0013-7227/90/1265-2642$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 5 Printed in U.S.A.
Effects of Castration on Luteinizing Hormone and Follicle-Stimulating Hormone Secretion by Pituitary Cells from Male Rats* SATOSHI KITAHARA, STEPHEN J. WINTERS, BARBARA ATTARDI, HIROYUKI OSHIMA, AND PHILIP TROEN Department of Medicine, Montefiore Hospital, and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
ABSTRACT. Because the role of the pituitary in the testicular control of gonadotropin secretion remains controversial, we examined the effects of castration on the release of LH and FSH under basal conditions and in response to GnRH stimulation by dispersed pituitary cells in monolayer culture as well as by cells perifused with pulses of GnRH. These effects were compared to changes in LH/3, FSH/3, and a-subunit mRNA levels determined by Northern blot analysis. Pituitary cells were prepared from 7week-old intact rats and rats orchidectomized 2 weeks previously. Castration increased basal FSH secretion from monolayer cultures, interpulse FSH release from perifused pituitary cells, FSH/3 mRNA concentrations and serum FSH levels each approximately 2-fold, whereas pituitary FSH contents were similar in cells from intact and castrated rats. Pituitary LH content rose 3-fold, LH/3 mRNA rose 5.6-fold, and basal LH secretion increased 6-fold, but serum LH levels increased 22-fold. Thus, the change in FSH synthesis inferred from the increase in FSH/3 mRNA was proportional to the increase in FSH secretion both in vitro and in vivo. Whereas the basal release of LH in vitro was also proportional to the change in LH/3 mRNA, secretion of LH in vivo exceeded these changes, underscoring the importance of increased GnRH to the serum LH castration response. Castration resulted in an increase in the sum of FSH content
and secretion during 10 days in culture in the absence of GnRH, indicating ongoing FSH synthesis. Total LH declined in cells from intact rats, and this decline was prevented by castration; this effect may be due to a castration-related decrease in intracellular LH degradation or increased LH synthesis in the absence of GnRH. Castration also augmented the GnRH-stimulated release of LH and FSH from monolayer cultures 4.5- and 1.8-fold, respectively, and increased the amplitude of GnRHstimulated LH and FSH pulses 5- and 2-fold in experiments with perifused pituitary cells. The EC60for GnRH was unaffected by castration. These data reveal that orchidectomy increases GnRH-independent FSH and LH secretion and also renders the pituitary more responsive to GnRH. Whereas changes in FSH secretion after orchidectomy are proportional to the increase in FSH/3 mRNA, and the increase in basal LH secretion is proportional to the change in LH/3 mRNA, the hypersecretion of LH after GnRH stimulation in vitro exceeds that of FSH because of increased LH/3 mRNA, increased LH content, greater responsivess to GnRH, as well as possible specific translational and posttranslational changes. LH secretion is further augmented in vivo by increased pulsatile GnRH secretion. (Endocrinology 126: 2642-2649,1990)
L
H AND FSH secretion increase after orchidectomy because testicular negative feedback signals are absent. The observation that LH pulse frequency is accelerated in male castrates of several species (1-4) and is slowed by testosterone (1, 2) leads to the proposal that testicular hormones influence the hypothalamic GnRH pulse generator. This effect has now been demonstrated conclusively in rams undergoing simultaneous hypophysial portal and peripheral blood sampling for GnRH and Received November 13,1989. Address all correspondence and requests for reprints to: Stephen J. Winters, M.D., Department of Medicine, Montefiore 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-12099 (to B.A.). A preliminary report of a portion of the data in this manuscript was presented at the 14th Annual Meeting of the American Society of Andrology, New Orleans, LA, 1989, and was published in J Androl 10:1, P-23, 1989.
LH (5) as well as in rats (6). Studies of isolated pituitary cells from intact rats, however, have identified a direct inhibitory effect of both testosterone (7, 8) and inhibin (9, 10) on gonadotropin release. Discrepant changes in GnRH and gonadotropin secretion further suggest that the pituitary is an additional important site of testicular negative feedback control. For example, after orchidectomy in rats, hypothalamic immunoreactive GnRH changed little until well after LH and FSH secretion rose (11), mean GnRH levels and GnRH pulse amplitude were similar in intact and short term castrates (6), androgen treatment increased the GnRH content of the medial basal hypothalamus (12) and increased GnRH release in vitro (13), and addition of androgens to isolated GnRH neurons increased GnRH release (14).
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CASTRATION EFFECTS ON LH AND FSH IN VITRO There is currently little information on the influence of castration directly on gonadotropin secretion by pituitary cells, and published data are conflicting. O'Conner et al. (15) showed that monolayer cultures of dispersed pituitary cells from rats castrated 1 week previously contained more LH and released more LH in response to 3-h GnRH stimulation than did cells from intact rats. In the same study, basal FSH release was unaffected by castration, whereas GnRH-stimulated FSH release and pituitary FSH content were increased. Schwartz and colleagues, using pituitary fragments from male rats castrated 6 days previously, reported reduced basal FSH and LH release (16), and reduced LH responses and unchanged FSH responses to hourly pulses of GnRH compared to responses in intact controls (17). In an effort to clarify the effects of orchidectomy directly on the pituitary, we have examined the release of FSH and LH in reponse to GnRH stimulation by both monolayer cell cultures and perifused pituitary cells from intact young adult male rats and rats castrated 2 weeks previously. We also compared these changes in secretion with changs in steady state mRNA levels of FSH/3, LH/3 and a-subunit in dispersed pituitary cells.
Materials and Methods Animals and treatments Male Sprague-Dawley rats, aged 5 weeks, were obtained from Zivic-Miller Laboratories, Inc. (Allison Park, PA). For each experiment the animals were divided into two groups. In group 1, bilateral orchidectomy was performed under methoxyfluorane anesthesia (Pitmanmore, Inc., Washington Crossing, NJ); group 2 was intact controls. Two weeks later all animals were killed by decapitation. Serum was saved for measurement of LH and FSH by RIA, as described previously (8, 10). RIA results are expressed in terms of LH and FSH RP-2 standards. Cell preparation Dispersed pituitary cells were prepared as described previously (8,10). In brief, pituitary glands were minced into several pieces and treated for 60 min with 0.4% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.003% DNase (Sigma Chemical Co., St. Louis, MO) in Hanks'-HEPES buffer (pH 7.3) containing 0.4% BSA and 0.2% glucose. Then, the cells were digested with 0.25% pancreatin (Sigma Chemical Co.) in Hanks'-HEPES for 8 min and washed three times with Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum. Cell yield was 7-12 x 105/rat, and viability, based upon trypan blue exclusion, was 91-95%. Monolayer cell cultures were prepared by plating dispersed pituitary cells at a density of 2 X 106 cells/ml in 24-well tissue culture dishes (Nunc, Roskilde, Denmark), which were incubated for 2-3 days at 37 C under an atmosphere of 5% CO2-95% air.
