University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Roman L. Hruska U.S. Meat Animal Research Center U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 1986 Regulation of Gonadotropin Secretion in the Male: Effect of an Aromatization Inhibitor in Estradiolimplanted, Orchidectomized Dogs C. Awoniyi T. Hasson V. Chandrashekar R. E. Falvo B. D. Schanbacher USDA-ARS Follow this and additional works at: http://digitalcommons.unl.edu/hruskareports Awoniyi, C.; Hasson, T.; Chandrashekar, V.; Falvo, R. E.; and Schanbacher, B. D., "Regulation of Gonadotropin Secretion in the Male: Effect of an Aromatization Inhibitor in Estradiol-implanted, Orchidectomized Dogs" (1986). Roman L. Hruska U.S. Meat Animal Research Center. 300. http://digitalcommons.unl.edu/hruskareports/300 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Roman L. Hruska U.S. Meat Animal Research Center by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.
Regulation of Gonadotropin Secretion in the Male: Effect of an Aromatization Inhibitor in Estradiol-implanted, Orchidectomized Dogs C. AWONIYI, T. HASSON, V. CHANDRASHEKAR, R. E. FALVO, AND B. D. SCHANBACHER* Testosterone is aromatized to estradiol in both peripheral tissues and the central nervous system. Various authors have suggested that this conversion in the male may be prerequisite for the regulation of gonadotropin secretion by testosterone. Previously, it was reported that inhibition of central nervous system aromatase caused a significant increase in plasma LH in the presence of physiologic testosterone levels (Winter et a!, 1983). In order to confirm whether aminoglutethimide, the aromatase inhibitor used in our previous study, either blocked aromatization, or the action of estradiol, the following study was conducted. Fifteen male mongrel dogs were equally divided into three groups. Group 1 dogs were implanted with estradiol-filled polydimethylsiloxane capsules only; Group 2 dogs were implanted with empty capsules and treated with 60 mg b.i.d. of aminoglutethimide; and Group 3 dogs were implanted with polydimethylsiloxane capsules filled with estradiol and treated with aminoglutethimide. Blood samples were drawn for 24 days during pretreatment, capsule implantation, castration, aminoglutethimide administration and capsule removal periods. The postcastration response of both plasma LH and FSH in dogs in groups I and 3 was suppressed in the presence of elevated estradiol, whereas that of Group 2 dogs was normal in the absence of estradiol. The results suggest that aminoglutethimide neither directly affects the plasma concentration of either LH or FSH nor blocks the effect of estradio! in inhibiting their release following castration. These data, taken together with our previous work, implicate aromatization of testosterone to estradiol in the control of gonadotropin secretion in the male. Key words: gonadotropins, testosterone, estradiol, aromatase, aminoglutethimide. J Andro! 1986; 7:234-239. Reprint requests: Richard E. Falvo, Ph.D., Department of Physiology, Southern Illinois University, School of Medicine, Lindegren Hall, Carbondale, Illinois 62901. Submitted for publication October 2, 1985; revised version received January 14, 1986; accepted for publication February 25, 1986. From the Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois and the 5Roman L. Hruska Meat Animal Research Center, U.S.D.A., Clay Center, Nebraska The role of testosterone (T) in the regulation of gonadotropin secretion has been studied in a large number of species, including rats (Swerdloff and Walsh, 1973; Block et a!, 1974), rams (Gamier et a!, 1977; D Occhio et a!, 1982), monkeys (Plant et al, 1978; Steiner et a!, 1978) and dogs (DePalatis et al, 1978; Falvo et al, 1979, 1982; Vincent et a!, 1979; Falvo and Vincent, 1980). However, the question as to whether T acts directly on the hypothalamus and pituitary or whether aromatization to E2, or reduction to dihydrotestosterone (DHT) are required for its action, remains unanswered (Worgul et al, 1981; Winter et al, 1983; Schanbacher, 1984). Although implicated in the control of gonadotropins by direct interaction with androgen receptors in the hypothalamic-pituitary unit (Swerdloff and Heber, 1980), these effects may be the result of T being converted to estradiol (E2) intracerebrally (Naftolin and Ryan, 1975). In the orchidectomized dog, both T and E2 are effective in reducing the high circulating levels of LH (Vincent et al, 1979) as well as FSH (Falvo and Vincent, 1980), whereas DHT is ineffective (Winter et a!, 1982). Previous work in our laboratory has indicated that physiologic levels of T in orchidectomized dogs can maintain LH chronically (Vincent et al, 1979) and FSH acutely (Falvo and Vincent, 1980) within the normal intact range. Furthermore, we also have shown that the aromatase inhibitor, aminoglutethi- 234
