Regulation of Pituitary Gonadotropin- releasing Hormone Receptors by Pulsatile Gonadotropin-releasing Hormone Injections in Male Rats Modulation by Testosterone Alfredo Garcia, Melissa Schiff, and John C. Marshall Division of Endocrinology and Metabolism, Department of Internal Medicine, University ofMichigan, Ann Arbor, Michigan 48109 Abstract. The pattern of the gonadotropin- on the serum T concentration. With one T implant, releasing hormone (GnRH) stimulus is critically impor- increasing GnRH doses per pulse stimulated GnRH-R tant in the regulation of pituitary gonadotropin secretion in a linear manner and the maximum receptor concen- and continuous infusions down-regulate secretion while tration (703±99 fmol/mg) was seen after the 250 ng intermittent pulses maintain luteinizing hormone (LH) GnRH dose. In the presence of two T implants, GnRH- and follicle-stimulating hormone (FSH) responsiveness. R was maximal (705±45 fmol/mg) after the 25-ng dose We examined the effects of pulsatile GnRH administra- and higher doses did not increase receptors above control tion on pituitary GnRH receptors (GnRH-R) and go- values. With four T implants, GnRH doses of 5 ng nadotropin secretion in the presence of physiological induced a maximum response, 17-50 ng/pulse did not concentrations of testosterone (T) to elucidate the mech- increase GnRH-R, but receptors were again increased anisms and sites of action of GnRH and T on the by the 250-ng dose (633±86 fmol/mg). pituitary gonadotroph. After 48 h of pulsatile GnRH administration there Castrate male rats received one, two, or four testos- was no correlation between the number of GnRH-R terone (T) implants (serum T concentrations of 1.1, 2.4, and LH responses to GnRH. In rats with one or two T and 5.2 ng/ml, respectively) to suppress endogenous implants, LH responses were absent after all but the GnRH secretion. Subsequently, intracarotid pulse injec- 250-ng doses. In contrast, LH responsiveness was not tions of GnRH (5-250 ng/pulse) or saline in controls impaired in the presence of four implants. Thus, low were given every 30 min for 48 h, after which gonado- dose GnRH pulses down-regulate LH secretion by an tropin responses and pituitary GnRH-R were measured. action at a post GnRH-R site, and this effect is regulated In control rats, the T implants prevented the rise in by testosterone. The results show that GnRH, given in GnRH-R that was seen in castrates (empty implant- a pulsatile manner, regulates its own receptor, and 600 fmol/mg protein) and maintained receptors at the physiological increases in serum T produce a 50-fold level that was present in intact animals (300 fmol/mg). increase in the sensitivity of GnRH-R stimulation by Pulsatile GnRH administration increased GnRH-R in GnRH. castrate T-implanted rats, but the response was dependent Introduction This work was presented in part at the Annual Meeting of the American Society for Clinical Investigation, 1983. Secretion of the pituitary gonadotropin hormones, luteinizing Address reprint requests to Dr. Marshall. hormone (LH)' and follicle-stimulating hormone (FSH), is Received for publication 20 January 1984 and in revised form 17 controlled by the hypothalamic gonadotropin-releasing hor- April 1984. mone (GnRH). In vivo, GnRH appears to be secreted in an J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: FSH, follicle-stimulating hormone; 0021-9738/84/09/0920/09 $ 1.00 GnRH, gonadotropin-releasing hormone; GnRH-R, GnRH receptors; Volume 74, September 1984, 920-928 LH, luteinizing hormone; PE 50, polyethylene cannula; T, testosterone. 920 A. Garcia, M. Schiff, and J. C. Marshall intermittent or pulsatile manner. This was initially suggested secretion is likely to be low in this model, as judged by by studies which demonstrated pulsatile secretion of LH, and infrequent LH pulses (27). Thus, the assessment of the effects by inference GnRH, in normal men and in castrated rats of the administration of exogenous GnRH pulses should not (1, 2). Subsequent studies confirmed this observation in other be unduly complicated by endogenous GnRH secretion. In species (3-5), and later direct measurement of GnRH in this manner we intended to delineate the effects of pulsatile hypothalamic-portal blood provided direct confirmation of GnRH in the modulation of GnRH-R with a view to deter- this suggestion. GnRH concentrations in portal blood were mining if alterations in receptor number may be part of the increased at the time of the proestrus gonadotropin surge and mechanisms involved in changing pituitary responsiveness. In in castrate females (6, 7), and the variable pattern of the addition, by performing the pulsatile GnRH studies in the elevated hormone concentrations in 10-15-min collections presence of varying concentrations of testosterone, we hoped suggested a pulsatile mode of secretion. Recently Clarke et al. to gain insight into the