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Gonadotropin subunit mRNA determinations Total RNA was extracted from pituitary cell cultures from 7-week-old intact rats and rats castrated 2 weeks previously; approximately 107 cells from 10 rat pituitaries were used for each determination. Northern blots were made by electrophoresis of 5 ng total pituitary RNA for LH/3, 10 ng RNA for FSH/3, and 3 ng RNA for a-subunit on 1.2% agarose-formaldehyde gels with transfer to GeneScreen membranes (NEN Research Products, Boston, MA), as described previously (18). Hybridization was performed to purified cDNA inserts for rat LH/3, FSH/3 and a (from Drs. J. Roberts, R. Maurer, and W. Chin, respectively). Membranes were autoradiographed, and slices were counted in a Packard 4530 scintillation counter (Packard, Downers Grove, IL). RNA concentrations were normalized by measuring the content of /3-actin mRNA using a chick cDNA probe. Exp 1. Dose-response curves relating GnRH concentrations to LH and FSH release by cultured pituitary cells from intact and castrated rats were performed after cells were precultured for 4 days, with one medium change on day 3. Cells were treated with 0-5 X 106 M GnRH for 6 h; media were removed and assayed for FSH and LH. Cells were then lysed in Dulbecco's PBS containing 0.1% Triton-X 100 (Bio-Rad Laboratories, Richmond, CA) at 37 C, and the extracts were assayed for FSH and LH. Exp 2. The effects of castration on intracellular and secreted FSH and LH under basal conditions were studied by culturing cells for 10 days and changing the medium every 2 days. Representative cell extracts were prepared at 2-day intervals. Exp 3. To examine the influence of castration on perifused pituitary cells, dispersed cells were cultured with Cytodex beads type 3 (Pharmacia, Piscataway, NJ) in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum (pH 7.3), as previously described (8,10), for 96 h, with a medium change at 72 h. The cell-bead mixtures containing 0.5-1.1 x 107 cells were packed into 1.5-ml microchambers and perifused at 0.3 ml/min using a computerized perifusion system (Endotronics, Inc., Coon Rapid, MN). Two chambers were perifused simultaneously: one chamber contained cells from castrated rats, and the second chamber contained cells from intact rats. Fractions of the column effluents were collected at 10-min intervals for FSH and LH assay. In selected experiments, the cell-bead mixtures were washed with 0.02% EDTA in PBS, and cells were detached using 0.25% trypsin (Sigma Chemical Co.). Trypan blue staining indicated approximately 95%Ncell viability in both groups. Data analysis Results are expressed as the mean ± SEM. RIA potency estimates were performed using the computer program of Rodbard (19). Dose-response curves were analyzed using the Allfit program of DeLean et al. (20). Differences between serum and pituitary gonadotropin levels were determined by Student's t test for nonpaired samples. For subunit mRNAs, the ratio between levels in cells from intact and castrate rats processed in parallel and run on the same gel was determined for each
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
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pair. The difference among the five paired RNA samples was analyzed by one-tailed Student's t test.
Results Table 1 indicates that serum levels of LH were increased 22-fold and FSH increased 2.4-fold 2 weeks after castration. The LH content of cells from castrated rats was increased 3-fold, whereas the FSH contents of cells from castrate and intact rats were similar. Dispersed pituitary cells from castrate and intact rats were maintained in culture for 4 days, total RNA was isolated, and the concentrations of LH/3, FSH/3, and asubunit mRNAs were determined by Northern analysis. Changes in gonadotropin subunit mRNA concentrations were intermediate between those in serum and pituitary LH and FSH levels; LH/3 mRNA was increased 5.6-fold (P < 0.005), while FSH/3 mRNA (P < 0.025) and asubunit mRNA (P < 0.005) were increased 1.7-fold (Table 1). Expl The effects of increasing doses of GnRH on FSH and LH release from dispersed pituitary cells from castrate and intact rats are shown in Fig. 1. In the absence of GnRH, castration increased LH release 6-fold from 1.3 ± 0.3 to 6.8 ± 0.6 ng/2 x 105 cells and FSH release 2fold from 3.1 ± 0.5 to 7.1 ± 0.8 ng/2 x 105 cells. Although GnRH produced classical sigmoidal-shaped dose-response curves for both FSH and LH release, FSH and LH responses to GnRH and the influence of castration on these responses were found to differ. GnRH maximally stimulated LH release by cells from intact rats 83fold, whereas FSH release rose 6.4-fold above basal after GnRH treatment. Castration augmented the maximum TABLE 1. Serum and pituitary concentrations of LH and FSH and subunit mRNA levels in dispersed pituitary cells from intact male rats and rats 2 weeks postcastration Intact
Castrate
Serum LH (ng/ml) Serum FSH (ng/ml)
0.28 ± 0.23 6.16 ± 1.0° (10) 11.7 ± 2.5 27.5 ± 3.2a (10)
Pituitary LH (^g/lO7 cells) Pituitary FSH (/ig/107 cells)
4.53 ± 0.34 13.4 ± 2.1" (10) 3.64 ± 0.35 3.41 ± 0.46 (10)
Endo • 1990 Vol 126 • No 5
LH response to 455 ± 57% that of intact cells, but the FSH response was augmented by only 179 ± 22% (P < 0.05 compared to the change in LH). The GnRH concentrations producing a half-maximal response were similar for LH and FSH (0.5 nM) and were not significantly influenced by castration. Figure 1 also shows that there was an inverse relationship between hormone released and intracellular content, such that total (content plus secretion) FSH or LH was unchanged after 6 h of GnRH treatment in cells from either castrate or intact rats (not shown).
Exp2 To examine further the importance of GnRH to FSH and LH release and to explore the influence of castration on basal gonadotropin release, cells were maintained in culture for 10 days in the absence of GnRH. Figure 2 reveals that LH release by cells from intact rats declined rapidly by days 2-4 and that this decline continued over the course of the experiment. For FSH, this dependence on GnRH was much less pronounced. By days 4-6 in culture, LH secretion fell to 9.2 ± 0.6% of that on days 0-2, whereas FSH secretion decreased to 33.8 ± 16.7% (P < 0.01 compared to the change in LH). As summarized in Table 2, these relative declines in LH and FSH release were comparable in cells from castrated and intact rats. The effects of castration on the time course of basal LH and FSH release are compared to changes in pituitary cell content in Fig. 3, A and B (LH) and C and D (FSH), and the results of three experiments are summarized in Fig. 4. These experiments were designed to provide insight into the influence of castration on gonadotropin synthesis and stability, as reflected by changes in total measurable FSH and LH with time in culture. For FSH, pituitary cell content decreased with time in culture to a similar extent in cells from castrate and intact rats; FSH cell content on day 10 was 16.3 ± 2.5% (castrate) and 21.8 ± 1.0% (intact) of that on day 0, respectively. By contrast, total FSH (content plus secretion) rose steadily with cells from castrate rats, since FSH release exceeded the fall in cellular content. However with cells from intact rats, total FSH did not increase during 10 days in culture. Total LH declined with cells from intact rats according to first order kinetics, with a half-time of 11 days. Castration eliminated this decline.
LH/3 mRNA (cpm bound/5 Mg RNA) 598 ± 61 3330 ± 244" (5) FSH/3 mRNA (cpm bound/10 ng RNA) 355 ± 30 594 ± 68C (5) a mRNA (cpm bound/3 Mg RNA) 4420 ± 634 7638 ± 562b (5)
Exp3
The number of samples is in parentheses. Levels of gonadotropin subunit mRNAs were adjusted based upon CPM /?-actin cDNA bound per lane to normalize for small differences in recovery of total pituitary RNA. a P < 0.001 vs. intact. b P < 0.005 vs. intact. c P < 0.025 vs. intact.
