0013-7227/90/1261-0653$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Luteinizing Hormone (LH)-Releasing Hormone: Effects on Maintenance of Immunoreactive Follicle-Stimulating Hormone and LH in Adenohypophysial Cells* MARK J. HORACEK, GARY T. CAMPBELL, AND CHARLES A. BLAKEt Department of Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208
ABSTRACT. We investigated the importance of LHRH on the maintenance of FSH and LH immunoreactivity in gonadotrophs. Hypophysectomized orchidectomized hamsters (hosts) each received an allograft of a 7-week-old male hamster pituitary gland beneath their right renal capsule. Starting 6 days after transplantation, hosts were injected sc, twice daily with 1 ng LHRH or vehicle for 16 days. Twelve hosts in each group were killed by decapitation 16 h after the last injection. Allografts from six of the hamsters in each group and pituitary glands in situ from 10-week-old normal males were prepared for histological examination. Sections of tissue were stained for FSH or LH and with hematoxylin. Allografts from the remaining hamsters were homogenized to measure FSH and LH concentrations. In allografts from the vehicle-treated hosts, 22.8% of adenohypophysial cells stained for LH, while only 16.9% stained for FSH. In allografts from LHRH-treated hosts, 22.6% and 23.8% of the adenohypophysial cells stained for LH and FSH, respectively.
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Adenohypophyses that developed for the same length of time in situ had 24.8% and 24.1% of the cells staining for LH and FSH, respectively. Matching of some of the FSH and LH cells in serial flip-flopped sections of tissue from all hamsters revealed that many if not all gonadotrophs contained LH. LH- and FSHcontaining cells in allografts were similar in size and shape, but were smaller and more circular in profile fhan those observed in situ. Treatment of hosts with LHRH did not alter gonadotroph size or shape, but it did reduce allograft LH concentration and elevate the serum FSH concentration compared to that in the vehicle-treated hamsters. These results suggest that in the hamster LHRH 1) plays a major role in maintaining FSH immunoreactiviry in adenohypophysial tissue, 2) does not play a role in maintaining numbers of immunoreactive LH cells in adult adenohypophysial tissue, and 3) functions to maintain FSH synthesis at least in part in cells that contain LH. (Endocrinology 126: 653-657, 1990)
physial cells. In the present study we assessed the importance of LHRH on the maintenance of numbers of immunoreactive FSH and LH cells in the hamster adenohypophysis.
E HAVE previously reported that in the hamster LHRH plays an important role in stimulating the formation of immunoreactive FSH in the developing pituitary gland (1, 2). FSH immunoreactivity was virtually absent in hypophysial tissue 3 weeks after transplantation of fetal or neonatal pituitary glands beneath the renal capsules of hypophysectomized orchidectomized hamsters unless LHRH was administered to the hosts. To date, evidence suggests that LHRH may not play a role in the appearance of immunoreactive LH during development (1-6). In contrast, less is known regarding the importance of LHRH in the maintenance of immunoreactive FSH and LH cells in the adenohypophysis. It has been observed that the number of FSH, but not LH, immunoreactive cells was markedly decreased in allografts of adult hypophysial tissue 8 weeks after grafting beneath the renal capsule of hypophysectomized orchidectomized hamsters (7). This suggests that some factor is necessary for the maintenance of FSH immunoreactivity in adenohypo-
Materials and Methods Animals Golden Syrian LVG hamsters to be employed as hosts were hypophysectomized and orchidectomized at 9 weeks of age and shipped 1 week later by Charles River Laboratories, Inc. (Wilmington, MA). The animals were housed individually in a room with controlled lighting (lights on, 0500-1900 h daily) and temperature (22-24 C). Food and 5% sucrose drinking water were constantly available. Three weeks after hypophysectomy and orchidectomy, hamsters were anesthetized with chloral hydrate (1.0 mg/100 g BW, ip) and exposure to ether fumes. The fur over the right flank was shaved, and the skin cleansed with 70% alcohol and betadine. A 1-cm incision was made in the skin and muscle dorsal to the right kidney. After gently exteriorizing the kidney, a pocket was created under the renal capsule by freeing the capsule from the underlying parenchymal tissue. Donor pituitary glands were removed from 7-week-old male hamsters that had been decapitated. One donor pituitary gland was placed in
Received August 17,1989. * This work was supported by a grant from the NIH (HD-22687). t To whom requests for reprints should be addressed. 653
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
the subcapsular pocket of the host, and the kidney was returned to its normal position. The muscle was closed with silk suture. The skin was closed with metal wound clips, and the animal was allowed to recuperate without any special postoperative care. Treatments were begun 6 days postgrafting. Treatment Twelve hosts were injected with LHRH (1 jug/injection in 100 fi\ vehicle) sc twice daily at approximately 0700 and 1900 h for 16 days. Another 12 hosts were injected with vehicle (0.1% gelatin in 0.9% saline) at these times. The LHRH was purchased from Peninsula Laboratories, Inc. (no. 7201, Belmont, CA). The hosts were decapitated 16 h after the last injection. No fragments of pituitary tissue were observed in the hypophysial fossa in any of the hosts. Trunk blood was collected, and serum was stored frozen at —70 C until assayed for LH and FSH concentrations. Six 10-week-old intact male hamsters also were decapitated to obtain normal