0013-7227/78/1026-1815$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 6 Printed in U.S.A.
Effect of Luteinizing Hormone-Releasing Hormone on the Secretion of Luteinizing Hormone, Follicle-Stimulating Hormone, and Testosterone in Adult Male Rhesus Monkeys* PERTTI T. K. TOIVOLA, WILLIAM E. BRIDSON, AND JERRY A. ROBINSON Wisconsin Regional Primate Research Center and Departments of Physiology and Medicine, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT. Plasma levels of radioimmunoreactive LH, FSH, and testosterone (T) were assayed before and after administration of synthetic gonadotropin-releasing hormone (GnRH) to five chair-restrained rhesus monkeys with chronic indwelling venous catheters. Intravenous injection of 1, 5, and 25 fig or infusion of 1 /ig/min for 25 min of GnRH resulted in a significant increase in plasma levels of LH. However, no significant increases in plasma FSH levels were detected. Plasma levels of T
T
HE DEMONSTRATION of a LH-releasing factor in hypothalamic tissue (1) precipitated an intensive search that culminated in the isolation of a decapeptide from porcine hypothalami which possessed LH-releasing activity (2, 3). Subsequently, the structures of porcine and ovine LH-releasing peptides were determined and found to be identical (3, 4). The relative ease of synthesizing large quantities of this peptide (5) has made its use possible in various physiological studies. In addition to promoting the secretion of LH, this synthetic material also possesses FSH-releasing activity, a finding which prompted the use of the terms gonadotropinreleasing hormone (GnRH) and LH/FSH-RH (6). Although the effects of synthetic GnRH have been extensively studied in females of many species, less emphasis has been directed toward its effects in the male. Kastin et al. (7) showed that administration of both native and Received August 18, 1977. Address all correspondence and requests for reprints to: Dr. P. T. K. Toivola, Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53706. * Publication 17-026 of the Wisconsin Regional Primate Research Center. This work was supported by NIH Grant RR-00167 to the Wisconsin Regional Primate Research Center and NIMH Grant MH-21312.
were also elevated after administration of 25 fig GnRH, but peak concentration of T lagged behind peak levels of LH by approximately 30 min. These studies indicate that the male rhesus responds to GnRH administration by increased secretion of LH, followed by an increase in T levels. A concomitant increase in plasma FSH was not observed after treatment with GnRH in the doses used. (Endocrinology 102: 1815, 1978)
synthetic GnRH promoted LH and FSH secretion in normal men; but, the synthetic material was much less effective in stimulating the secretion of FSH. Likewise, Yen et al. (8) have shown that administration of GnRH to normal men results in a marked increase of LH secretion but only a small increase in the levels of FSH. Continuous iv infusion of GnRH for 4 h in adult men resulted in a biphasic release of LH and again, relatively small changes in the plasma levels of FSH (9). Thus, it would seem that the GnRH in human males has a minimal effect on FSH secretion in adult human males. The effects of synthetic GnRH administration in the female rhesus monkey have been studied by Krey et al. (10) and Spies et al. (11) in a number of experimental situations, and in most cases was found to be an effective stimulus for LH secretion. The present report describes the effects of GnRH administration on LH, FSH, and testosterone secretion in normal adult male rhesus monkeys. Materials and Methods Five adult male rhesus (Macaca mulatto), weighing 6.5-9.5 kg, were used as experimental subjects. All monkeys were adapted to chronic re-
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TOIVOLA, BRIDSON, AND ROBINSON
straint in a primate chair for a minimum of 2 weeks, after which they were aseptically operated upon under general anesthesia for placement of a Silastic catheter (0.030 inches id x 0.065 inches od, Dow Corning) via the internal jugular vein into the right atrium. Postoperatively, the patency of the catheter was maintained by constant infusion of heparinized saline (10 U/ml, 24 ml/day) via a pulsatile pump. All subjects were routinely maintained on a 12-h light-12-h dark cycle with lights on from 0600-1800 h. Purina monkey chow supplemented with fresh fruits was fed once daily between 0730 and 0800 h and was available ad libitum, as was fresh water. During experimental sessions, two monkeys were housed together in an acoustically attenuated environmental chamber equipped with a white noise generator and a closed circuit video monitor. Catheters were passed through a port hole in the chamber wall which permitted blood sampling and drug administration from outside the chamber, obviating interaction with the monkey. Blood samples (3 ml) were drawn through the jugular catheter into sterile syringes, immediately placed into sterile heparinized centrifuge tubes, and centrifuged at 2500 rpm for 7 min. The plasma was aspirated with sterile pipettes and stored frozen at — 20 C until hormone assays could be performed. The packed red blood cells were resuspended in sterile isotonic saline and returned to the subjects immediately after withdrawal of the subsequent blood sample. Blood samples were collected at 15-min intervals for 60 min before and 180 min after the administration of GnRH. Synthetic GnRH (amide form; lot 21-103-DH; obtained from the Hormone Distribution Branch, NIAMDD, NIH) was diluted in sterile, pyrogenfree saline from the stock solution (100 jug/ml) to a final concentration of 1, 5, or 25 /xg/ml just before administration. Each of the five monkeys was given either 1 ml saline or 1 or 25 iig GnRH in 1 ml saline; four of these monkeys were also given 5 jag GnRH in 1 ml saline. To prolong the effects of the administered drug, 25 ug GnRH were also infused at the rate of 1 jug/min (1 ml/min) in all five monkeys, using a Harvard infusion pump and a precalibrated syringe. The order of administration of these doses was randomized. All experiments commenced at 1000 h with the drawing of the first blood sample. The administration of either saline as a control or one dose of GnRH constituted an experiment and 7 days were allowed to elapse between successive experiments in individual monkeys. The RIA of testosterone in the male rhesus monkey has previously been described in detail (12). After extraction and column chromatography, sam-
Kiulo 1978 Vol 102 . No 6