Three perifusions were performed in which cells from castrated rats were stimulated with increasing concentrations of GnRH (0.1-100 nM). The EC50 for both LH and FSH release was approximately 2.5 nM (data not shown). Once the range of effective concentrations was narrowed, cells from both intact and castrate rats were
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CASTRATION EFFECTS ON LH AND FSH IN VITRO Secretion Content
—•— Intact — o— Castrated k :i
3001
2645
" Intact " Castrated 60-1
B 50-
FIG. 1. Effects of castration on LH (A) and FSH (B) secretion and content of pituitary cells in monolayer culture stimulated with GnRH. Cells were precultured for 96 h, then stimulated with GnRH for 6 h at the indicated doses. Results are the mean ± SEM of four replicate wells. The experiment was repeated twice with similar results.
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LH
FSH
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DAYS IN CULTURE
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
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TABLE 2. Effects of castration on the time course of changes in LH and FSH release from pituitary cells cultures LH (% of control)
LH
FSH (% of control)
0-2 2-4 4-6 6-8 8-10
100 23.0 ± 9.2 ± 8.1 ± 6.2 ±
Castrate
2.6 0.6 1.5 1.0
100 27.7 ± 19.6 ± 8.9 ± 7.4 ±
2.6 2.6 1.0 0.5
Intact 100 58.3 ± 33.8 ± 39.0 ± 31.0 ±
2.7 16.7 3.5 4.6
FSH
1000 T
Day
Intact
Endo • 1990 Vol 126 • No 5
Castrate 100 64.0 ± 43.0 ± 31.6 ± 25.7 ±
INTACT
14.8 5.0 8.4 7.2
The data are a summary of the results (mean ± SE) of three experiments. Media were changed every 2 days, and each point was the mean of quadruplicate wells. 100% represents the concentration of LH and FSH in the medium after 2 days in culture.
CASTRATE
"55 o rX
c 100
CASTRATE
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200
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150
0 4-6 • 2-* • 0-2 •
10
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8
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10
0
2
4
6
8
10
DAYS IN CULTURE FIG. 4. Time course of changes in total LH and FSH (release plus cell content) of pituitary cells from intact and castrated rats. Results are the mean ± SEM of three separate experiments. A, P < 0.05 vs. day 0; B, P < 0.05 vs. day 2 (by one-way analysis of variance).
pulses were increased in amplitude in proportion to the GnRH dose with cells from both intact and castrated rats. Castration augmented the amplitude of GnRHinduced LH pulses approximately 5-fold and FSH pulses 2-fold above those in intact cells. Similar ratios were found with all doses of GnRH tested.
Pituitary Content
U
So s :
8
100
50-
Discussion 0
2 4 6 8 10 DAYS IN CULTURE
0
2 4 6 8 10 DAYS IN CULTURE
FlG. 3. Effects of castration on the time course of basal LH and FSH secretion compared with pituitary content of pituitary cell cultures. Media were changed every 2 days, at which time four replicate wells were harvested to determine LH and FSH cell content. The experiment was performed three times with similar results.
perifused simultaneously and were stimulated with hourly pulses of GnRH for 8 h. A representative experiment is shown in Fig. 5. The results of four experiments with three different GnRH concentrations are summarized in Table 3. Interpulse basal LH release was increased approximately 4-fold with castration, and both interpulse basal LH and FSH release were independent of GnRH concentration. GnRH-induced LH and FSH
We found that 2 weeks after castration of young adult male rats serum FSH levels, basal FSH secretion from monolayer cultures and FSH/3 mRNA concentrations had increased approximately 2-fold, whereas pituitary FSH content was similar to that of cells from intact rats. These comparable changes lend further support to the concept that FSH release is proportional to synthesis (9, 21) and that FSH secretion is less dependent on GnRH stimulation than is LH secretion (6, 22-24). Both the unchanged FSH content and the 2-fold increase in FSH/3 mRNA 2 weeks after castration agree with previous findings in intact pituitaries (11, 25, 26), but not with a previous study of FSH content in dispersed pituitary cells that reported increased concentrations 1 week after orchidectomy (15). After longer intervals postcastration,
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
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B 301 INTACT
75s
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FIG. 5. Effects of castration on LH and FSH secretion by perifused pituitary cells stimulated with pulses of GnRH. Cells were stimulated with 2.5 nM GnRH for 2 min every 1 h.
I 10
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10
20
30
40
FRACTION NUMBER TABLE 3. Effects of castration on GnRH-induced FSH and LH release from perifused pituitary cells GnRH (nM)
LH (ng/fraction)a Pulse amplitude'
FSH (ng/fraction)a Basal*
Pulse amplitude0
8.6 ± 1.5" " 9.2 ± 0.8" 7.4 ± 1.1 20.8 ± 1.2 6.6 ± 0.9". 37.0 ± 2.0e
7.8 ± 0.8 6.4 ± 0.8 6.3 ± 1.1
6.0 ± 0.4' 11.0 ± 0.8s 16.6 ± 1.3'*
28.3 ± 3.8d" 51.5 ± 3.8" 28.5 ± 4.0 88.9 ± 4.6' 10.0 25.7 ± 3.1 d . 145 ± 16.6'1-'
9.1 ± 1.4 10.0 ± 1.2 9.4 ± 1.0
11.4 ± 0.81 19.2 ± 1.6 26.7 ± 1.81
Basal*
Intact 0.1 1.0
10.0 Castrate 0.1 1.0
All data are the mean ± SEM. The results are from four experiments. For the purposes of statistical analysis, the data were transformed, such that the values for 0.1 nM GnRH for each experiment were set equal to 100%. Transformation was needed because of between-experiment differences in secretion due to cell growth characteristics and responsiveness to GnRH. Brackets indicate that, for the purposes of statistical analysis, basal hormone concentrations for all three GnRH doses were meaned. 0 Adjusted to nanograms per 107 cells. * Basal secretion is the mean of three basal levels between successive GnRH pulses. c The amplitude of a hormone pulse was 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. d ~' Values sharing a common letter are statistically different by oneway analysis of variance followed by the Bonferoni test.
the FSH content of pituitary homogenates has also been reported to be increased (27).
