0013-7227/90/1262-0992$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Luteinizing Hormone (LH)-Releasing Hormone: Chronic Effects on LH and Follicle-Stimulating Hormone Cells and Secretion in Adult Male Rats* LAURA L. GARNER, GARY T. CAMPBELL, AND CHARLES A. BLAKE Department of Anatomy, Cell Biology and Neurosciences, University of South Carolina School of Medicine, Columbia, South Carolina 29208; and the Department of Anatomy, University of Nebraska Medical Center (L.L.G.), Omaha, Nebraska 68105
at the light microscopic level were not accompanied by any apparent changes in LH cells at the ultrastructural level. However, they were accompanied by an approximate doubling of the basal serum LH and FSH concentrations, an increase in the APG FSH concentration, and an increase in the basal FSH release rate (measured in vitro). The results indicate that exogenous LHRH can be administered to increase numbers of gonadotrophs in the APG, synthesis of FSH in gonadotrophs, and basal serum LH and FSH concentrations. (Endocrinology 126: 992-1000, 1990)
ABSTRACT. We investigated whether chronic administration of LHRH to normal adult rats could increase the percentages of anterior pituitary gland (APG) cells that contain immunoreactive LH and/or FSH and gonadotropin secretion. Vehicle or 1 Hg LHRH was injected sc twice daily for 6 days, and rats were decapitated 16 h after the last injection. Treatment with LHRH caused nearly a doubling in the numerical density of LH and FSH cells and in the percentage of APG cells that contained LH or FSH. It also caused a shift in the gonadotroph population from LH and LH/FSH cells to LH/FSH cells. It did not change the mean size of gonadotrophs or APG weight. These changes
S
ical role in stimulating the appearance of immunoreactive FSH in LH cells in the developing hamster APG (8, 9). We also reported that during development, exogenous LHRH can increase the percentage of APG cells that are gonadotrophs, and that LHRH plays a role in the development of normally sized gonadotrophs (9). These studies were performed on neonatal adenohypophysial tissue allografted beneath the renal capsule of adult hypophysectomized-orchidectomized hamsters administered vehicle or LHRH. Other studies that employed adult male hamster pituitary glands for grafting in this model revealed that LHRH is important for the maintenance of immunoreactive FSH in LH cells (10). However, in contrast to the studies conducted on grafted neonatal tissue, LHRH did not increase gonadotroph number or maintain normal gonadotroph size in the adult grafted tissue (10). Studies of the APG in situ have shown that chronic administration of LHRH to normal hamsters (5) increases basal serum FSH concentrations, but FSH cells were not studied. Collectively, these investigations indicate that LHRH is important for the development and maintenance of immunoreactive FSH in LH cells and can be employed to increase basal serum FSH levels, but it is not known whether LHRH can be employed to increase gonadotroph number or size in the adult APG in situ.
INCE the elucidation of the structure of LHRH by Matsuo etal. (1), numerous studies have characterized the effects of LHRH in regulating the release of LH and FSH from anterior pituitary gland (APG) gonadotrophs. An acute injection of LHRH is more effective in elevating the level of circulating LH than circulating FSH (2). In contrast, multiple injections or prolonged infusions of LHRH are more effective in terms of elevating serum FSH levels and increasing the ratio of FSH to LH concentration in circulating blood (2-6). Attempts to explain differential effects of LHRH on LH and FSH release are complicated by the fact that both hormones often have been localized in the same cell. For example, in the strain of rat used in the present study, virtually all gonadotrophs of adult males contain LH, and about 89% of these cells contain FSH as well (7). In contrast to the large number of studies conducted on the effects of LHRH on LH and FSH release, relatively few studies have been conducted on the effects of LHRH on gonadotroph number and morphology. We have reported previously that LHRH plays a critReceived September 27, 1989. Address requests for reprints to: Dr. Charles A. Blake, Department of Anatomy, Cell Biology and Neurosciences, University of South Carolina School of Medicine, Columbia, South Carolina 29208. * This work was supported by a grant from the NIH (HD-22687).
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
In the present study we investigated whether chronic administration of LHRH to normal adult male rats could increase the percentages of APG cells that contain immunoreactive FSH or LH, the percentage of LH cells that contain FSH, and the size of gonadotrophs. We also investigated whether any such changes were accompanied by alterations in APG gonadotropin release rates, gonadotropin release in response to LHRH, and basal serum gonadotropin concentrations. Some of these results have been reported in abstract form (11).
Materials and Methods Animals Male Sprague-Dawley albino rats were purchased from Simonsen Laboratories, Inc. (Gilroy, CA), and kept in a temperature-controlled room (24-27 C) with automatically controlled lighting (lights on from 0500-1900 h daily). The rats had free access to Wayne laboratory rat chow and water. Rats were kept in the animal quarters for at least 2 weeks before experimentation. They were 11-15 weeks old at the time of decapitation. The animals received no treatment (naive controls) or were injected sc with 0.2 ml distilled water (vehicle controls) or 1 ng LHRH (Beckman, Palo Alto, CA; lot B10122) in 0.2 ml vehicle. All rats were decapitated for the collection of trunk blood, which was allowed to clot at room temperature. Serum was aspirated and stored frozen at —20 C until subsequent assay for LH and FSH concentrations. Exp 1: effects of a single injection of LHRH on serum LH and FSH concentrations These experiments were conducted to test the effectiveness of the dose of LHRH employed in causing release of APG LH and FSH. At approximately 0900 h, rats were decapitated before or 20 min after injection of water or 20, 40, or 60 min after injection of LHRH. Each group consisted of five rats. Exp 2: effects of single or multiple injections of LHRH on serum and APG LH and FSH concentrations These experiments were conducted to determine approximately how long serum LH and FSH concentrations remained elevated after a single injection of LHRH and the effects of chronic treatment with LHRH on serum and APG LH and FSH concentrations. Naive controls were decapitated at 0900 h (time zero, day 1). The remainder of the rats were injected with water or LHRH, with some decapitated at 2.5, 8, or 16 h after injection (day 1). The remaining rats received an injection at 1700 h (day 1) and then at 0900 and 1700 h daily. They were decapitated just before 1700 h on day 6 or 1, 8, or 16 h after the last injection at 1700 h on day 6. Each group consisted of five rats. The APG was separated from the posterior lobe in the waterand LHRH-treated rats killed on day 1 or 7, gently blotted, and weighed. The APG was homogenized in 0.01 M PBS and stored frozen at -20 C. Homogenates were later thawed and centrifuged, and the supernatants were assayed for LH and FSH concentrations.