pie recoveries of 76 ± 9.1% (SD) were obtained. The blank values for these assays were 1.7 ± 3.82 (SD) pg, resulting in an assay sensitivity of less than 10 pg. Within-assay precision and between-assay variability were found to be ± 9.4 and ± 14.2 (SD) pg, respectively. All samples were analyzed in duplicate utilizing 40-jul quantities of plasma. RIAs for protein hormones were performed utilizing l25I-labeled tracer, phosphosaline buffers (13), and double antibody precipitation for separation of free and bound hormones. Assay results were calculated by the use of statistical model II of Rodbard (14) and microprocessor programs developed in our laboratory. The influence of between-assay variance was minimized by including all sequential serum samples from a given subject in the same assay. Rhesus FSH was estimated utilizing methods and materials described by Hodgen et al. (13). Results are expressed as nanograms per ml in plasma of rhesus FSH reference preparation WDPX1-2-A [reported immunological activity of 1 mg equivalent to 1.02 mg NIH-FSH-Sl and biological activity of 1 mg equivalent to 1.10 mg of NIH-FSHSl (15)]. The immunological activity of 1 jug this preparation was equivalent to 20.1 /xg LER-1909-2 (rhesus gonadotrophin reference preparation) in our laboratory. All samples were assayed in duplicate using 200 ju.1 serum. The coefficient of variation for samples was 9.8% (mean concentration, 454.2 ng/ml). Assay sensitivity for reliable measures was 70 ng/tube (350 ng/ml serum, utilizing a 200-/xl sample.) Rabbit antiovine LH antibody (GDN-15), rat LH tracer, and rhesus LH reference preparation WDPX-81-1720 were utilized in a double antibody assay for rhesus LH. General unavailability of highly purified rhesus LH for radioiodination prompted the use of rat 125I-labeled LH (NIAMDD-rLH-I-3) as tracer, as has been previously described for a number of species (16). The biological potency of 1 mg WDP-X-81-1720 is equivalent to 2.90 mg NIHLS-S1 (15). In our laboratory, 1 ng WDP-X-81-1720 is, by RIA equivalent to 3.85 jug LER-1909-2. In this assay, the minimal detectable dose is 0.35 ng/tube. The between-assay coefficients of variation (CV) for LH standard concentrations of 6.15, 13.1, and 31.4 ng/ml were, respectively, 14.26, 12.15, and 11.44%, with 8.50, 6.31, and 6.47% being the respective CV for within-assay variability. Samples were analyzed utilizing duplicate 100-jwl quantities. Although this LH antibody has been reported (17) to be nonspecific for measurements of rhesus LH, it is the only generally available radioimmunological technique for estimating relative LH lev-
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EFFECT OF GnRH IN MALE RHESUS els in rhesus monkeys. On log dose curves, nonparallelism of sequentially diluted serum samples is noted when compared with the reference prepara/•* tion. This is expected because the LH-like interfering substance has a different slope from that of /^ pituitary-LH preparations (17). However, we have ^ found that assay interference with nonspecific LHlike substance (17) is minimized by utilizing small v sample volumes. We have found that this assay is generally useful to detect the preovulatory LH peak in cycling females and pharmacologically induced LH peaks in estrogenized castrate female monkeys (18). LH, as k analyzed by this method, also has been found to vary diurnally in concert with serum testosterone levels in male rhesus. This method appears to be most useful for assessing short term, acute changes in LH concentrations in the male rhesus and it is »» for this purpose that it has been utilized in this study. Because of the limitations of this assay, we have also included in this study testosterone deterx minations which provide direct in vivo evidence for increased LH activity. Statistical analysis was performed by using a one-way analysis of variance followed by Duncan's '' multiple range test. k.
Results
* "^
*
' -c " " k
Plasma levels of LH and FSH after iv administration of 0, 1, 5, and 25 /ig GnRH are shown in Fig. 1. Except for the saline control group, plasma levels of LH were elevated above the 1-h pretreatment LH levels at +15 min, with the maximal LH levels occurring by 15-30 min after treatment with all concentrations of GnRH. None of the dose levels tested resulted in a significant change of plasma FSH in any of the animals. A comparison of the mean integrated LH response above mean preinjection levels during the 3-h interval after administration of the various doses of GnRH is shown in Fig. 2. One-way analysis of variance revealed significant changes (P < 0.01, F = 22.1, df = 3, 16) in LH concentration after treatment and further analysis with Duncan's multiple range test revealed that the LH response to 1, 5, and 25 /ig GnRH differed significantly from the response to saline alone; significant differences were also found between the response to 1 and 25 /xg and 5 and 25 jug, but no significant difference could be found between the response to 1 and 5 /xg
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GnRH. In support of the LH changes observed, plasma testosterone levels were also found to be significantly elevated (P < 0.05, F = 10.82) after treatment with 25 fig GnRH. Testosterone rose from pretreatment levels of 2.7 ± 0.8 ng/ml to maximum levels of 10.4 ± 2.0 ng/ml at 60-75 min after treatment (Fig. 3). Although a lag of 30 min occurred between the time of observing peak LH levels (+30) and the peak levels of testosterone, a sustained elevation of plasma testosterone was observed for the duration of the sampling interval (180 min) which contrasted with the LH levels that fell to control levels within the sampling period. Once again, no stimulation of FSH levels was observed. In an attempt to determine whether the rate of delivery of GnRH to the anterior pituitary gland could possibly alter the secretory response, GnRH at a concentration of 1 /xg/ml saline was infused at a rate of 1 ml/min for a total of 25 min. As expected, LH levels rose significantly (P < 0.01, F = 16.26) and reached peak concentrations 45 min after initiation of drug delivery and fell slowly to pretreatment values by the end of the sampling interval (Fig. 4). Although the LH response was not significantly different from that after 25-jUg bolus treatment, there was a trend toward a more sustained elevation of LH and a slower decline to baseline values. Again, levels of FSH did not change in this series of experiments. Discussion This series of experiments shows that the adult male rhesus monkey, like humans (7, 8), rats (19, 20), and sheep (21), responds to GnRH with an increase in plasma concentration of LH; unlike other species, however, the adult male rhesus monkey does not respond to GnRH treatment with an increase in plasma levels of FSH. The time course in the rise of plasma LH closely parallels that reported for other species. Associated with the increase in LH levels is a marked elevation in plasma levels of testosterone which lags the peak LH levels by approximately 30 min but
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Kndo • 1978 Vol 102 • NoH
TOIVOLA, BRIDSON, AND ROBINSON 25 H
'LH/FSH-RH
20-
k 20FIG. 1. Plasma LH and FSH levels after treatment with saline or 1, 5, and 25 jug GnRH. Mean ± SEM; n = 5, except for the 5-fig
dose where n = 4.