50
0
10
20
30
40
50
FRACTION NUMBER
The finding of increased pituitary LH content after castration agrees with previous reports (11, 28) and has been explained at least partly by an increase in gonadotroph number and size with castration (29). For FSH content to remain unchanged, by contrast, the FSH concentration per cell must decline. The 2-fold increase in FSH/3 mRNA levels with castration parallels the reported 2- to 3-fold increase in the percentage of pituitary cells staining for FSH postorchidectomy (30). Thus, unlike the decline in FSH content, FSH/3 mRNA as well as basal FSH secretion, when expressed per gonadotroph, appear to have been unchanged 2 weeks after castration. Together these observation suggest that one aspect of the differential regulation of LH and FSH is a castrationrelated alteration in the dynamics of FSH storage and secretion. The relative increment we observed in LH/3 mRNA levels in isolated pituitary cells 2 weeks after orchidectomy (5- to 6-fold) is comparable to results using intact pituitaries (24, 31, 32) and exceeds the reported 2- to 3fold increase in the percentage of pituitary cells staining for LH postorchidectomy (30). Although castration increased LH content and LH/3 mRNA concentrations proportionately more than FSH, the withdrawal of GnRH in vitro severely impaired LH release, even though considerable LH remained within the pituitary cells. Together these findings underscore the important role of GnRH in regulating LH storage and secretion. Basal and interpulse LH secretion also increased with castration, however, suggesting that GnRH-independent LH release
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
contributes to the LH castration response. The further increase in total FSH with time in culture for cells from castrated rats indicates that greater FSH synthesis and release occurred in vitro for many days in the absence of GnRH. Similar results have been reported previously using pituitary cells from intact males and females of several species in long term culture (9, 21). Reduced intracellular degradation of FSH does not appear to have contributed to this hypersecretion of FSH after castration, since the decline in intracellular FSH content over the course of our study was similar in cells from intact and castrated rats. We observed that total LH declined in cells from intact rats with time in culture in the absence of GnRH . This decline indicates intracellular degradation and agrees with previous results (9). By contrast, total LH did not decline in cells from castrated rats. There are two possible explanations for these findings: castration may reduce the intracellular degradation of LH, or, alternatively, the increased LH synthesis that accompanies castration (33) may be sufficient to offset degradation. Which of these two possibilities is correct cannot be resolved by our experiments. Castration increased both LH and FSH release in response to GnRH stimulation in experiments using either monolayer cultures or perifused pituitary cells. This effect was not associated with a change in the EC50 for GnRH, providing biological support for the finding of identical affinity of a radiolabeled GnRH analog for binding sites in pituitary homogenates from intact and castrated rats (28). By contrast, addition of testosterone in vitro to dispersed pituitary cells from intact rats increased the EC50 value for the GnRH effect on LH secretion (7). The castration-associated increase in responsivess to GnRH no doubt results partly from an increase in GnRH receptor number (28), and the greater effect of castration on GnRH-stimulated LH than FSH release parallels the selective increased intracellular LH content. Our findings with GnRH-stimulated cells confirm and extend the results of previous studies using dispersed pituitary cells from 60-day-old rats castrated 1 week previously, in which significantly higher release of both LH and FSH after GnRH stimulation was found compared to that in intact controls (15). By contrast, pituitary fragments from 1-week castrate rats of similar age were less responsive to pulses of GnRH in a perifusion system than were pituitary fragments from intact rats (17). The use of pituitary fragments rather than dispersed cells in the latter study would appear to best reconcile these discrepancies. In summary, these data reveal that castration increases GnRH-independent FSH and LH secretion and renders the pituitary more responsive to GnRH. There
Endo• 1990 Vol 126 • No 5
is an excellent correlation between the change in FSH/3 mRNA levels with castration and increased FSH secretion, whereas the rise in LH secretion exceeds the increment in LH/3 mRNA in part because of increased pituitary LH content and in part because of the importance of the acceleration of the GnRH pulse generator to the hypersecretion of LH that accompanies orchidectomy. These results, however, indicate that pituitary changes also contribute to the castration response.
Acknowledgments The authors wish to thank Ms. Joyce Szczepanski and Theresa Fitzgerald for their skillful technical assistance. RIA reagents were provided by Dr. A. F. Parlow through the National Hormone and Pituitary Program.
References 1. D'Occhio MJ, Schanbacher BD, Kinder JE 1982 Relationship between serum testosterone concentration and patterns of luteinizing hormone secretion in male sheep. Endocrinology 110:1547 2. Plant TM 1982 Effects of orchidectomy and testosterone replacement treatment on pulsatile luteinizing hormone secretion in the adult rhesus monkey (Macaca mulatto). Endocrinology 110:1905 3. Winters SJ, Troen P 1983 A reexamination of pulsatile luteinizing hormone secretion in primary testicular failure. J Clin Endocrinol Metab 57:432 4. Ellis GB, Desjardins C 1984 Orchidectomy unleashes pulsatile luteinizing hormone secretion in the rat. Biol Reprod 30:619 5. Caraty A, Locatelli A 1988 Effect of time after castration on secretion of LHRH and LH in the ram. J Reprod Fertil 82:263 6. Levine JE, Duffy MT 1988 Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats. Endocrinology 122:2211 7. Drouin J, Labrie F 1976 Selective effect of androgens on LH and FSH release in anterior pituitary cells in culture. Endocrinology 98:1528 8. Kotsuji F, Winters SJ, Attardi B, Keeping HS, Oshima H, Troen P 1988 Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells. Endocrinology 123:2683 9. Farnworth PG, Robertson DM, de Kretser DM, Burger HG 1988 Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions. Endocrinology 122:207 10. Kotsuji F, Winters SJ, Keeping HS, Attardi B, Oshima H, Troen P 1988 Effects of inhibin from primate Sertoli cells on folliclestimulating hormone and luteinizing hormone release by perifused rat pituitary cells. Endocrinology 122:2796 11. Badger TM, Wilcox CE, Meyer ER, Bell RD, Cicero TJ 1978 Simultaneous changes in tissue and serum levels of luteinizing hormone, follicle-stimulating hormone, and luteinizing hormone/ follicle-stimulating hormone releasing factor after castration in the male rat. Endocrinology 102:136 12. Kalra PS, Kalra SP 1982 Discriminative effects of testosterone on hypothalamic luteinizing hormone-releasing hormone levels and luteinizing hormone secretion in castrated male rats: analyses of dose and duration characteristics. Endocrinology 111:24 13. Rudenstein RS, Bigdeli H, McDonald MH, Snyder PJ 1979 Administration of gonadal steroid to the castrated male rat prevents a decrease in the release of gonadotropin releasing hormone from the incubated hypothalamus. J Clin Invest 63:262 14. Melrose P, Gross L 1987 Steroid effects on the secretory modalities of gonadotropin-releasing hormone release. Endocrinology 121:190 15. O'Conner JL, Allen MB, Mahesh VB 1980 Castration effects on the response of rat pituitary cells to luteinizing hormone-releasing
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
16. 17. 18.
19. 20.
21. 22. 23.