993
Exp 3: effects of single or multiple injections of LHRH on the basal LH and FSH release rates in vitro These studies were conducted to repeat the previous experiment on the effects of chronic treatment with LHRH on serum LH and FSH concentrations and to determine the effects of the chronic treatment on basal LH and FSH release rates. Rats were injected with water or LHRH twice daily for 6 days and decapitated (16 h after the last injection) at 0900 h on day 7. A group of naive controls was decapitated at 0900 h on day 1. Each group consisted of eight rats. The APG was separated from the posterior lobe, bisected, and gently blotted, and the halves were weighed. Half of the APG was used to determine the basal LH and FSH release rates in vitro, as described previously in detail (12). The other half was used for pilot studies. In brief, hemiglands were incubated in medium 199 under humidified 95% O2-5% CO2 for a 30-min preincubation period. They were then incubated for 2 h, and the medium was assayed to determine the amount of LH and FSH released into the medium over the 2-h period. Exp 4: effects of multiple injections of LHRH on APG gonadotrophs These experiments were conducted to repeat the previous two experiments on the effects of chronic treatment with LHRH on serum LH and FSH concentrations and to determine the effects of the chronic treatment on gonadotroph numbers and morphology. Rats were injected with water or LHRH at 0900 and 1700 h for 6 days and decapitated 16 h after the last injection at 0900 h on day 7. An additional group of noninjected controls was decapitated at 0900 h on day 7. Trunk blood was collected from 21 rats in each group. APG tissue from 5 rats in each of the 3 groups was prepared for electron microscopic examination as described previously (13). Entire pituitary glands of 5 of the remaining 16 rats in the vehicle- and LHRHtreated groups were fixed and prepared for histological examination as described previously (7). Horizontal 7-/um thick sections were cut through each entire gland. Two adjacent sections were mounted on the same slide, with the dorsal section flipped over, from the dorsal, middle, and ventral portions of each gland as defined previously (7). Of a pair of flip-flopped sections mounted on the same slide, one was stained for LH using antirat LH S4 (1:2000), and the other was stained for FSH using antirat FSH S l l (1:250). All sections were counterstained with hematoxylin. Light microscopy: immunocytochemistry and morphometry The immunocytochemical method used was modified after the peroxidase-antiperoxidase complex technique of Sternberger et al. (14) and has been described previously in detail (7). We used antirat LH S4, as described previously (7). The method controls for the technique and the sensitivity and specificity controls for antirat FSH Sll were tested as described for antirat FSH S7 (7). Antirat FSH Sll was found to be just as specific for localizing FSH in cells as was antirat FSH S7 (7). The APG was divided into 18 approximately equal areas in the horizontal plane, and the area to be analyzed was chosen by using a systematic random numbers table. Once an area was
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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chosen, it was not analyzed again. Six areas were chosen to be analyzed in dorsal, middle, and ventral sections. Thus, all 18 areas were covered. No attempt was made to choose the best subarea within an area. A Reichert microscope with an E35 grid eyepiece reticle (E. F. Fullam, Schenectady, NY) was used to analyze each pair of serial flip-flopped sections at X400. The grid was calibrated using a slide micrometer. The total number of LH or FSH cells under the grid was calculated. The numerical density (Nv, the number of LH or FSH cells per unit volume of the APG) was calculated by the method of Weibel (15) as described previously (16). Photographs were taken of cells within the 18 areas for each rat. As the flip-flopped sections were mirror images of one another, one negative in each pair was reversed while printing, so that both prints had the same alignment. The percentages of cells in the APG that contained LH or FSH were determined using the photographs, as described previously (17). Each photograph contained a total of about 750 stained and unstained cells. The cross-sectional areas of the stained cells were determined from the photographs with the use of a Bioquant image analysis system (R and M Biometrics Corp., Nashville, TN). The maximum diameters of the cells and their shape factors also were calculated for determination of the Nv. We employed the sequential staining method described previously (7) to determine whether LHRH treatment increased the percentage of LH cells that contained FSH. Five slides for each group were chosen randomly from the 15 that had been stained for each hormone. Photographs were taken from 3 randomly chosen areas in each section. Then, the sections were stained for the other hormone, and photographs were taken from the same areas as before. Comparison between the same photographs revealed whether new immunoreactive cells appeared. As a control, four slides were chosen randomly from the control group, and sections were stained a second time with the same antiserum. Six areas within each section were photographed after the first and second stain, and the number of new immunoreactive cells was counted.
Endo • 1990 Vol 126 • No 2
RIAs For each experiment, all samples were run in single assays for serum, APG, or medium for LH or FSH as described previously (12). Intraassay coefficients of variation ranged from 3.9-11.6% for both the LH and FSH assays. Values are expressed in terms of NIDDK rat LH RP-1 or FSH RP-1. Statistics All light and ultrastructural observations made using sections from each APG were averaged, and statistical tests were carried out with five per group. Statistical comparisons of two sets of data were performed with Student's t test. Comparisons of more than two sets of data were made using one- or two-way analysis of variance (ANOVA). Post-hoc Newman-Keuls test was performed when the ANOVA indicated a significant differ-
Results Expl Subcutaneous injection of 1 ng LHRH (one-way ANOVA) but not of water (Student's t test) increased serum LH and FSH concentrations (P < 0.01) by 20 min after injection (Fig. 1). Serum LH and FSH levels decreased significantly (P < 0.05) between 40 and 60 min, but were still significantly elevated 60 min after injection.
Exp2 Serum LH and FSH concentrations were not significantly elevated (by two-way ANOVA) 2.5, 8, or 16 h after the first injection of LHRH compared to levels in naive rats (time zero, day 1) or water-injected controls (Fig. 2). Treatment with water for 6 days did not significantly SINGLE INJECTION OF LHRH 500 •
LEGEND
Blocks of tissue were sectioned (2 tissue sections/grid), and at least 6 grids were stained for LH from each of the 15 APGs. The procedures for staining with antirat LH/3 (AFP 2-11-27) and the controls employed have been described previously (13). The first 25 stained cells that were encountered in each gland were examined and classified in detail. Thus, 125 cells were analyzed in each group. For each rat, secretion granules were counted only in the first 10 cells that had a nuclear diameter greater than 2 cm when viewed at x 10,000. Cells were analyzed with respect to location on a blood vessel, cell shape, nuclear shape, nuclear outline, nuclear density (heterochromatic, euchromatic), presence of nucleolus, Golgi apparatus (presence, prominence), cell cytoplasm (homogeneous, vesiculated, signetring), cell density (light, dark), endoplasmic reticulum density, lysosomes and secretion granules per cell profile, granule size and granulation density, mitochondrial density, and light density bodies (number, size). More detailed descriptions of some of these classifications have been given previously (13, 18, 19).
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FIG. 1. Effects of a single injection of water (W) or 1 fig LHRH in water, sc, on mean (±SE) serum LH and FSH concentrations. Each group consists of five decapitated rats. *, P < 0.05; **, P < 0.01 [compared to values 0 or 20 min after injection of water (20-W)].
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION SINGLE OR MULTIPLE INJECTIONS OF LHRH A 80 LEGEND H H WATER
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LHRH at 1700 h on day 6 compared to values in watertreated controls (Fig. 3B). Two-way AN0VA revealed no significant differences in APG LH concentrations between groups at these times (Fig. 3A).
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Serum LH and FSH concentrations showed changes (by one-way ANOVA) similar to those observed in Exp 2. At 0900 h on day 7 (16 h after the last injection of LHRH), serum LH and FSH concentrations (48 ± 6 and 361 ± 40 ng/ml, respectively) were greater (P < 0.01) than those in naive (16 ± 3 and 171 ± 16 ng/ml, respectively) or vehicle-treated rats (22 ± 3 and 179 ± 7 ng/ ml, respectively). On day 7, 16 h after the last injection of LHRH at 1700 h on day 6, the basal FSH release rate
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FIG. 2. Effects of single or multiple injections of water or 1 fig LHRH in water, sc, on mean (±SE) serum LH (A) and FSH (B) concentrations. The 1st injection of water or LHRH was given at time zero, day 1, and the 12th injection of water or LHRH was given at time zero, day 6. Each group consists of five decapitated rats. Values for an untreated control group are plotted as an open bar at time zero on day 1. On day 6, *, P < 0.05 and • * , P < 0.01 compared to nontreated (naive; time zero, day 1) or water-injected controls killed at the same time.
alter basal serum LH or FSH concentrations (time zero, day 6 us. time zero, day 1). In contrast, treatment with LHRH for 6 days significantly elevated basal serum LH and FSH concentrations, as observed at time zero, day 6, which was 8 h after the 11th injection of LHRH. The 12th injection of LHRH at time zero on day 6 caused a further increase (P < 0.05) in serum LH, but not serum FSH, concentrations 1 h after injection. Serum LH and FSH concentrations 8 and 16 h after the 12th injection were not significantly different from preinjection values (time zero, day 6). APG FSH concentrations in LHRH-treated rats were elevated at 1700 h on day 6 (8 h after the 11th injection of LHRH) and 1, 8, and 16 h after the 12th injection of
6-7
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MULTIPLE INJECTIONS OF LHRH 30 T
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FIG. 3. Effects of multiple injections of water or LHRH in water sc on mean (±SE) APG LH (A) or FSH (B) concentrations per mg APG. The 12th injection of water or LHRH was given at time zero. Each group consists offivedecapitated rats. • * , P < 0.01 compared to rats injected with water.