300 -60
•60
•120
• 180
TIME IN MINUTES
is sustained at high levels for a longer period. Increases in testosterone after the LH elevation induced by administration of GnRH in men are of much smaller magnitudes and do not remain elevated for as prolonged a time period as they do in the rhesus. The time course of the testosterone increase is similar to that observed in an earlier study where hypothalamic median eminence extracts were injected into male monkeys to stimulate LH secretion (22). Data describing acute changes in testosterone secretion in response to admin-
istration of exogenous LH are not available for the male rhesus. Fetal rhesus monkeys treated with intraarterial hCG or GnRH show increased plasma levels of testosterone within 60 min after treatment (23). Rabbits injected iv with LH display a maximal testosterone increase within 60 min after treatment (24). Although not directly comparable to the present study, the studies cited above confirm that the time course between increases in LH and elevations of testosterone observed is consistent with the generally accepted role of LH as
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EFFECT OF GnRH IN MALE RHESUS
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9MEAN - SEM
Q X
B
"
?
7-
|
6-
h-
T
CO
r> _
c
NTEG
f>-
i
.H RESPONSE
1
500
-1-
ZERO
Ipg
5pg
25pg
FIG. 2. Integrated mean LH response above mean preinjection levels for each dose of LH/FSH-RH administered. Significant increases in LH were found for all dose levels when compared to zero (saline) administration (P < 0.01). Significant differences in response were found between 1 and 25 jug (P < 0.01) and 5 and 25 /xg (P < 0.01) but not between 1 and 5 ng GnRH.
the main hormonal stimulus for regulating steroidogenesis in Leydig cells. The heterologous LH assay used in the present study has recently come under criticism as being nonspecific for rhesus LH (17, 25); nonetheless, we feel that the use of this assay is valid in the present study. We have presented evidence that the increase in immunologically reactive LH in our assay also is biologically active, inasmuch as we were able to show a concomitant increase in the plasma levels of testosterone. In essence, we have a built-in bioassay for LH in the intact male monkey by being able to assay testosterone simultaneously. Furthermore, we know of no other substance, other than LH, capable of producing the increases in testosterone of the magnitude observed in the present study. We do, how-
-60
-30
0
«3O ' 6 0 TIME IN MINUTES
*I2O
M80
FIG. 3. Increase in plasma levels of LH followed by an increase in plasma testosterone after administration of 25 /xg GnRH. The highest concentrations of testosterone lags behind peak levels of LH by approximately 30 min but are elevated for the duration of the sampling period.
ever, recognize that this assay detects a nonspecific LH-like substance, but acute changes in this "LH-like substance" have not been reported. Our data, therefore, do suggest that our RIA system for LH is, in fact, measuring changes in biologically active LH. Of considerable surprise to us was the finding that we were unable to stimulate the secretion of FSH by administering GnRH over a 25-fold difference in dose level. This was especially surprising in light of the fact that normal men do secrete FSH, although sluggishly, in response to GnRH stimulation (7, 26). Several possibilities exist which may help explain the refractoriness of the male rhesus pituitary to secrete FSH when challenged. The first possibility is that the rhesus monkey has separate releasing hormones controlling LH and FSH secretion. Alternatively, the dose
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TOIVOLA, BRIDSON, AND ROBINSON
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Kndo • 1978 Vol 102 • No 6
LH/FSH-RH Ijjg/min.
25 n«5 MEAN- SEM 20
15
10
r700
•30
-60
•60
•120
•180
c
Lsoo £ * -1-300 *• ?
TIME IN MINUTES
FIG. 4. Infusion of GnRH (1 jug/min for 25 min) results in an increase of plasma LH but not plasma FSH. The magnitude of the LH increase is similar to that observed after 25-jng bolus injection (Fig. 1), but the elevated levels of LH were sustained longer.
of GnRH may not have been sufficiently high to elicit FSH secretion, as it is possible that a much higher threshold of stimulation may exist for FSH secretion compared to LH. Finally, FSH secretion may be under the influence of a strong negative feedback inhibition by testosterone and/or other testicular hormones. We have not yet had the opportunity to explore this possibility in castrate male monkeys, but the experiments of Krey et al. (10) show that castrate female rhesus monkeys are more sensitive to GnRH than their intact counterparts.
Acknowledgments The authors wish to express their appreciation to C. Bolme, G. Scheffler, M. J. Toivola, F. Wegner, and R. Webber for expert technical assistance in the performance of RIAs. We wish to also acknowledge the secretarial assistance of G. Dolphin, J. Haight, and S. Shepard. We are also grateful to Dr. G. D. Niswender and Dr. W. D. Peckham for generous gifts of reagents, and also the Hormone Distribution Branch, NIAMDD, NIH, for generous supplies of LH/FSH-RH and other reagents.