24.
hormone: retention in dispersed cell culture. Endocrinology 106:1706 Hiatt ES, Valadka RJ, Schwartz NB 1987 Sex differences following gonadectomy in basal gonadotropin secretion rate of rat pituitary fragments in vitro. Biol Reprod 37:1114 Fallest PC, Hiatt ES, Schwartz NB 1989 Effects of gonadectomy on the in vitro and in vivo gonadotropin responses to gonadotropinreleasing hormone in male and female rats. Endocrinology 124:1370 Attardi B, Keeping HS, Winters SJ, Kotsuji F, Maurer RA, Troen P 1989 Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone-/? by inhibin from primate Sertoli cells. Mol Endocrinol 3:280 Rodbard D 1971 Statistical aspects of radioimmunoassays. In: Odell WD, Daughaday WH (eds) Principles of Competitive Protein Binding Assays. Lippincott, Philadelphia, p 204 DeLean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97 Miller WL, Wu J 1981 Estrogen regulation of follicle-stimulating hormone production in vitro: species variation. Endocrinology 108:673 Culler MD, Negro-Vilar A 1986 Evidence that pulsatile folliclestimulating hormone secretion is independent of endogenous luteinizing hormone-releasing hormone. Endocrinology 118:609 Grady RR, Shin L, Charlesworth MC, Cohen-Becker IR, Smith M, Rivier C, Rivier J, Vale W, Schwartz NB 1985 Differential suppression of follicle-stimulating hormone and luteinizing hormone secretion in vivo by a gonadotropin-releasing hormone antagonist. Neuroendocrinology 40:246 Lincoln GA, Fraser HM 1987 Compensatory response of the luteinizing hormone (LH)-releasing hormone (LHRH)/LH pulse generator after administration of a potent LHRH antagonist in the ram. Endocrinology 120:2245
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25. Gharib SD, Wierman ME, Badger TM, Chin WW 1987 Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat. J Clin Invest 80:294 26. Vogel DL, Sherins RJ 1984 Orchiectomy in young rats results in differential regulation of follicle-stimulating hormone and luteinizing hormone content. J Androl 5:80 27. Spitzbarth TL, Horton TH, Lifka J, Schwartz NB 1988 Pituitary gonadotropin content in gonadectomized rats: immunoassay measurements influenced by extraction solvent and testosterone replacement. J Androl 9:294 28. Clayton RN, Catt KJ 1981 Regulation of pituitary gonadotropinreleasing hormone receptors by gonadal hormones. Endocrinology 108:887 29. Childs GV, Ellison DG, Lorenzen JR, Collins TJ, Schwartz NB 1982 Immunocytochemical studies of gonadotropin storage in developing castration cells. Endocrinology 111:1318 30. Ibrahim SN, Moussa SM, Childs GV 1986 Morphometric studies of rat anterior pituitary cells after gonadectomy: correlation of changes in gonadotropes with the serum levels of gonadotropins. Endocrinology 119:629 31. Papavasiliou SS, Zmeili S, Herbon L, Duncan-Weldon J, Marshall JC, Landefeld TD 1986 a and luteinizing hormone/3 messenger ribonucleic acid (RNA) of male and female rats after castration: quantitation using an optimized RNA dot blot hybridization assay. Endocrinology 119:691 32. Wierman ME, Gharib SD, LaRovere JM, Badger TM, Chin WW 1988 Selective failure of androgens to regulate follicle stimulating hormone /? messenger ribonucleic acid levels in the male rat. Mol Endocrinol 2:492 33. Vogel DL, Magner JA, Sherins RJ, Weintraub BD 1986 Biosynthesis, glycosylation, and secretion of rat luteinizing hormone aand /3-subunits: differential effects of orchiectomy and gonadotropin-releasing hormone. Endocrinology 119:202
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Vol. 126, No. 5 Printed in U.S.A.
Effects of Castration on Luteinizing Hormone and Follicle-Stimulating Hormone Secretion by Pituitary Cells from Male Rats* SATOSHI KITAHARA, STEPHEN J. WINTERS, BARBARA ATTARDI, HIROYUKI OSHIMA, AND PHILIP TROEN Department of Medicine, Montefiore Hospital, and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
ABSTRACT. Because the role of the pituitary in the testicular control of gonadotropin secretion remains controversial, we examined the effects of castration on the release of LH and FSH under basal conditions and in response to GnRH stimulation by dispersed pituitary cells in monolayer culture as well as by cells perifused with pulses of GnRH. These effects were compared to changes in LH/3, FSH/3, and a-subunit mRNA levels determined by Northern blot analysis. Pituitary cells were prepared from 7week-old intact rats and rats orchidectomized 2 weeks previously. Castration increased basal FSH secretion from monolayer cultures, interpulse FSH release from perifused pituitary cells, FSH/3 mRNA concentrations and serum FSH levels each approximately 2-fold, whereas pituitary FSH contents were similar in cells from intact and castrated rats. Pituitary LH content rose 3-fold, LH/3 mRNA rose 5.6-fold, and basal LH secretion increased 6-fold, but serum LH levels increased 22-fold. Thus, the change in FSH synthesis inferred from the increase in FSH/3 mRNA was proportional to the increase in FSH secretion both in vitro and in vivo. Whereas the basal release of LH in vitro was also proportional to the change in LH/3 mRNA, secretion of LH in vivo exceeded these changes, underscoring the importance of increased GnRH to the serum LH castration response. Castration resulted in an increase in the sum of FSH content
and secretion during 10 days in culture in the absence of GnRH, indicating ongoing FSH synthesis. Total LH declined in cells from intact rats, and this decline was prevented by castration; this effect may be due to a castration-related decrease in intracellular LH degradation or increased LH synthesis in the absence of GnRH. Castration also augmented the GnRH-stimulated release of LH and FSH from monolayer cultures 4.5- and 1.8-fold, respectively, and increased the amplitude of GnRHstimulated LH and FSH pulses 5- and 2-fold in experiments with perifused pituitary cells. The EC60for GnRH was unaffected by castration. These data reveal that orchidectomy increases GnRH-independent FSH and LH secretion and also renders the pituitary more responsive to GnRH. Whereas changes in FSH secretion after orchidectomy are proportional to the increase in FSH/3 mRNA, and the increase in basal LH secretion is proportional to the change in LH/3 mRNA, the hypersecretion of LH after GnRH stimulation in vitro exceeds that of FSH because of increased LH/3 mRNA, increased LH content, greater responsivess to GnRH, as well as possible specific translational and posttranslational changes. LH secretion is further augmented in vivo by increased pulsatile GnRH secretion. (Endocrinology 126: 2642-2649,1990)
L
H AND FSH secretion increase after orchidectomy because testicular negative feedback signals are absent. The observation that LH pulse frequency is accelerated in male castrates of several species (1-4) and is slowed by testosterone (1, 2) leads to the proposal that testicular hormones influence the hypothalamic GnRH pulse generator. This effect has now been demonstrated conclusively in rams undergoing simultaneous hypophysial portal and peripheral blood sampling for GnRH and Received November 13,1989. Address all correspondence and requests for reprints to: Stephen J. Winters, M.D., Department of Medicine, Montefiore 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-12099 (to B.A.). A preliminary report of a portion of the data in this manuscript was presented at the 14th Annual Meeting of the American Society of Andrology, New Orleans, LA, 1989, and was published in J Androl 10:1, P-23, 1989.
LH (5) as well as in rats (6). Studies of isolated pituitary cells from intact rats, however, have identified a direct inhibitory effect of both testosterone (7, 8) and inhibin (9, 10) on gonadotropin release. Discrepant changes in GnRH and gonadotropin secretion further suggest that the pituitary is an additional important site of testicular negative feedback control. For example, after orchidectomy in rats, hypothalamic immunoreactive GnRH changed little until well after LH and FSH secretion rose (11), mean GnRH levels and GnRH pulse amplitude were similar in intact and short term castrates (6), androgen treatment increased the GnRH content of the medial basal hypothalamus (12) and increased GnRH release in vitro (13), and addition of androgens to isolated GnRH neurons increased GnRH release (14).