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
996
was increased (by one-way ANOVA) compared to values in untreated controls (day 1) or vehicle-treated rats (Fig. 4). The total weights (mean ± SE) of the APG for the three groups of rats used to determine basal gonadotropin release rates were 8.1 ± 0.3 (naive), 8.7 ± 0.3 (vehicletreated), and 8.5 ± 0.4 mg (LHRH-treated). One-way ANOVA revealed no significant differences among the three groups. Exp4 Serum gonadotropin concentrations. Serum LH and FSH concentrations showed changes (by one-way ANOVA) similar to those observed in Exp 2 and 3. At 0900 h on day 7 (16 h after the last injection of LHRH), serum LH and FSH concentrations (18 ± 2 and 448 + 19 ng/ml, respectively) were elevated (P < 0.01) compared to values in the naive (7 ± 2 and 234 ± 7 ng/ml, respectively) or vehicle-injected controls (7 ± 2 and 248 ± 9 ng/ml, respectively). Light microscopy. Representative photographs of sections stained for LH and FSH in rats at 16 h after 6 days of treatment with water or LHRH are shown in Fig. 5. Treatment with LHRH for 6 days caused nearly a doubling (by two-way ANOVA) in the Nv of the LH and FSH cells (Fig. 6). Similarly, nearly a doubling of the percentage of APG cells that contained LH or FSH was observed after LHRH treatment (Fig. 7). These changes were not accompanied by any change in mean crosssectional area of cell profiles measured in the same rats (control, 113 ± 10 ^m2 for 344 LH cells, 108 ± 9 ^m2 for 357 FSH cells; experimental, 108 ± 9 /im2 for 722 LH cells, 99 ± 7 )um2for 652 FSH cells). BASAL GONADOTROPHIN RELEASE RATES 16 HOURS AFTER LAST INJECTION 4.0 • UJ
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DAY FlG. 4. Effects of 6 days of injection of water or LHRH in water sc on mean (±SE) basal gonadotropin release rates 16 h after the last injection. Each group consists of eight decapitated rats. **, P < 0.01 compared to the untreated controls (day 1) or rats injected with water for 6 days (7-W).
Endo • 1990 Vol 126 • No 2
Sequential staining of tissue from control rats for FSH and then for LH revealed 16.2% new cells, which was significantly greater (Student's t test; P < 0.05) than the 2.1% new cells observed when tissue from the LHRHtreated group was sequentially stained in an identical manner (Fig. 8). No differences in percentages of new cells were observed between groups after sequential staining of tissue for LH and then FSH (Fig. 8). Sequential staining of sections with the same antiserum revealed 2.3 ± 0.6% new LH cells and 3.8 ± 2.5% new FSH cells. Electron microscopy. Typical LH cells from naive and LHRH-treated rats are shown in Fig. 9. No statistically significant differences (by one-way ANOVA) were observed for the ultrastructural characteristics examined for LH cells among the three groups. Discussion It is clear from the data presented that synthetic LHRH can be employed to increase the numbers of gonadotrophs and basal serum gonadotropin concentrations. Our LHRH stimulation protocol was based on that of Gregerson and Campbell (5), who reported that twice daily injections of LHRH for 6 days elevated basal serum FSH concentrations in hamsters. We chose a dose of LHRH that would cause a greater than 2-fold elevation of the serum FSH concentration. The dose of LHRH administered was sufficient at the first injection to cause an acute increase in serum LH and FSH concentrations 20 min after injection. Serum LH and FSH concentrations returned to levels that were no longer significantly elevated by 2.5 h after injection. For this reason, serum LH and FSH concentrations 8 or 16 h after injection of LHRH in the present study are considered to represent basal levels, which reflect LH and FSH release independent of the immediate presence of exogenous LHRH. As observed 16 h after the end of twice daily injections of LHRH for as short a period as 6 days, there was approximately a doubling of both the number of APG gonadotrophs and the basal serum LH and FSH concentrations. This protocol provides an easy and effective method to elevate basal serum LH and FSH concentrations, which could be useful in research and clinical applications. It is possible that alterations in our protocol, such as a change in the dose of LHRH, the number of daily injections, and/or the number of days of injection, might be more effective in increasing gonadotroph numbers and basal serum gonadotropin concentrations on a chronic basis. Treatment of normal male hamsters with 100 ng or 15 /ig LHRH twice daily for 6 days caused approximately a 2.1- and 3.4-fold increase in the basal serum FSH concentration, respectively. In contrast to the results of the present study, the increases in the basal
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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FIG. 5. Sections of APG from male rats were stained for LH (A and B) or FSH (C and D) and counterstained with hematoxylin. Rats were killed 16 h after 6 days of sc injection of water (A and C) or 1 Mg LHRH (B and D). Bars = 10 /xm; magnification, X413.
serum FSH concentration with these doses of LHRH were accompanied by a decrease in the basal serum LH concentration (5). Our study did not determine how long basal serum gonadotropin levels will remain elevated after the 6 days of LHRH treatment or the effectiveness of administration of LHRH beyond 6 days on the maintenance of elevated basal serum gonadotropin concentrations on a chronic basis. Based on the observations that the in-
creased basal serum gonadotropin levels at 16 h after 6 days of treatment were associated with nearly a doubling of the number of gonadotrophs, we would predict that basal serum gonadotropin levels would remain elevated for some time without further administration of exogenous LHRH. Changes in serum LH and FSH concentrations in the present study were not associated with a change in APG weight or in the mean size of LH or FSH cells. These
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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REPEATED STAINING OF SECTIONS WITH DIFFERENT ANTISERA
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FIG. 6. Effects of 6 days of injection of LHRH in water sc on mean (± SE) NV of LH and FSH cells. In water-injected rats 1440 cells stained for LH and 1286 cells stained for FSH were counted. In LHRH-treated rats, 2141 cells stained for LH and 2427 cells stained for FSH were counted. Each mean represents 5 rats. *, P < 0.05; **, P < 0.01 (compared to water-injected controls). PERCENTAGE OF LH 8c FSH CELLS IN THE ANTERIOR PITUITARY 12
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Endo• 1990 Vol 126 • No 2
LHRH
FlG. 7. Effects of 6 days of injection of LHRH in water sc on mean (± SE) percentage of LH or FSH cells in the APG. Over 13,000 cells (stained and nonstained) were counted to determine the percentage of LH and FSH cells in the 5 rats in each group. *, P < 0.05 compared to water-injected controls.
data in association with the increases in both the Nv of LH and FSH cells and the percentage of APG cells containing LH or FSH indicate that LHRH-induced increased LH and FSH secretion was accompanied by an increase in the total number of APG gonadotrophs. The increase in gonadotrophs could have resulted from mitotic division of differentiated gonadotrophs, conversion of undifferentiated cells into gonadotrophs, or conversion of other differentiated cell types into gonadotrophs. It is not surprising that 6 days of treatment with LHRH in the present study did not increase the size of
LHRH
FlG. 8. Effects of 6 days of injection of water or LHRH in water sc on mean (±SE) new cells that stained after a second immunocytochemical stain was performed on the same tissue using a different antiserum. Sections were stained for LH and then for FSH (LH-FSH) or vice versa (FSH-LH). In water-treated rats, 557 cells initially stained for LH and 617 cells initially stained for FSH were counted. In LHRHtreated rats, 971 cells initially stained for LH and 977 cells initially stained for FSH were counted. Each mean represents 5 rats. •*, P < 0.05 compared to all other sequentially stained groups.