References 1. McCann, S. M., S. Taleisnik, and H. M. Friedman, LH-releasing activity in hypothalamic extracts, Proc
Soc Exp Biol Med 104: 432, 1960. 2. Matsuo, H., Y. Baba, R. M. G. Nair, A. Arimura, and A. V. Schally, Structure of the porcine LH- and FSHreleasing hormone. I. The proposed amino acid sequence, Biochem Biophys Res Commun 43: 1334, 1971. 3. Baba, Y., H. Matsuo, and A. V. Schally, Structure of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses, Biochem Biophys Res Commun 44: 459, 1971. 4. Burgus, R., M. Butcher, M. Amoss, N. Ling, M. Monahan, J. Rivier, R. Fellos, R. Blackwell, W. Vale, and R. Guillemin, Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF), Proc Natl Acad Sci 69: 278, 1972. 5. Matsuo, H., A. Arimura, R. M. Nair, and A. V. Schally, Synthesis of the porcine LH- and FSH-releasing hormone by the solid phase method, Biochem Biophys Res Commun 45: 822, 1971. 6. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. G. Nair, L. Debeljuk, and W. F. White, Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle stimulating hormones, Science 173: 1036. 1971. 7. Kastin, A. J., C. Gual, and A. V. Schally, Clinical experience with hypothalamic releasing hormones. II. Luteinizing hormone-releasing hormone and other hypophysiotropic releasing hormones, Recent Prog Horm Res 28: 201, 1972. 8. Yen, S. S. C, B. L. Lasley, C. F. Wang, H. Leblanc,
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EFFECT OF GnRH IN MALE RHESUS and M. Silert, The operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of biological action of somatostatin, Recent Prog Horm Res 31: 321, 1975. 9. Bremner, W. J., and C. A. Paulsen, Two pools of luteinizing hormone in the human pituitary: evidence from constant administration of luteinizing hormone releasing factor, J Clin Endocrinol Metab 39: 811, 1974. 10. Krey, L. C, W. R. Butler, G. Weiss, R. F. Weick, D. J. Dierschke, and E. Knobil, Influence of endogenous and exogenous steroids on the action of synthetic LRF in the rhesus monkey, In Gual, G., and E. Rosemberg (eds.), Hypothalamic Hypophysiotropic Hormones, Excerpta Medica Int. Congr. Series 263, 1973, p. 39. 11. Spies, H. G., and R. L. Norman, Interaction of estradiol and LHRH on LH release in rhesus monkeys: evidence of a neural site of action, Endocrinology 97: 685, 1975. 12. Robinson, J. A., G. Scheffler, S. G. Eisele, and R. W. Goy, Effects of age and season on sexual behavior and plasma testosterone and dihydrotestosterone concentrations of laboratory-housed male rhesus monkeys (Macaca mulatto), Biol Reprod 13: 203, 1975. 13. Hodgen, G. D., J. W. Wilks, J. L. Vaitukaitis, H. C. Chen, H. Papkoff, and G. T. Ross, A new radioimmunoassay for follicle stimulating hormone in macaques: ovulatory menstrual cycles, Endocrinology 99: 137, 1976. 14. Rodbard, D., and J. E. Lewald, Computer analysis of radioligand assay and radioimmunoassay data, Ada Endocrinol [Kbh] (Suppl) 147: 79, 1970. 15. Boorman, G. A., G. D. Niswender, V. L. Gay, L. E. Reichert, Jr., and A. R. Midgley, Jr., Radioimmunoassay for follicle stimulating hormone in the rhesus monkey using an anti-human FSH serum and rat FSH '"I, Endocrinology 92: 618, 1973. 16. Millar, R. P., and C. Aehnelt, Application of ovine luteinizing hormone (LH) radioimmunoassay in the quantitation of LH in different mammalian species, Endocrinology 101: 760, 1977. 17. Peckham, W. D., D. L. Foster, and E. Knobil, A new substance resembling luteinizing hormone in the blood of rhesus monkeys, Endocrinology 100: 826, 1977.
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18. Terasawa, E., J. A. Robinson, C. J. Mahoney, and W. E. Bridson, Effects of reserpine on ovulation and estrogen-induced LH surge in the rhesus monkey, Abstracts of the 58th Meeting of the Endocrine Society, San Francisco 1976, (Abstract 205). 19. Arimura, A., H. Matsuo, Y. Baba, L. Debeljuk, J. Sandow, and A. V. Schally, Stimulation of release of LH by synthetic LH-RH in vivo. I. A comparative study of natural and synthetic hormones, Endocrinology 90: 163, 1972. 20. Rennels, E. G., E. M. Bogdanove, A. Arimura, M. Saito, and A. V. Schally, Ultrastructural observations of rat pituitary gonadotrophs following injection of purified porcine LH-RH, Endocrinology 88: 1318, 1971. 21. Reeves, J. J., A. Arimura, A. V. Schally, C. L. Kragt, T. W. Beck, and J. M. Casey, Effects of synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone (LH-RH/FSH-RH) on serum LH, serum FSH, and ovulation in anestrous ewes, J Anim Sci 35: 84, 1972. 22. McCormack, S. A., and H. G. Spies, Response of the hypophyseal-testicular axis in monkeys to stalk-median eminence extract, Proc Soc Exp Biol Med 141: 263, 1972. 23. Huhtaniemi, I. T., C. C. Korenbrot, M. Seron-Ferre, D. B. Foster, J. T. Parer, and R. B. Jaffe, Stimulation of testosterone production in vivo and in vitro in the male rhesus monkey fetus in late gestation, Endocrinology 100: 839, 1977. 24. Smith, O. W., and H. D. Hafs, Competitive protein binding and radioimmunoassay for testosterone in bulls and rabbits; blood serum testosterone after injection of LH or prolactin in rabbits, Proc Soc Exp Biol Med 142: 804, 1973. 25. Neill, J. D., R. A. Dailey, R. C. Tsou, and L. E. Reichert, Jr., Immunoreactive LH-like substances in serum of hypophysectomized and prepubertal monkeys: inactive in an in vitro bioassay, Endocrinology 100: 856, 1977. 26. Rebar, R., S. S. C. Yen, G. Vandenberg, F. Naftolin, Y. Ehara, S. Engnblom, K. J. Ryan, J. Rivier, M. Amoss, and R. Guillemin, Gonadotropin responses to synthetic LRF: dose-response relationship in men, J Clin Endocrinol Metab 36: 10, 1973.
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Vol. 102, No. 6 Printed in U.S.A.