pituitary glands. Immunocytochemical staining Allografts from six vehicle- and six LHRH-treated hosts and pituitary glands in situ from 10-week-old hamsters were fixed by immersion in Bouin's solution for 48 h. They then were dehydrated in graded ethanols, stored in 70% cedarwood oil30% absolute ethanol for 4 days, cleared in xylene, and embedded in Paraplast Plus. Horizontal 5-^m thick serial sections were cut through each entire allograft or gland. Flip-flopped serial sections of tissue from the dorsal, middle, and ventral portions of each gland or allograft were stained, one section for LH and the adjacent section for FSH. The avidin-biotin-peroxidase complex (ABC) method was used (8). The sections were deparaffinized in xylene, rehydrated, and washed in PBS (pH 7.4). Sections were incubated in a moisture chamber at 24 C with the following solutions in sequence for the times indicated: 1:100 nonimmune goat serum for 1 h; antirat LH S10, antirat FSH Sll, or nonimmune rabbit serum for 36 h; 1:100 nonimmune goat serum for 1 h; biotinylated antirabbit 7-globulin for 1 h, and ABC for 1 h. The ABC reagents were purchased from and prepared as described by Vector Laboratories (Burlingame, CA). The sections were washed with PBS after all incubations. Peroxidase activity was demonstrated by reaction with a fresh solution of 3,3'-diaminobenzidine (0.25 mg/ml) in Tris (hydroxymethyl)aminomethane buffer, pH 7.6. This solution was stirred for 20 min and then filtered. Immediately before use, hydrogen peroxide was added to a final concentration of 0.03%. Sections were incubated for 4 min in the staining solution, washed, counterstained with hematoxylin, and mounted. Immunocytochemical controls The antirat SlO and antirat FSH Sll were gifts from the NIDDK, the National Hormone and Pituitary Program, and the University of Maryland School of Medicine. The antisera were diluted further with a 1:100 normal goat serum solution in 0.05 M EDTA-PBS to 1:100. We previously validated these
Endo • 1990 Voll26«Nol
antisera for immunocytochemical staining (2, 9). Method controls were run as reported previously (10). Photography and morphometry Photographs were taken of three randomly selected areas from each of the three sections of each graft or pituitary gland in situ stained for each hormone. All cells with visible nuclei that stained for either LH or FSH were counted from the photographs using a hand counter. Nuclei of unstained cells in the same area also were counted. The percentage of anterior pituitary gland cells containing LH or FSH was then calculated. Approximately 600 immunoreactive LH and 600 immunoreactive FSH cells were counted from each animal. Measurements of LH and FSH cell profiles were made as described previously (11). Pituitary homogenations Allografts from six vehicle- and six LHRH-treated hosts were dissected free from surrounding tissue, weighed, homogenized in RIA buffer, and stored at -70 C. Homogenates were thawed and centrifuged before assay of the supernatant for LH and FSH concentrations. RIAs Serum and pituitary LH and FSH concentrations were assayed in a single assay for each hormone as described previously (2). Values are expressed in terms of rat LH RP-2 and FSH RP-2. Statistics Cell percentages and morphometric measurements from a single hamster were averaged. These averages were combined with those of other animals in the same group to obtain a mean ± SE. Statistical comparisons of LH data or FSH data were performed by one-way analysis of variance. Scheffe's test was performed when the analysis of variance indicated a significant difference. Statistical comparisons of LH us. FSH data within the same group were performed with a paired t test. P < 0.05 was considered statistically significant. Pituitary and serum hormone concentrations in the two groups of hamsters with grafts were compared by Student's t test.
Results Immunocytochemical method controls No staining was observed when we used nonimmune rabbit serum instead of the anti-LH or anti-FSH serum or when a component of the immunocytochemical stain was omitted. Cell percentages
No differences in the percentage of adenohypophysial cells containing LH were observed among the three
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
groups (Table 1). In contrast, the percentage of cells containing FSH was less in allografts of vehicle-treated hamsters than in anterior pituitary glands in situ. Injections of LHRH into hosts prevented the decrease in the percentage of FSH cells in the transplanted tissue. Representative micrographs of adenohypophysial tissue from the three groups are shown in Fig. 1. Morphometric analyses Both LH and FSH cells were smaller in the allografts than in the glands in situ, and treatment of hosts with LHRH did not alter gonadotroph size (Table 2). No differences between the size of LH and FSH cells were observed in any of the groups. Associated with the decrease in size of the gonadotrophs in transplanted tissue was an increase in the value of the shape factor of LH and FSH cells (Table 2), indicating a more circular shape. Treatment of hosts with LHRH did not alter the shape of gonadotrophs in allografts, and no differences in shape were observed between LH and FSH cells in any group. TABLE 1. Percentages of adenohypophysial cells containing LH or FSH in tissue in situ and in allografts beneath the renal capsule of hypophysectomized orchidectomized hosts treated with vehicle or 1 ng LHRH twice daily for 16 days
LH FSH
In situ
Vehicle-treated (graft)
LHRH-treated (graft)
24.8 ± 1.0 24.1 ± 1.4
22.8 ± 1.2 16.9 ± 1.8°
22.6 ± 1.0 23.8 ± 1.8
All values are the mean ± SE (n = 6/group).
" Significantly different from FSH values in the other two groups; significantly different from the LH value within the same group.
