequently interact with specific receptors on gonadotropes within the anterior pituitary gland (13). Also well recognized is the fact that the secretion of LH is dependent on the gonadal hormone environment Received December 4,1989. Address all correspondence and requests for reprints to: Dr. Richard J. Krieg, Jr., Department of Anatomy, Medical College of Virginia, Box 709-MCV Station, Richmond, Virginia 23298. * This work was supported in part by NIH Grants HD-00711 and HD-19170 (to W.S.E.) and Diabetes and Hormone Action Training Grant DK-07320 and Clinical Investigator Award HD-00868 (to P.M.M.).f Current address: Department of Pediatrics, Baystate Medical Center, Springfield, Massachussets 01199.
dependent LH release. Pituitary cells from intact female rats showed an overall greater (P < 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P < 0.0001) or intact female (P < 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRHstimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P < 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact female rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens. {Endocrinology 126: 3022-3027,1990)
(4-9). Less well understood, however, are the mechanisms through which the gonadal hormones modulate the reproductive components of the hypothalamic-pituitary axis. A number of studies, primarily using sophisticated hormone pulse analysis algorithms, have suggested that the gonadal hormones may act directly on the hypothalamus to influence the activity of the GnRH pulse generator (10-17). In addition, significant investigative interest has been focussed on potential direct effects of testosterone and 17/?-estradiol on the anterior pituitary. In general, the latter studies have shown that GnRHstimulated LH release by cells from female animals is greater than that by cells from male animals (18, 19), that exposure of cells in vitro to testosterone diminishes GnRH-stimulated LH secretion (19-25), and that exposure to 17/3-estradiol may initially reduce (26, 27), but
3022
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE later increases (26-28), GnRH-stimulated LH secretion. In an effort to further our knowledge concerning gonadal hormone-modulated secretion of LH, we have undertaken studies using pituitary tissue from androgenresistant, testicular feminized (Tfm) rats. These animals represent a mutant strain in which the androgen receptors are functionally impaired (29), resulting in elevated circulating levels of LH (30, 31), presumably reflecting a lack of negative feedback by testosterone (32). Given that both the circulating concentrations of testosterone and 17/3-estradiol are elevated in the Tfm rat compared to those in normal male rats, but the pituitary is insensitive to the effects of the androgen, we designed the present series of experiments to characterize GnRHstimulated LH release by perifused cells from such animals and compared the results to those obtained using tissues from intact and gonadectomized rats of the same strain.
Materials and Methods Animals Male, female, and Tfm King x Holtzman rats (Stanley Gumbreck Tfm, Introgene, Oklahoma City, OK) were raised in an environmentally controlled animal facility (14 h of light, 10 h of darkness), with the temperature maintained at 21-23 C. The gonadectomized animals underwent orchiectomy/ovariectomy 3 weeks before use. All animals were age matched for each replication of the experiment, with the mean ± SD age being 112 ± 43 days. The mean ± SD body weights (grams) were 337.9 ± 68.3 for intact males, 231.3 ± 44.8 for intact females, 305.0 ± 33.4 for Tfm, 332.8 ± 45.1 for gonadectomized (GNX) males, and 244.3 ± 33.1 for GNX females. Standard laboratory chow and water were available ad libitum. Experimental design GnRH-induced LH release by cells from intact male, intact female, Tfm, GNX male, and GNX female rats was documented using a perifusion system previously described by Evans and colleagues (33). Three or four rats provided pituitaries for each experimental group, and for each group five to eight repetitions (on separate days) were performed. After rapid decapitation, the posterior lobe of the pituitary was removed, and the anterior pituitary was diced in Earle's Balanced Salt Solution (EBSS; Gibco, Grand Island, NY). Fragments were incubated for 20 min in 10 ml EBSS with 0.2% (wt/vol) trypsin and 0.15 mg DNAse (deoxyribonuclease-I, Sigma Chemical Co., St. Louis, MO) at 37 C. Fragments were washed with calcium- and magnesium-free EBSS, and cells were dispersed by gentle trituration with a 1.0-ml pipette (Pipetman, Rainin Instrument Co., Woburn, MA). Stranded DNA was removed, and an aliquot of cells was taken for determination of the number of viable cells by the trypan blue exclusion test, using a standard hemocytometer. Cells were gently mixed with Bio-Gel P2 (200400 mesh; Bio-Rad Laboratories, Richmond, CA), which had been preswollen overnight in normal saline. The pituitary cell-