Effect of Luteinizing Hormone-Releasing Hormone on the Secretion of Luteinizing Hormone, Follicle-Stimulating Hormone, and Testosterone in Adult Male Rhesus Monkeys* PERTTI T. K. TOIVOLA, WILLIAM E. BRIDSON, AND JERRY A. ROBINSON Wisconsin Regional Primate Research Center and Departments of Physiology and Medicine, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT. Plasma levels of radioimmunoreactive LH, FSH, and testosterone (T) were assayed before and after administration of synthetic gonadotropin-releasing hormone (GnRH) to five chair-restrained rhesus monkeys with chronic indwelling venous catheters. Intravenous injection of 1, 5, and 25 fig or infusion of 1 /ig/min for 25 min of GnRH resulted in a significant increase in plasma levels of LH. However, no significant increases in plasma FSH levels were detected. Plasma levels of T
T
HE DEMONSTRATION of a LH-releasing factor in hypothalamic tissue (1) precipitated an intensive search that culminated in the isolation of a decapeptide from porcine hypothalami which possessed LH-releasing activity (2, 3). Subsequently, the structures of porcine and ovine LH-releasing peptides were determined and found to be identical (3, 4). The relative ease of synthesizing large quantities of this peptide (5) has made its use possible in various physiological studies. In addition to promoting the secretion of LH, this synthetic material also possesses FSH-releasing activity, a finding which prompted the use of the terms gonadotropinreleasing hormone (GnRH) and LH/FSH-RH (6). Although the effects of synthetic GnRH have been extensively studied in females of many species, less emphasis has been directed toward its effects in the male. Kastin et al. (7) showed that administration of both native and Received August 18, 1977. Address all correspondence and requests for reprints to: Dr. P. T. K. Toivola, Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53706. * Publication 17-026 of the Wisconsin Regional Primate Research Center. This work was supported by NIH Grant RR-00167 to the Wisconsin Regional Primate Research Center and NIMH Grant MH-21312.
were also elevated after administration of 25 fig GnRH, but peak concentration of T lagged behind peak levels of LH by approximately 30 min. These studies indicate that the male rhesus responds to GnRH administration by increased secretion of LH, followed by an increase in T levels. A concomitant increase in plasma FSH was not observed after treatment with GnRH in the doses used. (Endocrinology 102: 1815, 1978)
synthetic GnRH promoted LH and FSH secretion in normal men; but, the synthetic material was much less effective in stimulating the secretion of FSH. Likewise, Yen et al. (8) have shown that administration of GnRH to normal men results in a marked increase of LH secretion but only a small increase in the levels of FSH. Continuous iv infusion of GnRH for 4 h in adult men resulted in a biphasic release of LH and again, relatively small changes in the plasma levels of FSH (9). Thus, it would seem that the GnRH in human males has a minimal effect on FSH secretion in adult human males. The effects of synthetic GnRH administration in the female rhesus monkey have been studied by Krey et al. (10) and Spies et al. (11) in a number of experimental situations, and in most cases was found to be an effective stimulus for LH secretion. The present report describes the effects of GnRH administration on LH, FSH, and testosterone secretion in normal adult male rhesus monkeys. Materials and Methods Five adult male rhesus (Macaca mulatto), weighing 6.5-9.5 kg, were used as experimental subjects. All monkeys were adapted to chronic re-
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TOIVOLA, BRIDSON, AND ROBINSON
straint in a primate chair for a minimum of 2 weeks, after which they were aseptically operated upon under general anesthesia for placement of a Silastic catheter (0.030 inches id x 0.065 inches od, Dow Corning) via the internal jugular vein into the right atrium. Postoperatively, the patency of the catheter was maintained by constant infusion of heparinized saline (10 U/ml, 24 ml/day) via a pulsatile pump. All subjects were routinely maintained on a 12-h light-12-h dark cycle with lights on from 0600-1800 h. Purina monkey chow supplemented with fresh fruits was fed once daily between 0730 and 0800 h and was available ad libitum, as was fresh water. During experimental sessions, two monkeys were housed together in an acoustically attenuated environmental chamber equipped with a white noise generator and a closed circuit video monitor. Catheters were passed through a port hole in the chamber wall which permitted blood sampling and drug administration from outside the chamber, obviating interaction with the monkey. Blood samples (3 ml) were drawn through the jugular catheter into sterile syringes, immediately placed into sterile heparinized centrifuge tubes, and centrifuged at 2500 rpm for 7 min. The plasma was aspirated with sterile pipettes and stored frozen at — 20 C until hormone assays could be performed. The packed red blood cells were resuspended in sterile isotonic saline and returned to the subjects immediately after withdrawal of the subsequent blood sample. Blood samples were collected at 15-min intervals for 60 min before and 180 min after the administration of GnRH. Synthetic GnRH (amide form; lot 21-103-DH; obtained from the Hormone Distribution Branch, NIAMDD, NIH) was diluted in sterile, pyrogenfree saline from the stock solution (100 jug/ml) to a final concentration of 1, 5, or 25 /xg/ml just before administration. Each of the five monkeys was given either 1 ml saline or 1 or 25 iig GnRH in 1 ml saline; four of these monkeys were also given 5 jag GnRH in 1 ml saline. To prolong the effects of the administered drug, 25 ug GnRH were also infused at the rate of 1 jug/min (1 ml/min) in all five monkeys, using a Harvard infusion pump and a precalibrated syringe. The order of administration of these doses was randomized. All experiments commenced at 1000 h with the drawing of the first blood sample. The administration of either saline as a control or one dose of GnRH constituted an experiment and 7 days were allowed to elapse between successive experiments in individual monkeys. The RIA of testosterone in the male rhesus monkey has previously been described in detail (12). After extraction and column chromatography, sam-
Kiulo 1978 Vol 102 . No 6