2642
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CASTRATION EFFECTS ON LH AND FSH IN VITRO There is currently little information on the influence of castration directly on gonadotropin secretion by pituitary cells, and published data are conflicting. O'Conner et al. (15) showed that monolayer cultures of dispersed pituitary cells from rats castrated 1 week previously contained more LH and released more LH in response to 3-h GnRH stimulation than did cells from intact rats. In the same study, basal FSH release was unaffected by castration, whereas GnRH-stimulated FSH release and pituitary FSH content were increased. Schwartz and colleagues, using pituitary fragments from male rats castrated 6 days previously, reported reduced basal FSH and LH release (16), and reduced LH responses and unchanged FSH responses to hourly pulses of GnRH compared to responses in intact controls (17). In an effort to clarify the effects of orchidectomy directly on the pituitary, we have examined the release of FSH and LH in reponse to GnRH stimulation by both monolayer cell cultures and perifused pituitary cells from intact young adult male rats and rats castrated 2 weeks previously. We also compared these changes in secretion with changs in steady state mRNA levels of FSH/3, LH/3 and a-subunit in dispersed pituitary cells.
Materials and Methods Animals and treatments Male Sprague-Dawley rats, aged 5 weeks, were obtained from Zivic-Miller Laboratories, Inc. (Allison Park, PA). For each experiment the animals were divided into two groups. In group 1, bilateral orchidectomy was performed under methoxyfluorane anesthesia (Pitmanmore, Inc., Washington Crossing, NJ); group 2 was intact controls. Two weeks later all animals were killed by decapitation. Serum was saved for measurement of LH and FSH by RIA, as described previously (8, 10). RIA results are expressed in terms of LH and FSH RP-2 standards. Cell preparation Dispersed pituitary cells were prepared as described previously (8,10). In brief, pituitary glands were minced into several pieces and treated for 60 min with 0.4% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.003% DNase (Sigma Chemical Co., St. Louis, MO) in Hanks'-HEPES buffer (pH 7.3) containing 0.4% BSA and 0.2% glucose. Then, the cells were digested with 0.25% pancreatin (Sigma Chemical Co.) in Hanks'-HEPES for 8 min and washed three times with Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum. Cell yield was 7-12 x 105/rat, and viability, based upon trypan blue exclusion, was 91-95%. Monolayer cell cultures were prepared by plating dispersed pituitary cells at a density of 2 X 106 cells/ml in 24-well tissue culture dishes (Nunc, Roskilde, Denmark), which were incubated for 2-3 days at 37 C under an atmosphere of 5% CO2-95% air.
2643
Gonadotropin subunit mRNA determinations Total RNA was extracted from pituitary cell cultures from 7-week-old intact rats and rats castrated 2 weeks previously; approximately 107 cells from 10 rat pituitaries were used for each determination. Northern blots were made by electrophoresis of 5 ng total pituitary RNA for LH/3, 10 ng RNA for FSH/3, and 3 ng RNA for a-subunit on 1.2% agarose-formaldehyde gels with transfer to GeneScreen membranes (NEN Research Products, Boston, MA), as described previously (18). Hybridization was performed to purified cDNA inserts for rat LH/3, FSH/3 and a (from Drs. J. Roberts, R. Maurer, and W. Chin, respectively). Membranes were autoradiographed, and slices were counted in a Packard 4530 scintillation counter (Packard, Downers Grove, IL). RNA concentrations were normalized by measuring the content of /3-actin mRNA using a chick cDNA probe. Exp 1. Dose-response curves relating GnRH concentrations to LH and FSH release by cultured pituitary cells from intact and castrated rats were performed after cells were precultured for 4 days, with one medium change on day 3. Cells were treated with 0-5 X 106 M GnRH for 6 h; media were removed and assayed for FSH and LH. Cells were then lysed in Dulbecco's PBS containing 0.1% Triton-X 100 (Bio-Rad Laboratories, Richmond, CA) at 37 C, and the extracts were assayed for FSH and LH. Exp 2. The effects of castration on intracellular and secreted FSH and LH under basal conditions were studied by culturing cells for 10 days and changing the medium every 2 days. Representative cell extracts were prepared at 2-day intervals. Exp 3. To examine the influence of castration on perifused pituitary cells, dispersed cells were cultured with Cytodex beads type 3 (Pharmacia, Piscataway, NJ) in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum (pH 7.3), as previously described (8,10), for 96 h, with a medium change at 72 h. The cell-bead mixtures containing 0.5-1.1 x 107 cells were packed into 1.5-ml microchambers and perifused at 0.3 ml/min using a computerized perifusion system (Endotronics, Inc., Coon Rapid, MN). Two chambers were perifused simultaneously: one chamber contained cells from castrated rats, and the second chamber contained cells from intact rats. Fractions of the column effluents were collected at 10-min intervals for FSH and LH assay. In selected experiments, the cell-bead mixtures were washed with 0.02% EDTA in PBS, and cells were detached using 0.25% trypsin (Sigma Chemical Co.). Trypan blue staining indicated approximately 95%Ncell viability in both groups. Data analysis Results are expressed as the mean ± SEM. RIA potency estimates were performed using the computer program of Rodbard (19). Dose-response curves were analyzed using the Allfit program of DeLean et al. (20). Differences between serum and pituitary gonadotropin levels were determined by Student's t test for nonpaired samples. For subunit mRNAs, the ratio between levels in cells from intact and castrate rats processed in parallel and run on the same gel was determined for each
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
2644
pair. The difference among the five paired RNA samples was analyzed by one-tailed Student's t test.
Results Table 1 indicates that serum levels of LH were increased 22-fold and FSH increased 2.4-fold 2 weeks after castration. The LH content of cells from castrated rats was increased 3-fold, whereas the FSH contents of cells from castrate and intact rats were similar. Dispersed pituitary cells from castrate and intact rats were maintained in culture for 4 days, total RNA was isolated, and the concentrations of LH/3, FSH/3, and asubunit mRNAs were determined by Northern analysis. Changes in gonadotropin subunit mRNA concentrations were intermediate between those in serum and pituitary LH and FSH levels; LH/3 mRNA was increased 5.6-fold (P < 0.005), while FSH/3 mRNA (P < 0.025) and asubunit mRNA (P < 0.005) were increased 1.7-fold (Table 1). Expl The effects of increasing doses of GnRH on FSH and LH release from dispersed pituitary cells from castrate and intact rats are shown in Fig. 1. In the absence of GnRH, castration increased LH release 6-fold from 1.3 ± 0.3 to 6.8 ± 0.6 ng/2 x 105 cells and FSH release 2fold from 3.1 ± 0.5 to 7.1 ± 0.8 ng/2 x 105 cells. Although GnRH produced classical sigmoidal-shaped dose-response curves for both FSH and LH release, FSH and LH responses to GnRH and the influence of castration on these responses were found to differ. GnRH maximally stimulated LH release by cells from intact rats 83fold, whereas FSH release rose 6.4-fold above basal after GnRH treatment. Castration augmented the maximum TABLE 1. Serum and pituitary concentrations of LH and FSH and subunit mRNA levels in dispersed pituitary cells from intact male rats and rats 2 weeks postcastration Intact
Castrate
Serum LH (ng/ml) Serum FSH (ng/ml)
0.28 ± 0.23 6.16 ± 1.0° (10) 11.7 ± 2.5 27.5 ± 3.2a (10)
Pituitary LH (^g/lO7 cells) Pituitary FSH (/ig/107 cells)
4.53 ± 0.34 13.4 ± 2.1" (10) 3.64 ± 0.35 3.41 ± 0.46 (10)
Endo • 1990 Vol 126 • No 5
LH response to 455 ± 57% that of intact cells, but the FSH response was augmented by only 179 ± 22% (P < 0.05 compared to the change in LH). The GnRH concentrations producing a half-maximal response were similar for LH and FSH (0.5 nM) and were not significantly influenced by castration. Figure 1 also shows that there was an inverse relationship between hormone released and intracellular content, such that total (content plus secretion) FSH or LH was unchanged after 6 h of GnRH treatment in cells from either castrate or intact rats (not shown).