3023
Bio-Gel mixture was packed into 2.0-ml plastic syringes (Sabre International Products Ltd., Reading, Berkshire, England), which served as the perifusion chambers. Cells were maintained at 37 C by submersion of the chambers in a water bath, and perifusion was performed with Medium 199 (Gibco), containing 0.25% BSA, 10 U/ml penicillin, 0.5 Mg/ml streptomycin, 187.5 ng/ml amphotericin-B, and 5 fig/m\ gentamicin at a mean flow rate of approximately 0.43 ml/min. Cells were allowed to equilibrate to perifusion conditions for 4 h, at which time GnRH was administered as 2.5-min pulses at 30-min intervals. The concentrations of GnRH (0.03, 0.1, 0.3,1.0, 3.0,10, 30, and 100 nM) were applied in a random order. Eluate was collected as 5min fractions and stored at —20 C until measurement of LH by R1A. RIA Concentrations of LH in the eluate fractions were measured by RIA, using reagents kindly supplied by Dr. A. F. Parlow and the National Pituitary Hormone Program of the NIDDK. Antirat LH serum (S-8) and rLH reference preparation RP-2 were used. The assay standards were set up in triplicate. Intraassay coefficients of variation were 4.3% and 5.7% at 0.2 and 1.0 ng/ tube, respectively, and the interassay coefficient of variation was 7.9% at 0.125 ng/tube. Data analysis The immunoactive LH concentrations (nanograms per ml) were converted to a secretory rate for each eluate fraction (nanograms per min) and expressed as secretion per 10 million cells (nanograms per min/107 cells). The secretory response to each concentration of GnRH was expressed as the mean secretory rate above baseline during the 30-min period immediately following administration of the appropriate concentration. The slope of the baseline was calculated using the first preinfusion value, the trough levels between GnRH-stimulated surges, and the last postinfusion LH value. The baseline value for each fraction was determined using the initial preinfusion value, the slope, and the sample number, and the amount of LH secreted above baseline was calculated by subtracting the individual baseline value from the total value of LH for that fraction. The resulting values were summed over the 30-min period following the initiation of the GnRH pulse, and the sum was divided by the number of fractions (six 5-min fractions per 30-min period) to produce a mean value for the response, expressed again as the secretory rate (nanograms per min/10 million cells). The sequence of concentrations was then rearranged from random to ascending order, and the overall means (±SEM) of these data were calculated for each experiment. Concentration-response curves were determined for each treatment group using univariate linear regression. All other statistical comparisons among group means were accomplished using analysis of variance with Duncan's multiple range test. Statistical significance was accepted at P < 0.05.
Results Number of pituitary cells and viability after dispersion The mean (±SEM) number (in millions) of cells recovered after trypsin dispersion per pituitary obtained
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE
3024
Endo • 1990 Voll26«No6
GnRH
from intact male rats (2.58 ± 0.30) was similar to that obtained from GNX male (2.19 ± 0.37) and GNX female (2.35 ± 0.21) rats, but significantly less than that obtained from intact female (4.03 ± 0.55; P = 0.016) rats. The number of cells recovered from the pituitaries of Tfm rats (3.81 ± 0.41) was intermediate between but not significantly different from the numbers obtained from intact male and intact female animals. Between 8-12 million cells were introduced into each perifusion chamber. Cell viability, as demonstrated by the ability to exclude trypan blue, was 90% in every case.
3
1
(nM) 30
100
10
0.1
O.3
I I
I I I
5.0)
I I
100
30
10
1.0
0.3
I
I
I
I
I
100 30 I , ,
10 I
0.1 I
0.3 I
Basal LH release
Unstimulated LH secretory rates, determined just before administration of the initial GnRH concentration, are shown in Table 1. As can be seen, basal LH secretory rates documented for cells from intact male and female rats were similar. Basal secretory rates for intact animals were significantly lower than those for cells from GNX male and female rats. Basal LH secretion by cells obtained from Tfm rats was higher than that by cells from intact rats, but lower than that by cells from GNX rats.