pie recoveries of 76 ± 9.1% (SD) were obtained. The blank values for these assays were 1.7 ± 3.82 (SD) pg, resulting in an assay sensitivity of less than 10 pg. Within-assay precision and between-assay variability were found to be ± 9.4 and ± 14.2 (SD) pg, respectively. All samples were analyzed in duplicate utilizing 40-jul quantities of plasma. RIAs for protein hormones were performed utilizing l25I-labeled tracer, phosphosaline buffers (13), and double antibody precipitation for separation of free and bound hormones. Assay results were calculated by the use of statistical model II of Rodbard (14) and microprocessor programs developed in our laboratory. The influence of between-assay variance was minimized by including all sequential serum samples from a given subject in the same assay. Rhesus FSH was estimated utilizing methods and materials described by Hodgen et al. (13). Results are expressed as nanograms per ml in plasma of rhesus FSH reference preparation WDPX1-2-A [reported immunological activity of 1 mg equivalent to 1.02 mg NIH-FSH-Sl and biological activity of 1 mg equivalent to 1.10 mg of NIH-FSHSl (15)]. The immunological activity of 1 jug this preparation was equivalent to 20.1 /xg LER-1909-2 (rhesus gonadotrophin reference preparation) in our laboratory. All samples were assayed in duplicate using 200 ju.1 serum. The coefficient of variation for samples was 9.8% (mean concentration, 454.2 ng/ml). Assay sensitivity for reliable measures was 70 ng/tube (350 ng/ml serum, utilizing a 200-/xl sample.) Rabbit antiovine LH antibody (GDN-15), rat LH tracer, and rhesus LH reference preparation WDPX-81-1720 were utilized in a double antibody assay for rhesus LH. General unavailability of highly purified rhesus LH for radioiodination prompted the use of rat 125I-labeled LH (NIAMDD-rLH-I-3) as tracer, as has been previously described for a number of species (16). The biological potency of 1 mg WDP-X-81-1720 is equivalent to 2.90 mg NIHLS-S1 (15). In our laboratory, 1 ng WDP-X-81-1720 is, by RIA equivalent to 3.85 jug LER-1909-2. In this assay, the minimal detectable dose is 0.35 ng/tube. The between-assay coefficients of variation (CV) for LH standard concentrations of 6.15, 13.1, and 31.4 ng/ml were, respectively, 14.26, 12.15, and 11.44%, with 8.50, 6.31, and 6.47% being the respective CV for within-assay variability. Samples were analyzed utilizing duplicate 100-jwl quantities. Although this LH antibody has been reported (17) to be nonspecific for measurements of rhesus LH, it is the only generally available radioimmunological technique for estimating relative LH lev-
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EFFECT OF GnRH IN MALE RHESUS els in rhesus monkeys. On log dose curves, nonparallelism of sequentially diluted serum samples is noted when compared with the reference prepara/•* tion. This is expected because the LH-like interfering substance has a different slope from that of /^ pituitary-LH preparations (17). However, we have ^ found that assay interference with nonspecific LHlike substance (17) is minimized by utilizing small v sample volumes. We have found that this assay is generally useful to detect the preovulatory LH peak in cycling females and pharmacologically induced LH peaks in estrogenized castrate female monkeys (18). LH, as k analyzed by this method, also has been found to vary diurnally in concert with serum testosterone levels in male rhesus. This method appears to be most useful for assessing short term, acute changes in LH concentrations in the male rhesus and it is »» for this purpose that it has been utilized in this study. Because of the limitations of this assay, we have also included in this study testosterone deterx minations which provide direct in vivo evidence for increased LH activity. Statistical analysis was performed by using a one-way analysis of variance followed by Duncan's '' multiple range test. k.
Results
* "^
*
' -c " " k
Plasma levels of LH and FSH after iv administration of 0, 1, 5, and 25 /ig GnRH are shown in Fig. 1. Except for the saline control group, plasma levels of LH were elevated above the 1-h pretreatment LH levels at +15 min, with the maximal LH levels occurring by 15-30 min after treatment with all concentrations of GnRH. None of the dose levels tested resulted in a significant change of plasma FSH in any of the animals. A comparison of the mean integrated LH response above mean preinjection levels during the 3-h interval after administration of the various doses of GnRH is shown in Fig. 2. One-way analysis of variance revealed significant changes (P < 0.01, F = 22.1, df = 3, 16) in LH concentration after treatment and further analysis with Duncan's multiple range test revealed that the LH response to 1, 5, and 25 /ig GnRH differed significantly from the response to saline alone; significant differences were also found between the response to 1 and 25 /xg and 5 and 25 jug, but no significant difference could be found between the response to 1 and 5 /xg
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GnRH. In support of the LH changes observed, plasma testosterone levels were also found to be significantly elevated (P < 0.05, F = 10.82) after treatment with 25 fig GnRH. Testosterone rose from pretreatment levels of 2.7 ± 0.8 ng/ml to maximum levels of 10.4 ± 2.0 ng/ml at 60-75 min after treatment (Fig. 3). Although a lag of 30 min occurred between the time of observing peak LH levels (+30) and the peak levels of testosterone, a sustained elevation of plasma testosterone was observed for the duration of the sampling interval (180 min) which contrasted with the LH levels that fell to control levels within the sampling period. Once again, no stimulation of FSH levels was observed. In an attempt to determine whether the rate of delivery of GnRH to the anterior pituitary gland could possibly alter the secretory response, GnRH at a concentration of 1 /xg/ml saline was infused at a rate of 1 ml/min for a total of 25 min. As expected, LH levels rose significantly (P < 0.01, F = 16.26) and reached peak concentrations 45 min after initiation of drug delivery and fell slowly to pretreatment values by the end of the sampling interval (Fig. 4). Although the LH response was not significantly different from that after 25-jUg bolus treatment, there was a trend toward a more sustained elevation of LH and a slower decline to baseline values. Again, levels of FSH did not change in this series of experiments. Discussion This series of experiments shows that the adult male rhesus monkey, like humans (7, 8), rats (19, 20), and sheep (21), responds to GnRH with an increase in plasma concentration of LH; unlike other species, however, the adult male rhesus monkey does not respond to GnRH treatment with an increase in plasma levels of FSH. The time course in the rise of plasma LH closely parallels that reported for other species. Associated with the increase in LH levels is a marked elevation in plasma levels of testosterone which lags the peak LH levels by approximately 30 min but
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Kndo • 1978 Vol 102 • NoH
TOIVOLA, BRIDSON, AND ROBINSON 25 H
'LH/FSH-RH
20-
k 20FIG. 1. Plasma LH and FSH levels after treatment with saline or 1, 5, and 25 jug GnRH. Mean ± SEM; n = 5, except for the 5-fig
dose where n = 4.