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Hormone concentrations Treatment of hosts with LHRH lowered the LH concentration, but was without effect on the FSH concentration in allografts, as observed 16 h after the last injection of LHRH (Table 3). Treatment of hosts with LHRH was without effect on serum LH concentrations, but caused a marked elevation in serum FSH concentrations, as observed 16 h after the last injection of LHRH (Table 4). Discussion The results of this study clearly indicate that LHRH plays a major role in maintaining FSH immunoreactivity in hamster adenohypophysial tissue. Transplantation of 7-week-old pituitary glands to a site distant from the hypothalamus and hypothalamic LHRH resulted in a decrease in the percentage of immunoreactive FSH cells within 22 days. This decrease was not a nonspecific effect associated with transplantation, because the percentage of LH cells did not change. Rather, the decrease can be attributed to inadequate stimulation of adenohypophysial tissue by LHRH, because administration of LHRH to hosts with pituitary gland allografts prevented the decrease in the percentage of FSH cells. It has been observed previously that there were about 9.6% immunoreactive FSH cells and 24.4% immunoreactive LH cells in allografts 8 weeks postgrafting (7). These observations and the results of the present study suggest that the number of gonadotrophs that synthesize
FSH decreases with time after cessation of adequate stimulation by LHRH. We chose to quantitate adeno-
FlG. 1. Representative sections of anterior pituitary gland tissue from male hamsters stained for antirat FSH S l l and counterstained with hematoxylin. Allografts of hypophysial tissue beneath the renal capsule of hypophysectomized orchidectomized hosts injected twice daily for 16 days with vehicle or LHRH are shown in A and B, respectively. Hypophysial tissue in situ is shown in C. Notice the diminished size of gonadotrophs in A and B and the reduced percentage of FSH-containing adenohypophysial cells in A. Bar = 20 nm. Magnification, X386.
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
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TABLE 2. Morphometric data for gonadotrophs in situ and in allografts in hosts treated with vehicle or LHRH
Area LH FSH Perimeter LH FSH
In situ
Vehicle-treated (graft)
LHRH-treated (graft)
139.5 ± 2.3 136.0 ± 3.0
84.6 ± 1.9° 83.1 ± 1.7°
87.8 ± 2.1° 89.2 ± 1.3°
45.2 ± 0.6 44.5 ± 0.4
34.5 ± 0.4° 34.4 ± 0.4"
35.0 ± 0.4" 35.4 ± 0.2°
Maximum diameter LH 11.8 ±0.1° 11.8 ± 0.2° 15.5 ± 0.2 12.0 ± 0.1° FSH 11.9 ±0.1° 15.3 ± 0.2 Shape LH 0.88 ± 0.01° 0.88 ± 0.01° 0.85 ± 0.01 FSH 0.87 ± 0.01° 0.88 ± 0.01° 0.85 ± 0.01 All values are the mean ± SE (n = 6/group). ° Significantly different from age-matched controls in situ. TABLE 3. Hormone concentrations (nanograms per mg tissue) in allografts of pituitary gland tissue in hosts treated with vehicle or LHRH
LH FSH
Vehicle-treated (graft)
LHRH-treated (graft)
350 ± 25 250 ± 31
242 ± 25° 266 ± 19
All values are the mean ± SE (n = 6/group). ° Significantly different from LH value in the vehicle-treated group. TABLE 4. Serum hormone concentrations (nanograms per ml) in hypophysectomized orchidectomized hamsters with allografts of pituitary glands
LH FSH
Vehicle-treated (graft)
LHRH-treated (graft)
0.15 ± 0.03 3.83 ± 0.31
0.11 ± 0.03 15.9 ± 1.38°
All values are the mean ± SE (n = 6/group). Hamsters were treated with vehicle or LHRH. 0 Significantly different from LH value in vehicle-treated group.
hypophysial cells 3 weeks after transplantation in the present study to simplify the experiment with respect to the length of time we administered LHRH. Thus, LHRH not only plays a critical role in the initial appearance of FSH immunoreactivity in the hamster anterior pituitary gland (1, 2), but it is important for maintaining immunoreactive FSH in hamster adenohypophysial tissue. LHRH treatment did not increase the FSH concentration in allografts even though there were about 41% more immunoreactive FSH cells in these grafts than in grafts in vehicle-treated hosts. However, LHRH did increase FSH release, because a greater than 4-fold increase in serum FSH levels was observed 16 h after the last injection of LHRH. These data collectively suggest that individual FSH cells in allografts of LHRHtreated hosts are synthesizing and releasing more FSH than FSH cells in allografts of vehicle-treated hosts. In
Endo • 1990 Vol 126 • No 1
a previous study, Gregerson and Campbell (12) obtained evidence to indicate that multiple injections of LHRH stimulate the hamster pituitary gland in situ to synthesize and release more FSH. Since the effects of LHRH on the number of FSH cells were not quantitated in that study (12), it is not possible to comment on FSH secretion by individual FSH cells. In this study serum FSH concentrations measured in blood collected 16 h after injection of LHRH are considered to represent basal values. We and others have observed previously that chronic treatment with LHRH elevates basal serum FSH levels in hamsters with pituitary glands in situ (12) or in hamsters (1, 13) or rats (14, 15) with allografts of adult pituitary glands beneath the renal capsule. We observed no evidence to suggest that LHRH plays a role in maintaining the percentage of LH cells within adenohypophysial tissue. The percentage of LH cells was not decreased in allografts in vehicle-treated hosts compared to the adenohypophysis in situ, and treatment of hosts with LHRH did not alter the percentage of LH cells. Although these data indicate that LHRH did not alter the percentage of LH cells, reduced LH concentrations in allografts in LHRH-treated hosts indicate that LHRH affected LH secretion. This probably reflects increased LH release at the expense of maintaining LH stores because LHRH causes an acute release of LH with serum LH concentrations returning to preinjection levels by 16 h after injection in hypophysectomized hamsters with pituitary gland allografts (12, 13). The decrease in the percentage of FSH cells in allografts of vehicle-treated hosts compared to that observed in glands in situ represents, at least in part, a decrease in FSH synthesis in cells containing immunoreactive LH. We did not conduct an exhaustive study to determine whether all of the FSH cells in both groups of grafts contained LH. However, we did match some of the FSH and LH cells in serial flip-flopped sections of adenohypophysial tissue in all hamsters and observed that the FSH cells did contain LH. Previously, we observed that many, if not all, of the small number of immunoreactive FSH cells that were present in allografts of neonatal hamster pituitaries contained LH as well (1, 2). Thus, all gonadotrophs may potentially synthesize both FSH and LH, but in the presence of little or no LHRH stimulation, the cells may synthesize and store primarily LH.