3 I
i DC
5
O
i
1 I
2.0
.-J\.
o.o L.
9.0]
GnRH-stimulated LH release Representative profiles of the LH secretory responses to several concentrations of GnRH administered in a random order are shown in Fig. 1. Visual inspection suggested that the responses occurred in a concentrationdependent manner and differed according to experimental group. Figure 2 illustrates the mean LH secretory responses, which have been rearranged into order of ascending GnRH concentration. As shown in Table 2, application of univariate linear regression analysis to these data demonstrated that the responses within each study group were strongly concentration dependent. Overall mean GnRH-stimulated LH secretory rates (picograms per min/10 million cells) were calculated using all responses to all GnRH concentrations (Table 1). As can be seen, overall mean GnRH-stimulated LH secretion by cells from intact male rats was significantly lower than that by all other expermental groups. Overall mean TABLE 1. Basal and overall mean GnRH-stimulated LH secretory rates (above basal) by perifused dispersed antior pituitary cells obtained from intact male and female, Tfm, and GNX male and female rats Group
Basal
GnRH-stimulated
Intact male Intact female Tfm GNX male GNX female
232 ± 11.6° 211 ± 9.1° 376 ± 11.66 569 ± 18.0c 608 ± 19.5C
37.8 ± 4.3° 167 ± 266 566 ± 63C 281 ± 23.2M 317 ± 58d
All values represent the mean ± SEM secretory rates (picograms per min/10 million cells). Secretory rates within columns identified by different superscripts differ significantly (P < 0.05).
100
30
1
0.3
0.1
10
I
I
I
I
|
|
JJ TIME
T (HOURS)
FIG. 1. LH secretion by perifused anterior pituitary cells obtained from intact male and female, Tfm, and GNX male and female rats. Individual data points are expressed as secretory rate (nanograms per min/10 million cells) for each 5-min fraction. The concentrations of GnRH were administered in random order.
secretion by cells from Tfm animals was higher than that in all other groups. Cells from GNX male and female rats displayed indistinguishable overall mean LH secretory rates. Although statistically intermediate between the intact male and Tfm groups, overall mean secretion by cells from intact female rats did not differ significantly (P = 0.079) from the rate for GNX male rats.
Discussion To our knowledge, the current in vitro study is the first in which both basal and GnRH-stimulated LH secretory rates have been compared using pituitary cells obtained from Tfm rats and their intact and gonadectomized littermates. Our results in the intact animals con-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 23 November 2015. at 18:06 For personal use only. No other uses without permission. . All rights reserved.
GnRH-STIMULATED LH RELEASE
GnRH RESPONSE 1.4 1.0
• Male • Female gS Gx Male 0 Gx Female • Tfm
.8
1
.6
c
.4
I
.2 0
0.1
0.3 1.0 3.0 10 30 100 GnRH Concentrations (nM) FIG. 2. Mean (±SEM) GnRH-stimulated LH release by pituitary cells from intact male (8 repetitions; n = 30), intact female (8 repetitions; n = 24), Tfm (10 repetitions; n = 35), GNX male (6 repetitions; n = 24), and GNX female (5 repetitions; n = 23) rats in response to several concentrations of GnRH. Although the GnRH challenges were given in random order, they have, for purposes of illustration, been rearranged into ascending order of concentration. The responses were concentration dependent and linear for all groups. TABLE 2. Concentration-response relationships between GnRH and LH release by cells from intact male and female, Tfm, and GNX male and female rats Slope (pg/min • 10 million cells/nM GnRH)a Intact male Intact female Tfm
GNX male GNX female 0 6
6.8 ± 45.5 ± 179.0 ± 83.3 ± 93.8 ±
0.8 8.5 20.2 8.7 19.9
r2
P"
0.45 0.45 0.62 0.76 0.59