300 -60
•60
•120
• 180
TIME IN MINUTES
is sustained at high levels for a longer period. Increases in testosterone after the LH elevation induced by administration of GnRH in men are of much smaller magnitudes and do not remain elevated for as prolonged a time period as they do in the rhesus. The time course of the testosterone increase is similar to that observed in an earlier study where hypothalamic median eminence extracts were injected into male monkeys to stimulate LH secretion (22). Data describing acute changes in testosterone secretion in response to admin-
istration of exogenous LH are not available for the male rhesus. Fetal rhesus monkeys treated with intraarterial hCG or GnRH show increased plasma levels of testosterone within 60 min after treatment (23). Rabbits injected iv with LH display a maximal testosterone increase within 60 min after treatment (24). Although not directly comparable to the present study, the studies cited above confirm that the time course between increases in LH and elevations of testosterone observed is consistent with the generally accepted role of LH as
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EFFECT OF GnRH IN MALE RHESUS
1819
9MEAN - SEM
Q X
B
"
?
7-
|
6-
h-
T
CO
r> _
c
NTEG
f>-
i
.H RESPONSE
1
500
-1-
ZERO
Ipg
5pg
25pg
FIG. 2. Integrated mean LH response above mean preinjection levels for each dose of LH/FSH-RH administered. Significant increases in LH were found for all dose levels when compared to zero (saline) administration (P < 0.01). Significant differences in response were found between 1 and 25 jug (P < 0.01) and 5 and 25 /xg (P < 0.01) but not between 1 and 5 ng GnRH.
the main hormonal stimulus for regulating steroidogenesis in Leydig cells. The heterologous LH assay used in the present study has recently come under criticism as being nonspecific for rhesus LH (17, 25); nonetheless, we feel that the use of this assay is valid in the present study. We have presented evidence that the increase in immunologically reactive LH in our assay also is biologically active, inasmuch as we were able to show a concomitant increase in the plasma levels of testosterone. In essence, we have a built-in bioassay for LH in the intact male monkey by being able to assay testosterone simultaneously. Furthermore, we know of no other substance, other than LH, capable of producing the increases in testosterone of the magnitude observed in the present study. We do, how-
-60
-30
0
«3O ' 6 0 TIME IN MINUTES
*I2O
M80
FIG. 3. Increase in plasma levels of LH followed by an increase in plasma testosterone after administration of 25 /xg GnRH. The highest concentrations of testosterone lags behind peak levels of LH by approximately 30 min but are elevated for the duration of the sampling period.
ever, recognize that this assay detects a nonspecific LH-like substance, but acute changes in this "LH-like substance" have not been reported. Our data, therefore, do suggest that our RIA system for LH is, in fact, measuring changes in biologically active LH. Of considerable surprise to us was the finding that we were unable to stimulate the secretion of FSH by administering GnRH over a 25-fold difference in dose level. This was especially surprising in light of the fact that normal men do secrete FSH, although sluggishly, in response to GnRH stimulation (7, 26). Several possibilities exist which may help explain the refractoriness of the male rhesus pituitary to secrete FSH when challenged. The first possibility is that the rhesus monkey has separate releasing hormones controlling LH and FSH secretion. Alternatively, the dose
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TOIVOLA, BRIDSON, AND ROBINSON
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Kndo • 1978 Vol 102 • No 6
LH/FSH-RH Ijjg/min.
25 n«5 MEAN- SEM 20
15
10
r700
•30
-60
•60
•120
•180
c
Lsoo £ * -1-300 *• ?
TIME IN MINUTES
FIG. 4. Infusion of GnRH (1 jug/min for 25 min) results in an increase of plasma LH but not plasma FSH. The magnitude of the LH increase is similar to that observed after 25-jng bolus injection (Fig. 1), but the elevated levels of LH were sustained longer.
of GnRH may not have been sufficiently high to elicit FSH secretion, as it is possible that a much higher threshold of stimulation may exist for FSH secretion compared to LH. Finally, FSH secretion may be under the influence of a strong negative feedback inhibition by testosterone and/or other testicular hormones. We have not yet had the opportunity to explore this possibility in castrate male monkeys, but the experiments of Krey et al. (10) show that castrate female rhesus monkeys are more sensitive to GnRH than their intact counterparts.
Acknowledgments The authors wish to express their appreciation to C. Bolme, G. Scheffler, M. J. Toivola, F. Wegner, and R. Webber for expert technical assistance in the performance of RIAs. We wish to also acknowledge the secretarial assistance of G. Dolphin, J. Haight, and S. Shepard. We are also grateful to Dr. G. D. Niswender and Dr. W. D. Peckham for generous gifts of reagents, and also the Hormone Distribution Branch, NIAMDD, NIH, for generous supplies of LH/FSH-RH and other reagents.