gonadotrophs. We have shown previously that LHRH plays a role in maintaining the normal size of gonadotrophs during development, but obtained no evidence to suggest that LHRH increases gonadotroph size above normal (9). We have also observed that orchidectomy increases gonadotroph size in the strain of rat used in the present study, but the hypertrophy was apparent 35, and not 7, days after castration (23). It is possible that more prolonged stimulation by LHRH using our protocol or a different regimen of LHRH administration might cause an increase in gonadotroph size. However, the putative release of testicular hormones after each bolus of LHRH may have precluded an effect of LHRH on gonadotroph size. Thus, studies designed to test the effects of LHRH on gonadotroph size will need a model in which the APG is exposed to the exogenous LHRH only and no testicular hormones. Whereas FSH was not detectable in all gonadotrophs in controls, virtually all gonadotrophs contained both FSH and LH after 6 days of treatment with LHRH. Sequential staining, first with FSH and then with LH, revealed new cells in control sections, but no statistically significant increase in stained cells was observed in sections from LHRH-treated rats. We have previously reported that LHRH is important for the maintenance of FSH in LH cells (10). Our data support the view that all gonadotrophs may potentially synthesize both FSH and LH and that exogenous LHRH combined with endogenous LHRH in the present study stimulates FSH synthesis in virtually all gonadotrophs. The effects of exog-
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
FIG. 9. These two electron micrographs are of typical cells that stained for LH/3. Upper panel, A polygonally shaped cell from an untreated rat with homogeneous cytoplasm and a polygonal, irregularly outlined nucleus. The cell contains a prominent Golgi zone. It is densely and uniformly granulated. A total of 1111 granules were counted, with 5 greater than 300 nm in diameter (X6050). Lower panel, A polygonal cell from a LHRH-treated rat with a homogeneous cytoplasm and a polygonal, irregularly outlined nucleus. Note the large prominent Golgi area. The cell is densely and uniformly granulated (1161 granules). It has 3 granules greater than 300 nm in diameter (X5650).
enous LHRH on endogenous LHRH release in the present study are not known. Correlation of the APG LH and FSH concentrations with the increases in gonadotroph cell numbers suggest that the average content of LH per cell decreased after LHRH treatment, whereas the average content of FSH per cell was better maintained after LHRH treatment. With the additional observation of increased basal levels of LH and FSH in serum after 6 days of LHRH treatment; one can conclude that, on the average, FSH cells were better able to maintain FSH stores while releasing FSH than LH cells were able to maintain LH stores
999
while releasing LH. Based on the observations that LHRH treatment increased LH cell number and was without effect on LH cell size and number of LH secretion granules, the data suggest that LH secretion granules contain less LH than those in LH cells of control rats. Previous studies have shown that the basal FSH release rate plays an important role in increasing the serum FSH concentration during the periovulatory FSH surge (12, 20, 21) and after ovariectomy (22). The increase in the basal serum FSH concentration after 6 days of LHRH treatment in the present study is probably due at least in part to an increase in the basal FSH release rate, as assessed in vitro. As the increase in FSH cell number after LHRH treatment was proportionately as great or greater than the increase in the basal FSH release rate, an increase in the basal secretion rate of FSH in vivo could, on the average, be due to an increase in the number of cells releasing FSH rather than to a higher basal FSH release rate per cell. In contrast, no evidence was obtained to suggest that the increase in the basal serum LH concentration was due to an increase in the basal LH release rate. The basal LH release rate in vitro did not increase. Coupled with the observation that LH cell number increased after LHRH treatment, it is likely that the basal LH release rate in vivo was reduced on a per cell basis. This is consistent with our suggestion that LHRH treatment did not alter secretion granule number per cell and that less LH was packaged per secretion granule. The ultrastructural observations revealed no other clues to elucidate the mechanisms of LHRH-induced gonadotroph cell number or increased gonadotropin secretion. In summary, the results of this study show for the first time that exogenous LHRH can be administered to increase the number of gonadotrophs. This appears to be an effective and efficient means to increase basal serum LH and FSH concentrations. Acknowledgments We thank Dr. Albert F. Parlow and the National Hormone and Pituitary Program for the antigens and antisera, Alison Rosenberg and Neda Osterman for assistance in making thefigures,and Carol A. Able for secretarial assistance. References 1. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally, AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334 2. Debeljuk L, Arimura A, Shiino M, Rennels EG, Schally AV 1973 Effects of chronic treatment with LH/FSH-RH in hypophysectomized pituitary-grafted male rats. Endocrinology 92:921 3. Johnson, DC, Mallampati RS 1975 Serum luteinizing hormone and follicle-stimulating hormone concentrations in immature female rats treated with multiple injections and various amounts of lu-
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
teinizing hormone releasing hormone. J Endocrinol 66:13 4. NcNeilly AS, deKretser DM, Sharpe RM 1979 Modulation of prolactin, luteinizing hormone (LH) and follicle stimulation hormone (FSH) secretion by LHRH and bromocriptine (CB154) in the hypophysectomized pituitary-grafted male rat and its effect on testicular LH receptors and testosterone output. Biol Reprod 21:141 5. Gregerson KA, Campbell GT 1984 Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster. Peptides 5:471 6. Campbell GT, Horacek MJ, Blake CA 1987 Effects of hypothalamic neurohormones on prolactin release from pituitary allografts in the hamster. Proc Soc Exp Biol Med 186:344 7. Dada MO, Campbell GT, Blake CA 1983 A quantitative immunocytochemical study on the luteinizing hormone and follicle-stimulating hormone cells in the adenohypophysis of adult male rats and of adult female rats throughout the estrous cycle. Endocrinology 113:970 8. Gregerson KA, Campbell GT 1982 Influences of luteinizing hormone releasing hormone, hypophysectomy, and orchidectomy on the differentiation of luteinizing hormone and follicle stimulating hormone cells in an ectopic pituitary in the hamster. Biol Reprod 27:169 9. Horacek MJ, Campbell GT, Blake CA 1989 Luteinizing hormone (LH)-releasing hormone: effects on induction of LH, follicle-stimulating hormone, and prolactin cell differentiation. Endocrinology 124:1800 10. Horacek MJ, Campbell GT, Blake CA 1990 Luteinizing hormone (LH)-Releasing Hormone: effects on maintenance of immunoreactive follicle- stimulating hormone and LH in adenohypophysial cells. Endocrinology 126:653 11. Garner LL, Campbell GT, Blake CA 1987 Gonadotroph number is increased by luteinizing hormone releasing hormone (LHRH). Fed Proc 46:1063 12. Elias KA, Kelch RP, Lipner H, Blake CA 1982 Relationships between basal gonadotropin secretion rates and serum gonadotro-
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pin concentrations in proestrous rats. Biol Reprod 27:1159 13. Garner LL, Blake CA 1979 Morphological correlates for LHRH self priming and anterior pituitary gland refractoriness to LHRH in proestrous rats: an immunocytochemical study. Biol Reprod 20:1055 14. Stemberger LA, Hardy Jr PH, Cuculis JJ, Meyer HG 1970 The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody (horseradish peroxidase anti-peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315 15. Weibel ER 1979 Stereological Methods. Academic Press, London, vol 1:9 16. Dada MO, Blake CA 1985 Monosodium L-glutamate administration: effects on gonadotrophin secretion, gonadotrophs and mammotrophs in prepubertal female rats. J Endocrinol 104:185 17. Dada MO, Campbell GT, Blake CA 1984 Pars distalis cell quantification in normal adult male and female rats. J Endocrinol 101:87 18. Blake CA 1980 Correlative study of changes in the morphology of the LH gonadotroph and anterior pituitary gland LH secretion during the 4-day rat estrous cycle. Biol Reprod 23:1097 19. Garner LL, Blake CA 1981 Ultrastructural, immunocytochemical study of the LH secreting cell of the rat anterior pituitary gland: changes occurring after ovariectomy. Biol Reprod 24:461 20. Elias KA, Blake CA 1980 A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat. Life Sci 26:749 21. Elias KA, Blake CA 1981 A detailed in vitro characterization of the basal follicle-stimulating hormone and luteinizing hormone secretion rates during the rat four-day estrous cycle. Endocrinology 109:708 22. Elias KA, Blake CA 1983 Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat. Biol Reprod 28:1107 23. Dada MO, Blake CA 1986 A detailed morphometric study of gonadotrophs in male rats after removal of negative feedback on the hypothalamic-pituitary axis. Soc Neurosci Abstr 12:1413
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Vol. 126, No. 2 Printed in U.S.A.