Recently, we observed that the size of gonadotrophs in allografts of fetal hamster pituitary glands transplanted beneath the renal capsule of adult hypophysectomized orchidectomized hosts was decreased compared to the size of gonadotrophs in situ. Gonadotrophs in allografts of hosts administered LHRH were increased in size compared to that observed in situ (2). These data suggest that LHRH may participate in regulating the size of
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY gonadotrophs during development. In the present study, which used adult pituitary glands for allografts, gonadotroph size also was decreased in the grafts of vehicletreated hosts. However, in contrast with the study using allografts of fetal glands, LHRH treatment did not increase gonadotroph size. This suggests that LHRH is more effective in regulating the size of gonadotrophs in glands in developing us. adult animals. Alternatively, the cells in allografts of adult tissue may not tolerate transplantation as well as the cells in allografts of glands of developing animals and, consequently, may not respond as well to LHRH. However, this is less likely because somatotrophs and corticotrophs in allografts of adult tissue were increased in size in response to GHRH and CRH, respectively (11,16), compared to the sizes of cells in vehicle-treated hosts. It also is of interest that gonadotroph size was reduced in allografts of 7-week-old transplanted pituitary glands in light of the fact that the hosts were orchidectomized. Castration is well known to increase the size of gonadotrophs in situ. We have observed an increase in gonadotroph size in rats 35 days, but not 7 days, after orchidectomy (17). It is possible that gonadotrophs in the allografts did not have sufficient time to increase in size in the present study. It also is possible that a particular frequency and amplitude of LHRH release occur after castration which are conducive to increasing gonadotroph size in situ and that our protocol for LHRH administration was ineffective in this regard. LHRH pulse frequency and amplitude are known to influence gonadotrophin secretion (18, 19). Alternatively, castration may alter other hypothalamic secretions, which, in turn, may alter gonadotroph size in the gland in situ. In summary, these data extend our previous observations that LHRH plays a major role in the initial appearance of immunoreactive FSH in cells in the developing pituitary gland (1, 2) to indicate that LHRH is also important for the retention of FSH immunoreactivity in gonadotrophs. This is in contrast to the development and maintenance of LH immunoreactivity in hamster anterior pituitary gland cells, which either do not require LHRH or do not require the amount of LHRH necessary for FSH synthesis and/or storage. Acknowledgments We thank Dr. Albert F. Parlow and the National Hormone and Pituitary Program for the antigens and antisera, Bonnie Livingston and Martha Steele for technical assistance, and Carol A. Able for secretarial assistance.
References 1. Gregerson KA, Campbell GT 1982 Influences of luteinizing hormone releasing hormone, hypophysectomy, and orchidectomy on
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the differentiation of luteinizing hormone and follicle stimulating hormone cells in an ectopic pituitary in the hamster. Biol Reprod 27:169 2. Horacek MJ, Campbell GT, Blake CA 1989 Luteinizing hormone (LH)- releasing hormone: Effects on induction of LH, folliclestimulating hormone, and prolactin cell differentiation. Endocrinology 124:1800 3. Watanabe YG 1987 Failure of luteinizing hormone-releasing hormone (LHRH) to affect the differentiation of LH cells in the rat hypophysial primordium in serum-free culture. Cell Tissue Res 250:35 4. Begeot M, Dupouy JP, Dubois MP, Dubois PM 1981 Immunocytological determination of gonadotropic and thyrotropic cells in fetal rat anterior pituitary during normal development and under experimental conditions. Neuroendocrinology 32:285 5. Gash D, Ahmad N, Schechter J 1982 Comparison of gonadotroph, thyrotroph and mammotroph development in situ, in transplants and in organ culture. Neuroendocrinology 34:222 6. Nemeskeri A, Halasz B, Kurcz M 1983 Ontogenesis of the rat hypothalamo-adenohypophyseal system and inherent capacity of the fetal pituitary to differentiate into hormone-synthesizing and releasing cells. In: Bhatnagar AS (ed) The Anterior Pituitary Gland. Raven Press, New York, p 341 7. Campbell GT, Wagoner J, Colosi P, Soares MJ, Talamantes F 1988 Development and retention of phenotypically specialized cells in pituitary allografts in the hamster (Mesocricetus auratus). Cell Tissue Res 251:215 8. Hsu S-M, Raine L, Fanger H 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577 9. Campbell GT, Kohn JD, Dada MO, Blake CA 1987 An immunohistochemical study of adenohypophysial cells containing folliclestimulating hormone and luteinizing hormone during the phase of selective follicle-stimulating hormone release in postnatal female rats. Cell Tissue Res 250:689 10. 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 11. Horacek MJ, Campbell GT, Blake CA 1988 Effects of growth hormone releasing hormone on somatotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters. Cell Tissue Res 253:287 12. Gregerson KA, Campbell GT 1984 Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster. Peptides 5:471 13. 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 14. 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 15. McNeilly 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 16. Horacek MJ, Campbell GT, Blake CA, 1989 Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters. Cell Tissue Res, 258:65 17. 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 18. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631 19. Wise PM, Ranee N, Barra GD, Barraclough CA 1979 Further evidence that LHRH also is FSH-RH. Endocrinology 104:940
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Vol. 126, No. 1 Printed in U.S.A.