References 1. McCann, S. M., S. Taleisnik, and H. M. Friedman, LH-releasing activity in hypothalamic extracts, Proc
Soc Exp Biol Med 104: 432, 1960. 2. Matsuo, H., Y. Baba, R. M. G. Nair, A. Arimura, and A. V. Schally, Structure of the porcine LH- and FSHreleasing hormone. I. The proposed amino acid sequence, Biochem Biophys Res Commun 43: 1334, 1971. 3. Baba, Y., H. Matsuo, and A. V. Schally, Structure of the porcine LH- and FSH-releasing hormone. II. Confirmation of the proposed structure by conventional sequential analyses, Biochem Biophys Res Commun 44: 459, 1971. 4. Burgus, R., M. Butcher, M. Amoss, N. Ling, M. Monahan, J. Rivier, R. Fellos, R. Blackwell, W. Vale, and R. Guillemin, Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF), Proc Natl Acad Sci 69: 278, 1972. 5. Matsuo, H., A. Arimura, R. M. Nair, and A. V. Schally, Synthesis of the porcine LH- and FSH-releasing hormone by the solid phase method, Biochem Biophys Res Commun 45: 822, 1971. 6. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. G. Nair, L. Debeljuk, and W. F. White, Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle stimulating hormones, Science 173: 1036. 1971. 7. Kastin, A. J., C. Gual, and A. V. Schally, Clinical experience with hypothalamic releasing hormones. II. Luteinizing hormone-releasing hormone and other hypophysiotropic releasing hormones, Recent Prog Horm Res 28: 201, 1972. 8. Yen, S. S. C, B. L. Lasley, C. F. Wang, H. Leblanc,
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EFFECT OF GnRH IN MALE RHESUS and M. Silert, The operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of biological action of somatostatin, Recent Prog Horm Res 31: 321, 1975. 9. Bremner, W. J., and C. A. Paulsen, Two pools of luteinizing hormone in the human pituitary: evidence from constant administration of luteinizing hormone releasing factor, J Clin Endocrinol Metab 39: 811, 1974. 10. Krey, L. C, W. R. Butler, G. Weiss, R. F. Weick, D. J. Dierschke, and E. Knobil, Influence of endogenous and exogenous steroids on the action of synthetic LRF in the rhesus monkey, In Gual, G., and E. Rosemberg (eds.), Hypothalamic Hypophysiotropic Hormones, Excerpta Medica Int. Congr. Series 263, 1973, p. 39. 11. Spies, H. G., and R. L. Norman, Interaction of estradiol and LHRH on LH release in rhesus monkeys: evidence of a neural site of action, Endocrinology 97: 685, 1975. 12. Robinson, J. A., G. Scheffler, S. G. Eisele, and R. W. Goy, Effects of age and season on sexual behavior and plasma testosterone and dihydrotestosterone concentrations of laboratory-housed male rhesus monkeys (Macaca mulatto), Biol Reprod 13: 203, 1975. 13. Hodgen, G. D., J. W. Wilks, J. L. Vaitukaitis, H. C. Chen, H. Papkoff, and G. T. Ross, A new radioimmunoassay for follicle stimulating hormone in macaques: ovulatory menstrual cycles, Endocrinology 99: 137, 1976. 14. Rodbard, D., and J. E. Lewald, Computer analysis of radioligand assay and radioimmunoassay data, Ada Endocrinol [Kbh] (Suppl) 147: 79, 1970. 15. Boorman, G. A., G. D. Niswender, V. L. Gay, L. E. Reichert, Jr., and A. R. Midgley, Jr., Radioimmunoassay for follicle stimulating hormone in the rhesus monkey using an anti-human FSH serum and rat FSH '"I, Endocrinology 92: 618, 1973. 16. Millar, R. P., and C. Aehnelt, Application of ovine luteinizing hormone (LH) radioimmunoassay in the quantitation of LH in different mammalian species, Endocrinology 101: 760, 1977. 17. Peckham, W. D., D. L. Foster, and E. Knobil, A new substance resembling luteinizing hormone in the blood of rhesus monkeys, Endocrinology 100: 826, 1977.
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18. Terasawa, E., J. A. Robinson, C. J. Mahoney, and W. E. Bridson, Effects of reserpine on ovulation and estrogen-induced LH surge in the rhesus monkey, Abstracts of the 58th Meeting of the Endocrine Society, San Francisco 1976, (Abstract 205). 19. Arimura, A., H. Matsuo, Y. Baba, L. Debeljuk, J. Sandow, and A. V. Schally, Stimulation of release of LH by synthetic LH-RH in vivo. I. A comparative study of natural and synthetic hormones, Endocrinology 90: 163, 1972. 20. Rennels, E. G., E. M. Bogdanove, A. Arimura, M. Saito, and A. V. Schally, Ultrastructural observations of rat pituitary gonadotrophs following injection of purified porcine LH-RH, Endocrinology 88: 1318, 1971. 21. Reeves, J. J., A. Arimura, A. V. Schally, C. L. Kragt, T. W. Beck, and J. M. Casey, Effects of synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone (LH-RH/FSH-RH) on serum LH, serum FSH, and ovulation in anestrous ewes, J Anim Sci 35: 84, 1972. 22. McCormack, S. A., and H. G. Spies, Response of the hypophyseal-testicular axis in monkeys to stalk-median eminence extract, Proc Soc Exp Biol Med 141: 263, 1972. 23. Huhtaniemi, I. T., C. C. Korenbrot, M. Seron-Ferre, D. B. Foster, J. T. Parer, and R. B. Jaffe, Stimulation of testosterone production in vivo and in vitro in the male rhesus monkey fetus in late gestation, Endocrinology 100: 839, 1977. 24. Smith, O. W., and H. D. Hafs, Competitive protein binding and radioimmunoassay for testosterone in bulls and rabbits; blood serum testosterone after injection of LH or prolactin in rabbits, Proc Soc Exp Biol Med 142: 804, 1973. 25. Neill, J. D., R. A. Dailey, R. C. Tsou, and L. E. Reichert, Jr., Immunoreactive LH-like substances in serum of hypophysectomized and prepubertal monkeys: inactive in an in vitro bioassay, Endocrinology 100: 856, 1977. 26. Rebar, R., S. S. C. Yen, G. Vandenberg, F. Naftolin, Y. Ehara, S. Engnblom, K. J. Ryan, J. Rivier, M. Amoss, and R. Guillemin, Gonadotropin responses to synthetic LRF: dose-response relationship in men, J Clin Endocrinol Metab 36: 10, 1973.
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