Exp2 To examine further the importance of GnRH to FSH and LH release and to explore the influence of castration on basal gonadotropin release, cells were maintained in culture for 10 days in the absence of GnRH. Figure 2 reveals that LH release by cells from intact rats declined rapidly by days 2-4 and that this decline continued over the course of the experiment. For FSH, this dependence on GnRH was much less pronounced. By days 4-6 in culture, LH secretion fell to 9.2 ± 0.6% of that on days 0-2, whereas FSH secretion decreased to 33.8 ± 16.7% (P < 0.01 compared to the change in LH). As summarized in Table 2, these relative declines in LH and FSH release were comparable in cells from castrated and intact rats. The effects of castration on the time course of basal LH and FSH release are compared to changes in pituitary cell content in Fig. 3, A and B (LH) and C and D (FSH), and the results of three experiments are summarized in Fig. 4. These experiments were designed to provide insight into the influence of castration on gonadotropin synthesis and stability, as reflected by changes in total measurable FSH and LH with time in culture. For FSH, pituitary cell content decreased with time in culture to a similar extent in cells from castrate and intact rats; FSH cell content on day 10 was 16.3 ± 2.5% (castrate) and 21.8 ± 1.0% (intact) of that on day 0, respectively. By contrast, total FSH (content plus secretion) rose steadily with cells from castrate rats, since FSH release exceeded the fall in cellular content. However with cells from intact rats, total FSH did not increase during 10 days in culture. Total LH declined with cells from intact rats according to first order kinetics, with a half-time of 11 days. Castration eliminated this decline.
LH/3 mRNA (cpm bound/5 Mg RNA) 598 ± 61 3330 ± 244" (5) FSH/3 mRNA (cpm bound/10 ng RNA) 355 ± 30 594 ± 68C (5) a mRNA (cpm bound/3 Mg RNA) 4420 ± 634 7638 ± 562b (5)
Exp3
The number of samples is in parentheses. Levels of gonadotropin subunit mRNAs were adjusted based upon CPM /?-actin cDNA bound per lane to normalize for small differences in recovery of total pituitary RNA. a P < 0.001 vs. intact. b P < 0.005 vs. intact. c P < 0.025 vs. intact.
Three perifusions were performed in which cells from castrated rats were stimulated with increasing concentrations of GnRH (0.1-100 nM). The EC50 for both LH and FSH release was approximately 2.5 nM (data not shown). Once the range of effective concentrations was narrowed, cells from both intact and castrate rats were
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CASTRATION EFFECTS ON LH AND FSH IN VITRO Secretion Content
—•— Intact — o— Castrated k :i
3001
2645
" Intact " Castrated 60-1
B 50-
FIG. 1. Effects of castration on LH (A) and FSH (B) secretion and content of pituitary cells in monolayer culture stimulated with GnRH. Cells were precultured for 96 h, then stimulated with GnRH for 6 h at the indicated doses. Results are the mean ± SEM of four replicate wells. The experiment was repeated twice with similar results.
40-
10-
o
id10
id8
GnRH (M)
LH
FSH
150 i •
INTACT
•
(3 CASTRATE
INTACT
E3 CASTRATE
> (0
•o CM ^100 FlG. 2. Effects of castration on the time course of basal LH and FSH secretion from rat pituitary cell cultures. Media were changed every 2 days. Results are the mean ± SEM of four replicate wells from a representative experiment.
o
O)
c UJ 0)
50
UJ UJ
V, 0-2
Jl 2 - 44 - 66-8 8-10
DAYS IN CULTURE
0-2
2-4 4 - 66-8 8 - 1 0
DAYS IN CULTURE
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
2646
TABLE 2. Effects of castration on the time course of changes in LH and FSH release from pituitary cells cultures LH (% of control)
LH
FSH (% of control)
0-2 2-4 4-6 6-8 8-10
100 23.0 ± 9.2 ± 8.1 ± 6.2 ±
Castrate
2.6 0.6 1.5 1.0
100 27.7 ± 19.6 ± 8.9 ± 7.4 ±
2.6 2.6 1.0 0.5
Intact 100 58.3 ± 33.8 ± 39.0 ± 31.0 ±
2.7 16.7 3.5 4.6
FSH
1000 T
Day
Intact
Endo • 1990 Vol 126 • No 5
Castrate 100 64.0 ± 43.0 ± 31.6 ± 25.7 ±
INTACT
14.8 5.0 8.4 7.2
The data are a summary of the results (mean ± SE) of three experiments. Media were changed every 2 days, and each point was the mean of quadruplicate wells. 100% represents the concentration of LH and FSH in the medium after 2 days in culture.
CASTRATE
"55 o rX
c 100
CASTRATE
INTACT
200
o Z U.
_J
150
0 4-6 • 2-* • 0-2 •
10
CASTRATE
INTACT
200
8
FSH RtUiud Par 2 Oaya
10
0
2
4
6
8
10
DAYS IN CULTURE FIG. 4. Time course of changes in total LH and FSH (release plus cell content) of pituitary cells from intact and castrated rats. Results are the mean ± SEM of three separate experiments. A, P < 0.05 vs. day 0; B, P < 0.05 vs. day 2 (by one-way analysis of variance).
pulses were increased in amplitude in proportion to the GnRH dose with cells from both intact and castrated rats. Castration augmented the amplitude of GnRHinduced LH pulses approximately 5-fold and FSH pulses 2-fold above those in intact cells. Similar ratios were found with all doses of GnRH tested.
Pituitary Content
U
So s :
8
100
50-
Discussion 0
2 4 6 8 10 DAYS IN CULTURE
0
2 4 6 8 10 DAYS IN CULTURE
FlG. 3. Effects of castration on the time course of basal LH and FSH secretion compared with pituitary content of pituitary cell cultures. Media were changed every 2 days, at which time four replicate wells were harvested to determine LH and FSH cell content. The experiment was performed three times with similar results.
perifused simultaneously and were stimulated with hourly pulses of GnRH for 8 h. A representative experiment is shown in Fig. 5. The results of four experiments with three different GnRH concentrations are summarized in Table 3. Interpulse basal LH release was increased approximately 4-fold with castration, and both interpulse basal LH and FSH release were independent of GnRH concentration. GnRH-induced LH and FSH
We found that 2 weeks after castration of young adult male rats serum FSH levels, basal FSH secretion from monolayer cultures and FSH/3 mRNA concentrations had increased approximately 2-fold, whereas pituitary FSH content was similar to that of cells from intact rats. These comparable changes lend further support to the concept that FSH release is proportional to synthesis (9, 21) and that FSH secretion is less dependent on GnRH stimulation than is LH secretion (6, 22-24). Both the unchanged FSH content and the 2-fold increase in FSH/3 mRNA 2 weeks after castration agree with previous findings in intact pituitaries (11, 25, 26), but not with a previous study of FSH content in dispersed pituitary cells that reported increased concentrations 1 week after orchidectomy (15). After longer intervals postcastration,
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CASTRATION EFFECTS ON LH AND FSH IN VITRO
2647
B 301 INTACT
75s
9
jo
c E « 50 H
CASTRATE
;
j
:
1 ?