Luteinizing Hormone (LH)-Releasing Hormone: Effects on Maintenance of Immunoreactive Follicle-Stimulating Hormone and LH in Adenohypophysial Cells* MARK J. HORACEK, GARY T. CAMPBELL, AND CHARLES A. BLAKEt Department of Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208
ABSTRACT. We investigated the importance of LHRH on the maintenance of FSH and LH immunoreactivity in gonadotrophs. Hypophysectomized orchidectomized hamsters (hosts) each received an allograft of a 7-week-old male hamster pituitary gland beneath their right renal capsule. Starting 6 days after transplantation, hosts were injected sc, twice daily with 1 ng LHRH or vehicle for 16 days. Twelve hosts in each group were killed by decapitation 16 h after the last injection. Allografts from six of the hamsters in each group and pituitary glands in situ from 10-week-old normal males were prepared for histological examination. Sections of tissue were stained for FSH or LH and with hematoxylin. Allografts from the remaining hamsters were homogenized to measure FSH and LH concentrations. In allografts from the vehicle-treated hosts, 22.8% of adenohypophysial cells stained for LH, while only 16.9% stained for FSH. In allografts from LHRH-treated hosts, 22.6% and 23.8% of the adenohypophysial cells stained for LH and FSH, respectively.
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Adenohypophyses that developed for the same length of time in situ had 24.8% and 24.1% of the cells staining for LH and FSH, respectively. Matching of some of the FSH and LH cells in serial flip-flopped sections of tissue from all hamsters revealed that many if not all gonadotrophs contained LH. LH- and FSHcontaining cells in allografts were similar in size and shape, but were smaller and more circular in profile fhan those observed in situ. Treatment of hosts with LHRH did not alter gonadotroph size or shape, but it did reduce allograft LH concentration and elevate the serum FSH concentration compared to that in the vehicle-treated hamsters. These results suggest that in the hamster LHRH 1) plays a major role in maintaining FSH immunoreactiviry in adenohypophysial tissue, 2) does not play a role in maintaining numbers of immunoreactive LH cells in adult adenohypophysial tissue, and 3) functions to maintain FSH synthesis at least in part in cells that contain LH. (Endocrinology 126: 653-657, 1990)
physial cells. In the present study we assessed the importance of LHRH on the maintenance of numbers of immunoreactive FSH and LH cells in the hamster adenohypophysis.
E HAVE previously reported that in the hamster LHRH plays an important role in stimulating the formation of immunoreactive FSH in the developing pituitary gland (1, 2). FSH immunoreactivity was virtually absent in hypophysial tissue 3 weeks after transplantation of fetal or neonatal pituitary glands beneath the renal capsules of hypophysectomized orchidectomized hamsters unless LHRH was administered to the hosts. To date, evidence suggests that LHRH may not play a role in the appearance of immunoreactive LH during development (1-6). In contrast, less is known regarding the importance of LHRH in the maintenance of immunoreactive FSH and LH cells in the adenohypophysis. It has been observed that the number of FSH, but not LH, immunoreactive cells was markedly decreased in allografts of adult hypophysial tissue 8 weeks after grafting beneath the renal capsule of hypophysectomized orchidectomized hamsters (7). This suggests that some factor is necessary for the maintenance of FSH immunoreactivity in adenohypo-
Materials and Methods Animals Golden Syrian LVG hamsters to be employed as hosts were hypophysectomized and orchidectomized at 9 weeks of age and shipped 1 week later by Charles River Laboratories, Inc. (Wilmington, MA). The animals were housed individually in a room with controlled lighting (lights on, 0500-1900 h daily) and temperature (22-24 C). Food and 5% sucrose drinking water were constantly available. Three weeks after hypophysectomy and orchidectomy, hamsters were anesthetized with chloral hydrate (1.0 mg/100 g BW, ip) and exposure to ether fumes. The fur over the right flank was shaved, and the skin cleansed with 70% alcohol and betadine. A 1-cm incision was made in the skin and muscle dorsal to the right kidney. After gently exteriorizing the kidney, a pocket was created under the renal capsule by freeing the capsule from the underlying parenchymal tissue. Donor pituitary glands were removed from 7-week-old male hamsters that had been decapitated. One donor pituitary gland was placed in
Received August 17,1989. * This work was supported by a grant from the NIH (HD-22687). t To whom requests for reprints should be addressed. 653
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654
LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
the subcapsular pocket of the host, and the kidney was returned to its normal position. The muscle was closed with silk suture. The skin was closed with metal wound clips, and the animal was allowed to recuperate without any special postoperative care. Treatments were begun 6 days postgrafting. Treatment Twelve hosts were injected with LHRH (1 jug/injection in 100 fi\ vehicle) sc twice daily at approximately 0700 and 1900 h for 16 days. Another 12 hosts were injected with vehicle (0.1% gelatin in 0.9% saline) at these times. The LHRH was purchased from Peninsula Laboratories, Inc. (no. 7201, Belmont, CA). The hosts were decapitated 16 h after the last injection. No fragments of pituitary tissue were observed in the hypophysial fossa in any of the hosts. Trunk blood was collected, and serum was stored frozen at —70 C until assayed for LH and FSH concentrations. Six 10-week-old intact male hamsters also were decapitated to obtain normal pituitary