Luteinizing Hormone (LH)-Releasing Hormone: Chronic Effects on LH and Follicle-Stimulating Hormone Cells and Secretion in Adult Male Rats* LAURA L. GARNER, GARY T. CAMPBELL, AND CHARLES A. BLAKE Department of Anatomy, Cell Biology and Neurosciences, University of South Carolina School of Medicine, Columbia, South Carolina 29208; and the Department of Anatomy, University of Nebraska Medical Center (L.L.G.), Omaha, Nebraska 68105
at the light microscopic level were not accompanied by any apparent changes in LH cells at the ultrastructural level. However, they were accompanied by an approximate doubling of the basal serum LH and FSH concentrations, an increase in the APG FSH concentration, and an increase in the basal FSH release rate (measured in vitro). The results indicate that exogenous LHRH can be administered to increase numbers of gonadotrophs in the APG, synthesis of FSH in gonadotrophs, and basal serum LH and FSH concentrations. (Endocrinology 126: 992-1000, 1990)
ABSTRACT. We investigated whether chronic administration of LHRH to normal adult rats could increase the percentages of anterior pituitary gland (APG) cells that contain immunoreactive LH and/or FSH and gonadotropin secretion. Vehicle or 1 Hg LHRH was injected sc twice daily for 6 days, and rats were decapitated 16 h after the last injection. Treatment with LHRH caused nearly a doubling in the numerical density of LH and FSH cells and in the percentage of APG cells that contained LH or FSH. It also caused a shift in the gonadotroph population from LH and LH/FSH cells to LH/FSH cells. It did not change the mean size of gonadotrophs or APG weight. These changes
S
ical role in stimulating the appearance of immunoreactive FSH in LH cells in the developing hamster APG (8, 9). We also reported that during development, exogenous LHRH can increase the percentage of APG cells that are gonadotrophs, and that LHRH plays a role in the development of normally sized gonadotrophs (9). These studies were performed on neonatal adenohypophysial tissue allografted beneath the renal capsule of adult hypophysectomized-orchidectomized hamsters administered vehicle or LHRH. Other studies that employed adult male hamster pituitary glands for grafting in this model revealed that LHRH is important for the maintenance of immunoreactive FSH in LH cells (10). However, in contrast to the studies conducted on grafted neonatal tissue, LHRH did not increase gonadotroph number or maintain normal gonadotroph size in the adult grafted tissue (10). Studies of the APG in situ have shown that chronic administration of LHRH to normal hamsters (5) increases basal serum FSH concentrations, but FSH cells were not studied. Collectively, these investigations indicate that LHRH is important for the development and maintenance of immunoreactive FSH in LH cells and can be employed to increase basal serum FSH levels, but it is not known whether LHRH can be employed to increase gonadotroph number or size in the adult APG in situ.
INCE the elucidation of the structure of LHRH by Matsuo etal. (1), numerous studies have characterized the effects of LHRH in regulating the release of LH and FSH from anterior pituitary gland (APG) gonadotrophs. An acute injection of LHRH is more effective in elevating the level of circulating LH than circulating FSH (2). In contrast, multiple injections or prolonged infusions of LHRH are more effective in terms of elevating serum FSH levels and increasing the ratio of FSH to LH concentration in circulating blood (2-6). Attempts to explain differential effects of LHRH on LH and FSH release are complicated by the fact that both hormones often have been localized in the same cell. For example, in the strain of rat used in the present study, virtually all gonadotrophs of adult males contain LH, and about 89% of these cells contain FSH as well (7). In contrast to the large number of studies conducted on the effects of LHRH on LH and FSH release, relatively few studies have been conducted on the effects of LHRH on gonadotroph number and morphology. We have reported previously that LHRH plays a critReceived September 27, 1989. Address requests for reprints to: Dr. Charles A. Blake, Department of Anatomy, Cell Biology and Neurosciences, University of South Carolina School of Medicine, Columbia, South Carolina 29208. * This work was supported by a grant from the NIH (HD-22687).
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
In the present study we investigated whether chronic administration of LHRH to normal adult male rats could increase the percentages of APG cells that contain immunoreactive FSH or LH, the percentage of LH cells that contain FSH, and the size of gonadotrophs. We also investigated whether any such changes were accompanied by alterations in APG gonadotropin release rates, gonadotropin release in response to LHRH, and basal serum gonadotropin concentrations. Some of these results have been reported in abstract form (11).
Materials and Methods Animals Male Sprague-Dawley albino rats were purchased from Simonsen Laboratories, Inc. (Gilroy, CA), and kept in a temperature-controlled room (24-27 C) with automatically controlled lighting (lights on from 0500-1900 h daily). The rats had free access to Wayne laboratory rat chow and water. Rats were kept in the animal quarters for at least 2 weeks before experimentation. They were 11-15 weeks old at the time of decapitation. The animals received no treatment (naive controls) or were injected sc with 0.2 ml distilled water (vehicle controls) or 1 ng LHRH (Beckman, Palo Alto, CA; lot B10122) in 0.2 ml vehicle. All rats were decapitated for the collection of trunk blood, which was allowed to clot at room temperature. Serum was aspirated and stored frozen at —20 C until subsequent assay for LH and FSH concentrations. Exp 1: effects of a single injection of LHRH on serum LH and FSH concentrations These experiments were conducted to test the effectiveness of the dose of LHRH employed in causing release of APG LH and FSH. At approximately 0900 h, rats were decapitated before or 20 min after injection of water or 20, 40, or 60 min after injection of LHRH. Each group consisted of five rats. Exp 2: effects of single or multiple injections of LHRH on serum and APG LH and FSH concentrations These experiments were conducted to determine approximately how long serum LH and FSH concentrations remained elevated after a single injection of LHRH and the effects of chronic treatment with LHRH on serum and APG LH and FSH concentrations. Naive controls were decapitated at 0900 h (time zero, day 1). The remainder of the rats were injected with water or LHRH, with some decapitated at 2.5, 8, or 16 h after injection (day 1). The remaining rats received an injection at 1700 h (day 1) and then at 0900 and 1700 h daily. They were decapitated just before 1700 h on day 6 or 1, 8, or 16 h after the last injection at 1700 h on day 6. Each group consisted of five rats. The APG was separated from the posterior lobe in the waterand LHRH-treated rats killed on day 1 or 7, gently blotted, and weighed. The APG was homogenized in 0.01 M PBS and stored frozen at -20 C. Homogenates were later thawed and centrifuged, and the supernatants were assayed for LH and FSH concentrations.