\b \ o
H ?
7«
FIG. 5. Effects of castration on LH and FSH secretion by perifused pituitary cells stimulated with pulses of GnRH. Cells were stimulated with 2.5 nM GnRH for 2 min every 1 h.
I 10
£ 25H
I
•c :
10
20
30
40
FRACTION NUMBER TABLE 3. Effects of castration on GnRH-induced FSH and LH release from perifused pituitary cells GnRH (nM)
LH (ng/fraction)a Pulse amplitude'
FSH (ng/fraction)a Basal*
Pulse amplitude0
8.6 ± 1.5" " 9.2 ± 0.8" 7.4 ± 1.1 20.8 ± 1.2 6.6 ± 0.9". 37.0 ± 2.0e
7.8 ± 0.8 6.4 ± 0.8 6.3 ± 1.1
6.0 ± 0.4' 11.0 ± 0.8s 16.6 ± 1.3'*
28.3 ± 3.8d" 51.5 ± 3.8" 28.5 ± 4.0 88.9 ± 4.6' 10.0 25.7 ± 3.1 d . 145 ± 16.6'1-'
9.1 ± 1.4 10.0 ± 1.2 9.4 ± 1.0
11.4 ± 0.81 19.2 ± 1.6 26.7 ± 1.81
Basal*
Intact 0.1 1.0
10.0 Castrate 0.1 1.0
All data are the mean ± SEM. The results are from four experiments. For the purposes of statistical analysis, the data were transformed, such that the values for 0.1 nM GnRH for each experiment were set equal to 100%. Transformation was needed because of between-experiment differences in secretion due to cell growth characteristics and responsiveness to GnRH. Brackets indicate that, for the purposes of statistical analysis, basal hormone concentrations for all three GnRH doses were meaned. 0 Adjusted to nanograms per 107 cells. * Basal secretion is the mean of three basal levels between successive GnRH pulses. c The amplitude of a hormone pulse was 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. d ~' Values sharing a common letter are statistically different by oneway analysis of variance followed by the Bonferoni test.
the FSH content of pituitary homogenates has also been reported to be increased (27).
50
0
10
20
30
40
50
FRACTION NUMBER
The finding of increased pituitary LH content after castration agrees with previous reports (11, 28) and has been explained at least partly by an increase in gonadotroph number and size with castration (29). For FSH content to remain unchanged, by contrast, the FSH concentration per cell must decline. The 2-fold increase in FSH/3 mRNA levels with castration parallels the reported 2- to 3-fold increase in the percentage of pituitary cells staining for FSH postorchidectomy (30). Thus, unlike the decline in FSH content, FSH/3 mRNA as well as basal FSH secretion, when expressed per gonadotroph, appear to have been unchanged 2 weeks after castration. Together these observation suggest that one aspect of the differential regulation of LH and FSH is a castrationrelated alteration in the dynamics of FSH storage and secretion. The relative increment we observed in LH/3 mRNA levels in isolated pituitary cells 2 weeks after orchidectomy (5- to 6-fold) is comparable to results using intact pituitaries (24, 31, 32) and exceeds the reported 2- to 3fold increase in the percentage of pituitary cells staining for LH postorchidectomy (30). Although castration increased LH content and LH/3 mRNA concentrations proportionately more than FSH, the withdrawal of GnRH in vitro severely impaired LH release, even though considerable LH remained within the pituitary cells. Together these findings underscore the important role of GnRH in regulating LH storage and secretion. Basal and interpulse LH secretion also increased with castration, however, suggesting that GnRH-independent LH release
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2648
CASTRATION EFFECTS ON LH AND FSH IN VITRO
contributes to the LH castration response. The further increase in total FSH with time in culture for cells from castrated rats indicates that greater FSH synthesis and release occurred in vitro for many days in the absence of GnRH. Similar results have been reported previously using pituitary cells from intact males and females of several species in long term culture (9, 21). Reduced intracellular degradation of FSH does not appear to have contributed to this hypersecretion of FSH after castration, since the decline in intracellular FSH content over the course of our study was similar in cells from intact and castrated rats. We observed that total LH declined in cells from intact rats with time in culture in the absence of GnRH . This decline indicates intracellular degradation and agrees with previous results (9). By contrast, total LH did not decline in cells from castrated rats. There are two possible explanations for these findings: castration may reduce the intracellular degradation of LH, or, alternatively, the increased LH synthesis that accompanies castration (33) may be sufficient to offset degradation. Which of these two possibilities is correct cannot be resolved by our experiments. Castration increased both LH and FSH release in response to GnRH stimulation in experiments using either monolayer cultures or perifused pituitary cells. This effect was not associated with a change in the EC50 for GnRH, providing biological support for the finding of identical affinity of a radiolabeled GnRH analog for binding sites in pituitary homogenates from intact and castrated rats (28). By contrast, addition of testosterone in vitro to dispersed pituitary cells from intact rats increased the EC50 value for the GnRH effect on LH secretion (7). The castration-associated increase in responsivess to GnRH no doubt results partly from an increase in GnRH receptor number (28), and the greater effect of castration on GnRH-stimulated LH than FSH release parallels the selective increased intracellular LH content. Our findings with GnRH-stimulated cells confirm and extend the results of previous studies using dispersed pituitary cells from 60-day-old rats castrated 1 week previously, in which significantly higher release of both LH and FSH after GnRH stimulation was found compared to that in intact controls (15). By contrast, pituitary fragments from 1-week castrate rats of similar age were less responsive to pulses of GnRH in a perifusion system than were pituitary fragments from intact rats (17). The use of pituitary fragments rather than dispersed cells in the latter study would appear to best reconcile these discrepancies. In summary, these data reveal that castration increases GnRH-independent FSH and LH secretion and renders the pituitary more responsive to GnRH. There
Endo• 1990 Vol 126 • No 5
is an excellent correlation between the change in FSH/3 mRNA levels with castration and increased FSH secretion, whereas the rise in LH secretion exceeds the increment in LH/3 mRNA in part because of increased pituitary LH content and in part because of the importance of the acceleration of the GnRH pulse generator to the hypersecretion of LH that accompanies orchidectomy. These results, however, indicate that pituitary changes also contribute to the castration response.
Acknowledgments The authors wish to thank Ms. Joyce Szczepanski and Theresa Fitzgerald for their skillful technical assistance. RIA reagents were provided by Dr. A. F. Parlow through the National Hormone and Pituitary Program.
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