glands. Immunocytochemical staining Allografts from six vehicle- and six LHRH-treated hosts and pituitary glands in situ from 10-week-old hamsters were fixed by immersion in Bouin's solution for 48 h. They then were dehydrated in graded ethanols, stored in 70% cedarwood oil30% absolute ethanol for 4 days, cleared in xylene, and embedded in Paraplast Plus. Horizontal 5-^m thick serial sections were cut through each entire allograft or gland. Flip-flopped serial sections of tissue from the dorsal, middle, and ventral portions of each gland or allograft were stained, one section for LH and the adjacent section for FSH. The avidin-biotin-peroxidase complex (ABC) method was used (8). The sections were deparaffinized in xylene, rehydrated, and washed in PBS (pH 7.4). Sections were incubated in a moisture chamber at 24 C with the following solutions in sequence for the times indicated: 1:100 nonimmune goat serum for 1 h; antirat LH S10, antirat FSH Sll, or nonimmune rabbit serum for 36 h; 1:100 nonimmune goat serum for 1 h; biotinylated antirabbit 7-globulin for 1 h, and ABC for 1 h. The ABC reagents were purchased from and prepared as described by Vector Laboratories (Burlingame, CA). The sections were washed with PBS after all incubations. Peroxidase activity was demonstrated by reaction with a fresh solution of 3,3'-diaminobenzidine (0.25 mg/ml) in Tris (hydroxymethyl)aminomethane buffer, pH 7.6. This solution was stirred for 20 min and then filtered. Immediately before use, hydrogen peroxide was added to a final concentration of 0.03%. Sections were incubated for 4 min in the staining solution, washed, counterstained with hematoxylin, and mounted. Immunocytochemical controls The antirat SlO and antirat FSH Sll were gifts from the NIDDK, the National Hormone and Pituitary Program, and the University of Maryland School of Medicine. The antisera were diluted further with a 1:100 normal goat serum solution in 0.05 M EDTA-PBS to 1:100. We previously validated these
Endo • 1990 Voll26«Nol
antisera for immunocytochemical staining (2, 9). Method controls were run as reported previously (10). Photography and morphometry Photographs were taken of three randomly selected areas from each of the three sections of each graft or pituitary gland in situ stained for each hormone. All cells with visible nuclei that stained for either LH or FSH were counted from the photographs using a hand counter. Nuclei of unstained cells in the same area also were counted. The percentage of anterior pituitary gland cells containing LH or FSH was then calculated. Approximately 600 immunoreactive LH and 600 immunoreactive FSH cells were counted from each animal. Measurements of LH and FSH cell profiles were made as described previously (11). Pituitary homogenations Allografts from six vehicle- and six LHRH-treated hosts were dissected free from surrounding tissue, weighed, homogenized in RIA buffer, and stored at -70 C. Homogenates were thawed and centrifuged before assay of the supernatant for LH and FSH concentrations. RIAs Serum and pituitary LH and FSH concentrations were assayed in a single assay for each hormone as described previously (2). Values are expressed in terms of rat LH RP-2 and FSH RP-2. Statistics Cell percentages and morphometric measurements from a single hamster were averaged. These averages were combined with those of other animals in the same group to obtain a mean ± SE. Statistical comparisons of LH data or FSH data were performed by one-way analysis of variance. Scheffe's test was performed when the analysis of variance indicated a significant difference. Statistical comparisons of LH us. FSH data within the same group were performed with a paired t test. P < 0.05 was considered statistically significant. Pituitary and serum hormone concentrations in the two groups of hamsters with grafts were compared by Student's t test.
Results Immunocytochemical method controls No staining was observed when we used nonimmune rabbit serum instead of the anti-LH or anti-FSH serum or when a component of the immunocytochemical stain was omitted. Cell percentages
No differences in the percentage of adenohypophysial cells containing LH were observed among the three
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
groups (Table 1). In contrast, the percentage of cells containing FSH was less in allografts of vehicle-treated hamsters than in anterior pituitary glands in situ. Injections of LHRH into hosts prevented the decrease in the percentage of FSH cells in the transplanted tissue. Representative micrographs of adenohypophysial tissue from the three groups are shown in Fig. 1. Morphometric analyses Both LH and FSH cells were smaller in the allografts than in the glands in situ, and treatment of hosts with LHRH did not alter gonadotroph size (Table 2). No differences between the size of LH and FSH cells were observed in any of the groups. Associated with the decrease in size of the gonadotrophs in transplanted tissue was an increase in the value of the shape factor of LH and FSH cells (Table 2), indicating a more circular shape. Treatment of hosts with LHRH did not alter the shape of gonadotrophs in allografts, and no differences in shape were observed between LH and FSH cells in any group. TABLE 1. Percentages of adenohypophysial cells containing LH or FSH in tissue in situ and in allografts beneath the renal capsule of hypophysectomized orchidectomized hosts treated with vehicle or 1 ng LHRH twice daily for 16 days
LH FSH
In situ
Vehicle-treated (graft)
LHRH-treated (graft)
24.8 ± 1.0 24.1 ± 1.4
22.8 ± 1.2 16.9 ± 1.8°
22.6 ± 1.0 23.8 ± 1.8
All values are the mean ± SE (n = 6/group).
" Significantly different from FSH values in the other two groups; significantly different from the LH value within the same group.