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Exp 3: effects of single or multiple injections of LHRH on the basal LH and FSH release rates in vitro These studies were conducted to repeat the previous experiment on the effects of chronic treatment with LHRH on serum LH and FSH concentrations and to determine the effects of the chronic treatment on basal LH and FSH release rates. Rats were injected with water or LHRH twice daily for 6 days and decapitated (16 h after the last injection) at 0900 h on day 7. A group of naive controls was decapitated at 0900 h on day 1. Each group consisted of eight rats. The APG was separated from the posterior lobe, bisected, and gently blotted, and the halves were weighed. Half of the APG was used to determine the basal LH and FSH release rates in vitro, as described previously in detail (12). The other half was used for pilot studies. In brief, hemiglands were incubated in medium 199 under humidified 95% O2-5% CO2 for a 30-min preincubation period. They were then incubated for 2 h, and the medium was assayed to determine the amount of LH and FSH released into the medium over the 2-h period. Exp 4: effects of multiple injections of LHRH on APG gonadotrophs These experiments were conducted to repeat the previous two experiments on the effects of chronic treatment with LHRH on serum LH and FSH concentrations and to determine the effects of the chronic treatment on gonadotroph numbers and morphology. Rats were injected with water or LHRH at 0900 and 1700 h for 6 days and decapitated 16 h after the last injection at 0900 h on day 7. An additional group of noninjected controls was decapitated at 0900 h on day 7. Trunk blood was collected from 21 rats in each group. APG tissue from 5 rats in each of the 3 groups was prepared for electron microscopic examination as described previously (13). Entire pituitary glands of 5 of the remaining 16 rats in the vehicle- and LHRHtreated groups were fixed and prepared for histological examination as described previously (7). Horizontal 7-/um thick sections were cut through each entire gland. Two adjacent sections were mounted on the same slide, with the dorsal section flipped over, from the dorsal, middle, and ventral portions of each gland as defined previously (7). Of a pair of flip-flopped sections mounted on the same slide, one was stained for LH using antirat LH S4 (1:2000), and the other was stained for FSH using antirat FSH S l l (1:250). All sections were counterstained with hematoxylin. Light microscopy: immunocytochemistry and morphometry The immunocytochemical method used was modified after the peroxidase-antiperoxidase complex technique of Sternberger et al. (14) and has been described previously in detail (7). We used antirat LH S4, as described previously (7). The method controls for the technique and the sensitivity and specificity controls for antirat FSH Sll were tested as described for antirat FSH S7 (7). Antirat FSH Sll was found to be just as specific for localizing FSH in cells as was antirat FSH S7 (7). The APG was divided into 18 approximately equal areas in the horizontal plane, and the area to be analyzed was chosen by using a systematic random numbers table. Once an area was
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chosen, it was not analyzed again. Six areas were chosen to be analyzed in dorsal, middle, and ventral sections. Thus, all 18 areas were covered. No attempt was made to choose the best subarea within an area. A Reichert microscope with an E35 grid eyepiece reticle (E. F. Fullam, Schenectady, NY) was used to analyze each pair of serial flip-flopped sections at X400. The grid was calibrated using a slide micrometer. The total number of LH or FSH cells under the grid was calculated. The numerical density (Nv, the number of LH or FSH cells per unit volume of the APG) was calculated by the method of Weibel (15) as described previously (16). Photographs were taken of cells within the 18 areas for each rat. As the flip-flopped sections were mirror images of one another, one negative in each pair was reversed while printing, so that both prints had the same alignment. The percentages of cells in the APG that contained LH or FSH were determined using the photographs, as described previously (17). Each photograph contained a total of about 750 stained and unstained cells. The cross-sectional areas of the stained cells were determined from the photographs with the use of a Bioquant image analysis system (R and M Biometrics Corp., Nashville, TN). The maximum diameters of the cells and their shape factors also were calculated for determination of the Nv. We employed the sequential staining method described previously (7) to determine whether LHRH treatment increased the percentage of LH cells that contained FSH. Five slides for each group were chosen randomly from the 15 that had been stained for each hormone. Photographs were taken from 3 randomly chosen areas in each section. Then, the sections were stained for the other hormone, and photographs were taken from the same areas as before. Comparison between the same photographs revealed whether new immunoreactive cells appeared. As a control, four slides were chosen randomly from the control group, and sections were stained a second time with the same antiserum. Six areas within each section were photographed after the first and second stain, and the number of new immunoreactive cells was counted.
Endo • 1990 Vol 126 • No 2
RIAs For each experiment, all samples were run in single assays for serum, APG, or medium for LH or FSH as described previously (12). Intraassay coefficients of variation ranged from 3.9-11.6% for both the LH and FSH assays. Values are expressed in terms of NIDDK rat LH RP-1 or FSH RP-1. Statistics All light and ultrastructural observations made using sections from each APG were averaged, and statistical tests were carried out with five per group. Statistical comparisons of two sets of data were performed with Student's t test. Comparisons of more than two sets of data were made using one- or two-way analysis of variance (ANOVA). Post-hoc Newman-Keuls test was performed when the ANOVA indicated a significant differ-
Results Expl Subcutaneous injection of 1 ng LHRH (one-way ANOVA) but not of water (Student's t test) increased serum LH and FSH concentrations (P < 0.01) by 20 min after injection (Fig. 1). Serum LH and FSH levels decreased significantly (P < 0.05) between 40 and 60 min, but were still significantly elevated 60 min after injection.
Exp2 Serum LH and FSH concentrations were not significantly elevated (by two-way ANOVA) 2.5, 8, or 16 h after the first injection of LHRH compared to levels in naive rats (time zero, day 1) or water-injected controls (Fig. 2). Treatment with water for 6 days did not significantly SINGLE INJECTION OF LHRH 500 •
LEGEND
Blocks of tissue were sectioned (2 tissue sections/grid), and at least 6 grids were stained for LH from each of the 15 APGs. The procedures for staining with antirat LH/3 (AFP 2-11-27) and the controls employed have been described previously (13). The first 25 stained cells that were encountered in each gland were examined and classified in detail. Thus, 125 cells were analyzed in each group. For each rat, secretion granules were counted only in the first 10 cells that had a nuclear diameter greater than 2 cm when viewed at x 10,000. Cells were analyzed with respect to location on a blood vessel, cell shape, nuclear shape, nuclear outline, nuclear density (heterochromatic, euchromatic), presence of nucleolus, Golgi apparatus (presence, prominence), cell cytoplasm (homogeneous, vesiculated, signetring), cell density (light, dark), endoplasmic reticulum density, lysosomes and secretion granules per cell profile, granule size and granulation density, mitochondrial density, and light density bodies (number, size). More detailed descriptions of some of these classifications have been given previously (13, 18, 19).
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FIG. 1. Effects of a single injection of water (W) or 1 fig LHRH in water, sc, on mean (±SE) serum LH and FSH concentrations. Each group consists of five decapitated rats. *, P < 0.05; **, P < 0.01 [compared to values 0 or 20 min after injection of water (20-W)].
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION SINGLE OR MULTIPLE INJECTIONS OF LHRH A 80 LEGEND H H WATER
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LHRH at 1700 h on day 6 compared to values in watertreated controls (Fig. 3B). Two-way AN0VA revealed no significant differences in APG LH concentrations between groups at these times (Fig. 3A).
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Serum LH and FSH concentrations showed changes (by one-way ANOVA) similar to those observed in Exp 2. At 0900 h on day 7 (16 h after the last injection of LHRH), serum LH and FSH concentrations (48 ± 6 and 361 ± 40 ng/ml, respectively) were greater (P < 0.01) than those in naive (16 ± 3 and 171 ± 16 ng/ml, respectively) or vehicle-treated rats (22 ± 3 and 179 ± 7 ng/ ml, respectively). On day 7, 16 h after the last injection of LHRH at 1700 h on day 6, the basal FSH release rate
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FIG. 2. Effects of single or multiple injections of water or 1 fig LHRH in water, sc, on mean (±SE) serum LH (A) and FSH (B) concentrations. The 1st injection of water or LHRH was given at time zero, day 1, and the 12th injection of water or LHRH was given at time zero, day 6. Each group consists of five decapitated rats. Values for an untreated control group are plotted as an open bar at time zero on day 1. On day 6, *, P < 0.05 and • * , P < 0.01 compared to nontreated (naive; time zero, day 1) or water-injected controls killed at the same time.
alter basal serum LH or FSH concentrations (time zero, day 6 us. time zero, day 1). In contrast, treatment with LHRH for 6 days significantly elevated basal serum LH and FSH concentrations, as observed at time zero, day 6, which was 8 h after the 11th injection of LHRH. The 12th injection of LHRH at time zero on day 6 caused a further increase (P < 0.05) in serum LH, but not serum FSH, concentrations 1 h after injection. Serum LH and FSH concentrations 8 and 16 h after the 12th injection were not significantly different from preinjection values (time zero, day 6). APG FSH concentrations in LHRH-treated rats were elevated at 1700 h on day 6 (8 h after the 11th injection of LHRH) and 1, 8, and 16 h after the 12th injection of
6-7
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MULTIPLE INJECTIONS OF LHRH 30 T
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FIG. 3. Effects of multiple injections of water or LHRH in water sc on mean (±SE) APG LH (A) or FSH (B) concentrations per mg APG. The 12th injection of water or LHRH was given at time zero. Each group consists offivedecapitated rats. • * , P < 0.01 compared to rats injected with water.