action of androgens at the pituitary (8) obtained simultaneous samples of portal and jugular venous level and to determine if part of the feedback action of steroids blood in sheep and showed that each LH pulse was preceded was exerted at the level of the pituitary cell and, more by a pulse of GnRH. A pulsatile GnRH stimulus appears to specifically, on the GnRH-R. be essential for gonadotropin secretion to be manifest. If GnRH is given to GnRH-deficient humans or hypothalamic Methods lesioned monkeys in a pulsatile manner, the normal hormonal changes of puberty or the menstrual cycle can be reproduced Experimental procedures. Adult Sprague-Dawley rats (275-300 g) were (9-16). On the other hand, continuous infusion of GnRH to castrated under ether anesthesia and one, two, or four 20-mm silastic lesioned monkeys results in "down-regulation" of gonadotropin implants that contained testosterone were implanted subcutaneously. secretion, which is restored by reinstitution of a pulsatile A polyethylene cannula (PE 50) with a silastic tip was placed in the stimulus (17). Similarly, "down-regulation" of LH secretion right carotid artery for subsequent GnRH injections. Control animals were anesthetized and sham operated or sham implanted in an has been observed in man and in other animal species when identical manner. constant infusions of GnRH or injections of long-acting GnRH After surgery and recovery from anesthesia rats were placed in agonists have been used (18-23). Thus, the pulsatile nature of immobilization cages which allowed anterior mobility but restricted GnRH secretion appears to be critical in determining LH and turning movements in order to prevent kinking or chewing of the FSH responsiveness, and some evidence suggests that changes cannula. The cannula was connected to a 10-ml syringe that was in frequency may be one mechanism by which steroid hormones attached to a pump (Autosyringe AS-2C), and during the subsequent exert feedback effects on the hypothalamic-pituitary axis. LH 48-h normal saline or different GnRH concentrations were delivered pulse frequency is increased in castrate animals and both via the cannula as intermittent pulses at a frequency of every 30 min progesterone and testosterone (T) can reduce LH pulse fre- to mimic LH pulse frequency in castrate rats. GnRH was diluted in quency in castrate sheep and rats, respectively 0.1% bovine serum albumin (BSA) in normal saline and the syringes (24-27). had been presoaked in 0.1% BSA for 24 h before each experiment. These studies indicate the importance of the manner of Syringes were refilled with a fresh GnRH solution every 12 h and the GnRH delivery to the pituitary, but little information is residual GnRH solution was assayed for GnRH as a check on the available regarding the nature of the cellular mechanisms that concentration delivered. At the end of this period, rats were decapitated are responsive to the stimulus. GnRH acts by an initial step 20 min after the last pulse injection and blood was collected for assay of binding to a plasma membrane receptor (28, 29), and a of LH, FSH, and testosterone. The anterior pituitary was removed, considerable body of evidence has shown that GnRH is the placed in 0.25 M sucrose, snap-frozen in ethanol-dry ice, and then main regulator of its own receptor on the gonadotroph. stored at -70°C before use in the binding assay. Castration results in an increase in GnRH receptors (GnRH- In initial studies we performed experiments to determine the R), and this effect can be abolished by hypothalamic lesions, maximum plasma levels of GnRH and the initial LH response to the different GnRH doses that were used in the or of subsequent pulsatile administration of GnRH anti-sera, replacement testos- studies. This was done in acutely castrated rats where an additional terone (30-33). These manipulations prevent the rise in GnRH PE 50 cannula was placed in the aorta. GnRH was injected 30 min after castration, and injections of GnRH given subcutaneously after castration, and I min later blood was collected from the other every 8 h increased GnRH-R in these animals (30, 32). Low PE 50 cannula for measurement of GnRH and 20 min later for dose infusions of GnRH can also increase GnRH-R, whereas measurement of LH. GnRH doses > 250 ng were not used in view of high dose infusions reduce receptor number (34). These data the plasma half life of GnRH (-3-4 min). Higher doses might not be clearly show the importance of GnRH in the regulation of its completely cleared from the circulation within 30 min and this would own receptor, but studies to date have not examined the effect alter the pulsatile nature of the GnRH signal. of pulsatile administration of GnRH in a Assessment ofpituitary GnRH receptors. The affinity of the GnRH- given manner to R is unchanged in different physiological and experimental conditions mimic the physiological mode of secretion.