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Hormone concentrations Treatment of hosts with LHRH lowered the LH concentration, but was without effect on the FSH concentration in allografts, as observed 16 h after the last injection of LHRH (Table 3). Treatment of hosts with LHRH was without effect on serum LH concentrations, but caused a marked elevation in serum FSH concentrations, as observed 16 h after the last injection of LHRH (Table 4). Discussion The results of this study clearly indicate that LHRH plays a major role in maintaining FSH immunoreactivity in hamster adenohypophysial tissue. Transplantation of 7-week-old pituitary glands to a site distant from the hypothalamus and hypothalamic LHRH resulted in a decrease in the percentage of immunoreactive FSH cells within 22 days. This decrease was not a nonspecific effect associated with transplantation, because the percentage of LH cells did not change. Rather, the decrease can be attributed to inadequate stimulation of adenohypophysial tissue by LHRH, because administration of LHRH to hosts with pituitary gland allografts prevented the decrease in the percentage of FSH cells. It has been observed previously that there were about 9.6% immunoreactive FSH cells and 24.4% immunoreactive LH cells in allografts 8 weeks postgrafting (7). These observations and the results of the present study suggest that the number of gonadotrophs that synthesize
FSH decreases with time after cessation of adequate stimulation by LHRH. We chose to quantitate adeno-
FlG. 1. Representative sections of anterior pituitary gland tissue from male hamsters stained for antirat FSH S l l and counterstained with hematoxylin. Allografts of hypophysial tissue beneath the renal capsule of hypophysectomized orchidectomized hosts injected twice daily for 16 days with vehicle or LHRH are shown in A and B, respectively. Hypophysial tissue in situ is shown in C. Notice the diminished size of gonadotrophs in A and B and the reduced percentage of FSH-containing adenohypophysial cells in A. Bar = 20 nm. Magnification, X386.
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY
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TABLE 2. Morphometric data for gonadotrophs in situ and in allografts in hosts treated with vehicle or LHRH
Area LH FSH Perimeter LH FSH
In situ
Vehicle-treated (graft)
LHRH-treated (graft)
139.5 ± 2.3 136.0 ± 3.0
84.6 ± 1.9° 83.1 ± 1.7°
87.8 ± 2.1° 89.2 ± 1.3°
45.2 ± 0.6 44.5 ± 0.4
34.5 ± 0.4° 34.4 ± 0.4"
35.0 ± 0.4" 35.4 ± 0.2°
Maximum diameter LH 11.8 ±0.1° 11.8 ± 0.2° 15.5 ± 0.2 12.0 ± 0.1° FSH 11.9 ±0.1° 15.3 ± 0.2 Shape LH 0.88 ± 0.01° 0.88 ± 0.01° 0.85 ± 0.01 FSH 0.87 ± 0.01° 0.88 ± 0.01° 0.85 ± 0.01 All values are the mean ± SE (n = 6/group). ° Significantly different from age-matched controls in situ. TABLE 3. Hormone concentrations (nanograms per mg tissue) in allografts of pituitary gland tissue in hosts treated with vehicle or LHRH
LH FSH
Vehicle-treated (graft)
LHRH-treated (graft)
350 ± 25 250 ± 31
242 ± 25° 266 ± 19
All values are the mean ± SE (n = 6/group). ° Significantly different from LH value in the vehicle-treated group. TABLE 4. Serum hormone concentrations (nanograms per ml) in hypophysectomized orchidectomized hamsters with allografts of pituitary glands
LH FSH
Vehicle-treated (graft)
LHRH-treated (graft)
0.15 ± 0.03 3.83 ± 0.31
0.11 ± 0.03 15.9 ± 1.38°
All values are the mean ± SE (n = 6/group). Hamsters were treated with vehicle or LHRH. 0 Significantly different from LH value in vehicle-treated group.
hypophysial cells 3 weeks after transplantation in the present study to simplify the experiment with respect to the length of time we administered LHRH. Thus, LHRH not only plays a critical role in the initial appearance of FSH immunoreactivity in the hamster anterior pituitary gland (1, 2), but it is important for maintaining immunoreactive FSH in hamster adenohypophysial tissue. LHRH treatment did not increase the FSH concentration in allografts even though there were about 41% more immunoreactive FSH cells in these grafts than in grafts in vehicle-treated hosts. However, LHRH did increase FSH release, because a greater than 4-fold increase in serum FSH levels was observed 16 h after the last injection of LHRH. These data collectively suggest that individual FSH cells in allografts of LHRHtreated hosts are synthesizing and releasing more FSH than FSH cells in allografts of vehicle-treated hosts. In
Endo • 1990 Vol 126 • No 1
a previous study, Gregerson and Campbell (12) obtained evidence to indicate that multiple injections of LHRH stimulate the hamster pituitary gland in situ to synthesize and release more FSH. Since the effects of LHRH on the number of FSH cells were not quantitated in that study (12), it is not possible to comment on FSH secretion by individual FSH cells. In this study serum FSH concentrations measured in blood collected 16 h after injection of LHRH are considered to represent basal values. We and others have observed previously that chronic treatment with LHRH elevates basal serum FSH levels in hamsters with pituitary glands in situ (12) or in hamsters (1, 13) or rats (14, 15) with allografts of adult pituitary glands beneath the renal capsule. We observed no evidence to suggest that LHRH plays a role in maintaining the percentage of LH cells within adenohypophysial tissue. The percentage of LH cells was not decreased in allografts in vehicle-treated hosts compared to the adenohypophysis in situ, and treatment of hosts with LHRH did not alter the percentage of LH cells. Although these data indicate that LHRH did not alter the percentage of LH cells, reduced LH concentrations in allografts in LHRH-treated hosts indicate that LHRH affected LH secretion. This probably reflects increased LH release at the expense of maintaining LH stores because LHRH causes an acute release of LH with serum LH concentrations returning to preinjection levels by 16 h after injection in hypophysectomized hamsters with pituitary gland allografts (12, 13). The decrease in the percentage of FSH cells in allografts of vehicle-treated hosts compared to that observed in glands in situ represents, at least in part, a decrease in FSH synthesis in cells containing immunoreactive LH. We did not conduct an exhaustive study to determine whether all of the FSH cells in both groups of grafts contained LH. However, we did match some of the FSH and LH cells in serial flip-flopped sections of adenohypophysial tissue in all hamsters and observed that the FSH cells did contain LH. Previously, we observed that many, if not all, of the small number of immunoreactive FSH cells that were present in allografts of neonatal hamster pituitaries contained LH as well (1, 2). Thus, all gonadotrophs may potentially synthesize both FSH and LH, but in the presence of little or no LHRH stimulation, the cells may synthesize and store primarily LH.