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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was increased (by one-way ANOVA) compared to values in untreated controls (day 1) or vehicle-treated rats (Fig. 4). The total weights (mean ± SE) of the APG for the three groups of rats used to determine basal gonadotropin release rates were 8.1 ± 0.3 (naive), 8.7 ± 0.3 (vehicletreated), and 8.5 ± 0.4 mg (LHRH-treated). One-way ANOVA revealed no significant differences among the three groups. Exp4 Serum gonadotropin concentrations. Serum LH and FSH concentrations showed changes (by one-way ANOVA) similar to those observed in Exp 2 and 3. At 0900 h on day 7 (16 h after the last injection of LHRH), serum LH and FSH concentrations (18 ± 2 and 448 + 19 ng/ml, respectively) were elevated (P < 0.01) compared to values in the naive (7 ± 2 and 234 ± 7 ng/ml, respectively) or vehicle-injected controls (7 ± 2 and 248 ± 9 ng/ml, respectively). Light microscopy. Representative photographs of sections stained for LH and FSH in rats at 16 h after 6 days of treatment with water or LHRH are shown in Fig. 5. Treatment with LHRH for 6 days caused nearly a doubling (by two-way ANOVA) in the Nv of the LH and FSH cells (Fig. 6). Similarly, nearly a doubling of the percentage of APG cells that contained LH or FSH was observed after LHRH treatment (Fig. 7). These changes were not accompanied by any change in mean crosssectional area of cell profiles measured in the same rats (control, 113 ± 10 ^m2 for 344 LH cells, 108 ± 9 ^m2 for 357 FSH cells; experimental, 108 ± 9 /im2 for 722 LH cells, 99 ± 7 )um2for 652 FSH cells). BASAL GONADOTROPHIN RELEASE RATES 16 HOURS AFTER LAST INJECTION 4.0 • UJ
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DAY FlG. 4. Effects of 6 days of injection of water or LHRH in water sc on mean (±SE) basal gonadotropin release rates 16 h after the last injection. Each group consists of eight decapitated rats. **, P < 0.01 compared to the untreated controls (day 1) or rats injected with water for 6 days (7-W).
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Sequential staining of tissue from control rats for FSH and then for LH revealed 16.2% new cells, which was significantly greater (Student's t test; P < 0.05) than the 2.1% new cells observed when tissue from the LHRHtreated group was sequentially stained in an identical manner (Fig. 8). No differences in percentages of new cells were observed between groups after sequential staining of tissue for LH and then FSH (Fig. 8). Sequential staining of sections with the same antiserum revealed 2.3 ± 0.6% new LH cells and 3.8 ± 2.5% new FSH cells. Electron microscopy. Typical LH cells from naive and LHRH-treated rats are shown in Fig. 9. No statistically significant differences (by one-way ANOVA) were observed for the ultrastructural characteristics examined for LH cells among the three groups. Discussion It is clear from the data presented that synthetic LHRH can be employed to increase the numbers of gonadotrophs and basal serum gonadotropin concentrations. Our LHRH stimulation protocol was based on that of Gregerson and Campbell (5), who reported that twice daily injections of LHRH for 6 days elevated basal serum FSH concentrations in hamsters. We chose a dose of LHRH that would cause a greater than 2-fold elevation of the serum FSH concentration. The dose of LHRH administered was sufficient at the first injection to cause an acute increase in serum LH and FSH concentrations 20 min after injection. Serum LH and FSH concentrations returned to levels that were no longer significantly elevated by 2.5 h after injection. For this reason, serum LH and FSH concentrations 8 or 16 h after injection of LHRH in the present study are considered to represent basal levels, which reflect LH and FSH release independent of the immediate presence of exogenous LHRH. As observed 16 h after the end of twice daily injections of LHRH for as short a period as 6 days, there was approximately a doubling of both the number of APG gonadotrophs and the basal serum LH and FSH concentrations. This protocol provides an easy and effective method to elevate basal serum LH and FSH concentrations, which could be useful in research and clinical applications. It is possible that alterations in our protocol, such as a change in the dose of LHRH, the number of daily injections, and/or the number of days of injection, might be more effective in increasing gonadotroph numbers and basal serum gonadotropin concentrations on a chronic basis. Treatment of normal male hamsters with 100 ng or 15 /ig LHRH twice daily for 6 days caused approximately a 2.1- and 3.4-fold increase in the basal serum FSH concentration, respectively. In contrast to the results of the present study, the increases in the basal
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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FIG. 5. Sections of APG from male rats were stained for LH (A and B) or FSH (C and D) and counterstained with hematoxylin. Rats were killed 16 h after 6 days of sc injection of water (A and C) or 1 Mg LHRH (B and D). Bars = 10 /xm; magnification, X413.
serum FSH concentration with these doses of LHRH were accompanied by a decrease in the basal serum LH concentration (5). Our study did not determine how long basal serum gonadotropin levels will remain elevated after the 6 days of LHRH treatment or the effectiveness of administration of LHRH beyond 6 days on the maintenance of elevated basal serum gonadotropin concentrations on a chronic basis. Based on the observations that the in-
creased basal serum gonadotropin levels at 16 h after 6 days of treatment were associated with nearly a doubling of the number of gonadotrophs, we would predict that basal serum gonadotropin levels would remain elevated for some time without further administration of exogenous LHRH. Changes in serum LH and FSH concentrations in the present study were not associated with a change in APG weight or in the mean size of LH or FSH cells. These
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
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REPEATED STAINING OF SECTIONS WITH DIFFERENT ANTISERA
NUMERICAL DENSITY 3OOOO
LEGEND
25000 •
H I LH H I FSH
# •
25.
LEGEND
22.
H I LH —FSH H i FSH-LH
20. 2
20000
LLS/
o en o 15000 LLJ
10000
5OOO
I
.
•
1 •
WATER
••
17. 15. 12.
1
10. 7. 5
1
2 WATER
LHRH
FIG. 6. Effects of 6 days of injection of LHRH in water sc on mean (± SE) NV of LH and FSH cells. In water-injected rats 1440 cells stained for LH and 1286 cells stained for FSH were counted. In LHRH-treated rats, 2141 cells stained for LH and 2427 cells stained for FSH were counted. Each mean represents 5 rats. *, P < 0.05; **, P < 0.01 (compared to water-injected controls). PERCENTAGE OF LH 8c FSH CELLS IN THE ANTERIOR PITUITARY 12
LEGEND
m
LH FSH
9
CELL
00
* T
Li_
[RCEfvJTAGE
O
LJLJ
6
•
3•
0
WATER
Endo• 1990 Vol 126 • No 2
LHRH
FlG. 7. Effects of 6 days of injection of LHRH in water sc on mean (± SE) percentage of LH or FSH cells in the APG. Over 13,000 cells (stained and nonstained) were counted to determine the percentage of LH and FSH cells in the 5 rats in each group. *, P < 0.05 compared to water-injected controls.
data in association with the increases in both the Nv of LH and FSH cells and the percentage of APG cells containing LH or FSH indicate that LHRH-induced increased LH and FSH secretion was accompanied by an increase in the total number of APG gonadotrophs. The increase in gonadotrophs could have resulted from mitotic division of differentiated gonadotrophs, conversion of undifferentiated cells into gonadotrophs, or conversion of other differentiated cell types into gonadotrophs. It is not surprising that 6 days of treatment with LHRH in the present study did not increase the size of
LHRH
FlG. 8. Effects of 6 days of injection of water or LHRH in water sc on mean (±SE) new cells that stained after a second immunocytochemical stain was performed on the same tissue using a different antiserum. Sections were stained for LH and then for FSH (LH-FSH) or vice versa (FSH-LH). In water-treated rats, 557 cells initially stained for LH and 617 cells initially stained for FSH were counted. In LHRHtreated rats, 971 cells initially stained for LH and 977 cells initially stained for FSH were counted. Each mean represents 5 rats. •*, P < 0.05 compared to all other sequentially stained groups.