Recently, we observed that the size of gonadotrophs in allografts of fetal hamster pituitary glands transplanted beneath the renal capsule of adult hypophysectomized orchidectomized hosts was decreased compared to the size of gonadotrophs in situ. Gonadotrophs in allografts of hosts administered LHRH were increased in size compared to that observed in situ (2). These data suggest that LHRH may participate in regulating the size of
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LHRH AND MAINTENANCE OF LH AND FSH IMMUNOREACTIVITY gonadotrophs during development. In the present study, which used adult pituitary glands for allografts, gonadotroph size also was decreased in the grafts of vehicletreated hosts. However, in contrast with the study using allografts of fetal glands, LHRH treatment did not increase gonadotroph size. This suggests that LHRH is more effective in regulating the size of gonadotrophs in glands in developing us. adult animals. Alternatively, the cells in allografts of adult tissue may not tolerate transplantation as well as the cells in allografts of glands of developing animals and, consequently, may not respond as well to LHRH. However, this is less likely because somatotrophs and corticotrophs in allografts of adult tissue were increased in size in response to GHRH and CRH, respectively (11,16), compared to the sizes of cells in vehicle-treated hosts. It also is of interest that gonadotroph size was reduced in allografts of 7-week-old transplanted pituitary glands in light of the fact that the hosts were orchidectomized. Castration is well known to increase the size of gonadotrophs in situ. We have observed an increase in gonadotroph size in rats 35 days, but not 7 days, after orchidectomy (17). It is possible that gonadotrophs in the allografts did not have sufficient time to increase in size in the present study. It also is possible that a particular frequency and amplitude of LHRH release occur after castration which are conducive to increasing gonadotroph size in situ and that our protocol for LHRH administration was ineffective in this regard. LHRH pulse frequency and amplitude are known to influence gonadotrophin secretion (18, 19). Alternatively, castration may alter other hypothalamic secretions, which, in turn, may alter gonadotroph size in the gland in situ. In summary, these data extend our previous observations that LHRH plays a major role in the initial appearance of immunoreactive FSH in cells in the developing pituitary gland (1, 2) to indicate that LHRH is also important for the retention of FSH immunoreactivity in gonadotrophs. This is in contrast to the development and maintenance of LH immunoreactivity in hamster anterior pituitary gland cells, which either do not require LHRH or do not require the amount of LHRH necessary for FSH synthesis and/or storage. Acknowledgments We thank Dr. Albert F. Parlow and the National Hormone and Pituitary Program for the antigens and antisera, Bonnie Livingston and Martha Steele for technical assistance, and Carol A. Able for secretarial assistance.
References 1. Gregerson KA, Campbell GT 1982 Influences of luteinizing hormone releasing hormone, hypophysectomy, and orchidectomy on
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the differentiation of luteinizing hormone and follicle stimulating hormone cells in an ectopic pituitary in the hamster. Biol Reprod 27:169 2. Horacek MJ, Campbell GT, Blake CA 1989 Luteinizing hormone (LH)- releasing hormone: Effects on induction of LH, folliclestimulating hormone, and prolactin cell differentiation. Endocrinology 124:1800 3. Watanabe YG 1987 Failure of luteinizing hormone-releasing hormone (LHRH) to affect the differentiation of LH cells in the rat hypophysial primordium in serum-free culture. Cell Tissue Res 250:35 4. Begeot M, Dupouy JP, Dubois MP, Dubois PM 1981 Immunocytological determination of gonadotropic and thyrotropic cells in fetal rat anterior pituitary during normal development and under experimental conditions. Neuroendocrinology 32:285 5. Gash D, Ahmad N, Schechter J 1982 Comparison of gonadotroph, thyrotroph and mammotroph development in situ, in transplants and in organ culture. Neuroendocrinology 34:222 6. Nemeskeri A, Halasz B, Kurcz M 1983 Ontogenesis of the rat hypothalamo-adenohypophyseal system and inherent capacity of the fetal pituitary to differentiate into hormone-synthesizing and releasing cells. In: Bhatnagar AS (ed) The Anterior Pituitary Gland. Raven Press, New York, p 341 7. Campbell GT, Wagoner J, Colosi P, Soares MJ, Talamantes F 1988 Development and retention of phenotypically specialized cells in pituitary allografts in the hamster (Mesocricetus auratus). Cell Tissue Res 251:215 8. Hsu S-M, Raine L, Fanger H 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577 9. Campbell GT, Kohn JD, Dada MO, Blake CA 1987 An immunohistochemical study of adenohypophysial cells containing folliclestimulating hormone and luteinizing hormone during the phase of selective follicle-stimulating hormone release in postnatal female rats. Cell Tissue Res 250:689 10. 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 11. Horacek MJ, Campbell GT, Blake CA 1988 Effects of growth hormone releasing hormone on somatotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters. Cell Tissue Res 253:287 12. Gregerson KA, Campbell GT 1984 Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster. Peptides 5:471 13. 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 14. 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 15. McNeilly 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 16. Horacek MJ, Campbell GT, Blake CA, 1989 Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters. Cell Tissue Res, 258:65 17. 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 18. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631 19. Wise PM, Ranee N, Barra GD, Barraclough CA 1979 Further evidence that LHRH also is FSH-RH. Endocrinology 104:940
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