gonadotrophs. We have shown previously that LHRH plays a role in maintaining the normal size of gonadotrophs during development, but obtained no evidence to suggest that LHRH increases gonadotroph size above normal (9). We have also observed that orchidectomy increases gonadotroph size in the strain of rat used in the present study, but the hypertrophy was apparent 35, and not 7, days after castration (23). It is possible that more prolonged stimulation by LHRH using our protocol or a different regimen of LHRH administration might cause an increase in gonadotroph size. However, the putative release of testicular hormones after each bolus of LHRH may have precluded an effect of LHRH on gonadotroph size. Thus, studies designed to test the effects of LHRH on gonadotroph size will need a model in which the APG is exposed to the exogenous LHRH only and no testicular hormones. Whereas FSH was not detectable in all gonadotrophs in controls, virtually all gonadotrophs contained both FSH and LH after 6 days of treatment with LHRH. Sequential staining, first with FSH and then with LH, revealed new cells in control sections, but no statistically significant increase in stained cells was observed in sections from LHRH-treated rats. We have previously reported that LHRH is important for the maintenance of FSH in LH cells (10). Our data support the view that all gonadotrophs may potentially synthesize both FSH and LH and that exogenous LHRH combined with endogenous LHRH in the present study stimulates FSH synthesis in virtually all gonadotrophs. The effects of exog-
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
FIG. 9. These two electron micrographs are of typical cells that stained for LH/3. Upper panel, A polygonally shaped cell from an untreated rat with homogeneous cytoplasm and a polygonal, irregularly outlined nucleus. The cell contains a prominent Golgi zone. It is densely and uniformly granulated. A total of 1111 granules were counted, with 5 greater than 300 nm in diameter (X6050). Lower panel, A polygonal cell from a LHRH-treated rat with a homogeneous cytoplasm and a polygonal, irregularly outlined nucleus. Note the large prominent Golgi area. The cell is densely and uniformly granulated (1161 granules). It has 3 granules greater than 300 nm in diameter (X5650).
enous LHRH on endogenous LHRH release in the present study are not known. Correlation of the APG LH and FSH concentrations with the increases in gonadotroph cell numbers suggest that the average content of LH per cell decreased after LHRH treatment, whereas the average content of FSH per cell was better maintained after LHRH treatment. With the additional observation of increased basal levels of LH and FSH in serum after 6 days of LHRH treatment; one can conclude that, on the average, FSH cells were better able to maintain FSH stores while releasing FSH than LH cells were able to maintain LH stores
999
while releasing LH. Based on the observations that LHRH treatment increased LH cell number and was without effect on LH cell size and number of LH secretion granules, the data suggest that LH secretion granules contain less LH than those in LH cells of control rats. Previous studies have shown that the basal FSH release rate plays an important role in increasing the serum FSH concentration during the periovulatory FSH surge (12, 20, 21) and after ovariectomy (22). The increase in the basal serum FSH concentration after 6 days of LHRH treatment in the present study is probably due at least in part to an increase in the basal FSH release rate, as assessed in vitro. As the increase in FSH cell number after LHRH treatment was proportionately as great or greater than the increase in the basal FSH release rate, an increase in the basal secretion rate of FSH in vivo could, on the average, be due to an increase in the number of cells releasing FSH rather than to a higher basal FSH release rate per cell. In contrast, no evidence was obtained to suggest that the increase in the basal serum LH concentration was due to an increase in the basal LH release rate. The basal LH release rate in vitro did not increase. Coupled with the observation that LH cell number increased after LHRH treatment, it is likely that the basal LH release rate in vivo was reduced on a per cell basis. This is consistent with our suggestion that LHRH treatment did not alter secretion granule number per cell and that less LH was packaged per secretion granule. The ultrastructural observations revealed no other clues to elucidate the mechanisms of LHRH-induced gonadotroph cell number or increased gonadotropin secretion. In summary, the results of this study show for the first time that exogenous LHRH can be administered to increase the number of gonadotrophs. This appears to be an effective and efficient means to increase basal serum LH and FSH concentrations. Acknowledgments We thank Dr. Albert F. Parlow and the National Hormone and Pituitary Program for the antigens and antisera, Alison Rosenberg and Neda Osterman for assistance in making thefigures,and Carol A. Able for secretarial assistance. References 1. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally, AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334 2. Debeljuk L, Arimura A, Shiino M, Rennels EG, Schally AV 1973 Effects of chronic treatment with LH/FSH-RH in hypophysectomized pituitary-grafted male rats. Endocrinology 92:921 3. Johnson, DC, Mallampati RS 1975 Serum luteinizing hormone and follicle-stimulating hormone concentrations in immature female rats treated with multiple injections and various amounts of lu-
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LHRH EFFECTS ON LH AND FSH CELLS AND SECRETION
teinizing hormone releasing hormone. J Endocrinol 66:13 4. NcNeilly AS, deKretser DM, Sharpe RM 1979 Modulation of prolactin, luteinizing hormone (LH) and follicle stimulation hormone (FSH) secretion by LHRH and bromocriptine (CB154) in the hypophysectomized pituitary-grafted male rat and its effect on testicular LH receptors and testosterone output. Biol Reprod 21:141 5. Gregerson KA, Campbell GT 1984 Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster. Peptides 5:471 6. Campbell GT, Horacek MJ, Blake CA 1987 Effects of hypothalamic neurohormones on prolactin release from pituitary allografts in the hamster. Proc Soc Exp Biol Med 186:344 7. Dada MO, Campbell GT, Blake CA 1983 A quantitative immunocytochemical study on the luteinizing hormone and follicle-stimulating hormone cells in the adenohypophysis of adult male rats and of adult female rats throughout the estrous cycle. Endocrinology 113:970 8. Gregerson KA, Campbell GT 1982 Influences of luteinizing hormone releasing hormone, hypophysectomy, and orchidectomy on the differentiation of luteinizing hormone and follicle stimulating hormone cells in an ectopic pituitary in the hamster. Biol Reprod 27:169 9. Horacek MJ, Campbell GT, Blake CA 1989 Luteinizing hormone (LH)-releasing hormone: effects on induction of LH, follicle-stimulating hormone, and prolactin cell differentiation. Endocrinology 124:1800 10. Horacek MJ, Campbell GT, Blake CA 1990 Luteinizing hormone (LH)-Releasing Hormone: effects on maintenance of immunoreactive follicle- stimulating hormone and LH in adenohypophysial cells. Endocrinology 126:653 11. Garner LL, Campbell GT, Blake CA 1987 Gonadotroph number is increased by luteinizing hormone releasing hormone (LHRH). Fed Proc 46:1063 12. Elias KA, Kelch RP, Lipner H, Blake CA 1982 Relationships between basal gonadotropin secretion rates and serum gonadotro-
Endo• 1990 Voll26«No2
pin concentrations in proestrous rats. Biol Reprod 27:1159 13. Garner LL, Blake CA 1979 Morphological correlates for LHRH self priming and anterior pituitary gland refractoriness to LHRH in proestrous rats: an immunocytochemical study. Biol Reprod 20:1055 14. Stemberger LA, Hardy Jr PH, Cuculis JJ, Meyer HG 1970 The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody (horseradish peroxidase anti-peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315 15. Weibel ER 1979 Stereological Methods. Academic Press, London, vol 1:9 16. Dada MO, Blake CA 1985 Monosodium L-glutamate administration: effects on gonadotrophin secretion, gonadotrophs and mammotrophs in prepubertal female rats. J Endocrinol 104:185 17. Dada MO, Campbell GT, Blake CA 1984 Pars distalis cell quantification in normal adult male and female rats. J Endocrinol 101:87 18. Blake CA 1980 Correlative study of changes in the morphology of the LH gonadotroph and anterior pituitary gland LH secretion during the 4-day rat estrous cycle. Biol Reprod 23:1097 19. Garner LL, Blake CA 1981 Ultrastructural, immunocytochemical study of the LH secreting cell of the rat anterior pituitary gland: changes occurring after ovariectomy. Biol Reprod 24:461 20. Elias KA, Blake CA 1980 A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat. Life Sci 26:749 21. Elias KA, Blake CA 1981 A detailed in vitro characterization of the basal follicle-stimulating hormone and luteinizing hormone secretion rates during the rat four-day estrous cycle. Endocrinology 109:708 22. Elias KA, Blake CA 1983 Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat. Biol Reprod 28:1107 23. Dada MO, Blake CA 1986 A detailed morphometric study of gonadotrophs in male rats after removal of negative feedback on the hypothalamic-pituitary axis. Soc Neurosci Abstr 12:1413
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