No.4 REGULATION OF GONADOTROPIN SECRETION Awoniyi et al 235 mide, prevents the ability of T to inhibit the postorchidectomy rise of LH in T-imp!anted orchidectomized dogs despite the presence of normal plasma concentrations of T and E2 (Winter et al, 1983). These data suggest that aromatizaiton of T to E2 is a necessary step in the regulation of LH secretion in the male dog. The lack of change in the peripheral concentration of E2, in the presence of aminoglutethimide, led led us to conclude indirectly that inhibition of aromatization occurred centrally, rather than peripherally (Winter et al, 1983). Data regarding FSH were inconclusive since in the absence of aminoglutethimide, T was able to maintain intact levels of FSH in the orchidectomized dog only acutely (approximately 6 days). To confirm whether the aromatase inhibitor (aminoglutethimide) used in our previous study either blocked the aromatization of T to E2 or the action of E2, we have repeated the protocol of our previous study (Winter et al, 1983), substituting Ez-imp!antation for T in both the presence or absence of aminoglutethimide following orchidectomy in the male dog. These data, with those we reported previously, further substantiate that the aromatization of T toe2 may be required for the control of gonadotropin secretion in the male dog. Design Methods and Materials Fifteen healthy, sexually mature male mongrel dogs were used in this study. They were housed indoors individually under controlled lighting (12 h light: 12 h dark) and temperature. Water and dog chow were available ad libitum. The dogs were equally divided into three groups. Group 1 was implanted with E2-filled polydimethylsiloxane capsules (PDS-E2). Group 2 was implanted with empty PDS capsules and treated with aminoglutethimide (Cytadren-AG, Ciba-Geigy Pharmaceuticals, Summit, NJ), while group 3 was implanted with PDS-E2 and treated with aminoglutethimide. This aromatase inhibitor (Graves and Salhanich, 1979) has been used in similar doses in both dogs (Worgul et al. 1981) and sheep (Schanbacher 1984). During the 24-day study, the protocol was as follows for each group: days 1 to 4, intact (untreated); day 4 capsule implantation; day 8, castration (capsules left in situ); days 14 to 24, aminoglutethimide administration to groups 2 and 3; and day 19, capsule removal. The oral dose of aminoglutethimide (60 mg bid.) was administered at 1000 hours following blood sampling and again at 1700 hours. Blood samples were drawn from all dogs for 1 hour at 20-minute intervals. Sampling took place daily for 24 days via saphenous or cephalic vein puncture. The plasma was separated by centrifugation, aliquoted, and pooled for each dog over the 1-hour period, and stored in three separate plasma vials at -20 C until assayed for E2, LH, and FSH. Capsule Preparation PDS capsule preparation and implantation was done according to a method previously published by this laboratory (Vincent et al, 1979). The capsules (3.35 mm LD X 4.65 mm OD) were implanted subcutaneously in the shoulder region under general anesthesia at the rate of 7.0-cm capsule length/b kg body weight. Hormone Assays Plasma LH was measured by RIA as described previously by this laboratory (DePalatis et al, 1978). Ovine LH (LER-1056-62) was used as the radiolabeled antigen. The first antibody was No. 15 of G.D. Niswender and the canine standards were purified canine LH (LER-1685-1). The sensitivity of the assay was 0.02 ng/tube with an interassay variation of 11%. Plasma FSH was measured by RIA as described previously by this laboratory. (Winter et a!, 1982). Ovine FSH (LER-1181-3) was used as the radiolabeled antigen. The first antibody was M91 (rabbit antiserum to human FSH) and the standards were purified canine FSH (LER-1685-3A). The sensitivity of the assay was 12.5 ng/tube with intra-assay and interassay values of 5.3 and 8.7, respectively. Plasma E2 was measured in duplicate 500-l volumes following extraction in freshly opened diethyl ether as described previously by this laboratory (Winter et al, 1983). The antiserum, GDN-E2-6 provided by GD. Niswender, has 30% displacing ability for estrone and virtually none for estriol when compared with E2. Extraction of canine steroid-stripped plasma was consistently undetectable. When 31.2 pg of unlabelled E2 was added to 1 ml of stripped canine plasma, the mean recovery was 25.7 ± 1.1 (SEM) pg for ten assays. The sensitivity of this assay was 0.85 pg/tube, and blank values were not subtracted from obtained values. Following overnight incubation at 4 C, bound steroid was separated from free steriod using dextran-coated charcoal. Statistical Analysis The significance of treatment effects among groups and within each group was tested. The statistical analysis included an analysis of variance followed by comparison of means by Tukey and Scheffe post-hoc tests at the 95% confidence limits. Results The mean concentration of E2, LH, and FSH obtained from daily blood samples for the five dogs in each group are shown in Fig. 1. Tables 1 to 3 show the mean plasma concentration of these same hormones for each entire treatment period. There were significant treatment effects both between and within the groups during the experiment. Table 1 shows the plasma E2 concentrations of the three groups of dogs. In the dogs implanted with PDS-E2 capsules (groups 1 and 3), the mean concentrations of E2 rose immediately after PDS-E2 capsule
236 Journal of Andrology. July/August 1986 Vol. 7 12. E 0, C I E U, -J 0 following castration on day 8 and remained low throughout the experiment. Table 2 shows the plasma LH concentrations of the three groups of dogs during the different treatment periods. In group 1, the only significant differences were found between the period of PDS-E2 implantation and following castration. In dogs implanted with empty PDS capsules and treated with aminog!utethimide (group 2), the plasma LH levels rose significantly following castration and continued to increase until the end of the experiment. In group 3 (PDS-E2 and treated with aminoglutethimide), the plasma levels of LH fell following capsule implantation and remained low until termination of the experiment. The capsule implantation and castration periods were not different from each other but differed significantly from all the other periods. The aminoglutethimide treatment and capsule removal periods were not significantly different from each other. The mean plasma concentrations of FSH are shown in Table 3. The plasma FSH levels in group 1 (PDS-E2) and group 3 (PDS-E2 and treated with aminoglutethimide) followed a similar trend in that the plasma FSH values were not affected by E2 treatments. Following capsule removal, FSH concentrations rose in group 1 but not in group 3. In dogs implanted with empty PDS capsules and treated with aminog!utethimide (group 2), plasma FSH concentrations rose immediately after castration and kept rising until the end of the experiment. Discussion a. 6 10 14 DAYS OF TREATMENTS Fig. 1. Profiles of plasma E2, LH, and FSH in dogs implanted with E2-filled polydimethylsiloxane capsules (PDS-E2) (group I *...*..._.*), with empty PDS and treated with aminoglutethimide (group 20-0-0), and with PDS-E2 and treated with aminoglutethimide (group 3--). Means ± SEM are shown in Tables 1, 2, and 3. Dogs were implanted with PDS on day 4, castrated on day 8, aminoglutethimide administered (groups 2 and 3) twice daily from days 14 to 19, and the PDS capsules removed on day 19. All treatments were performed following blood sampling. implantation, and remained elevated until they fell on day 20 following capsule removal. However, in group 2 (empty PDS capsule and treated with aminoglutethimide), the mean plasma E2 concentration fell Data collected in various species indicate that androgens and estrogens regulate LH secretion in the male (Santen, 1981). It has been shown further that some of the effects of T on the regulation of LH secretion are due to T conversion to E2 either centrally and/or peripherally (Worgul et a!, 1981). Previously, we reported that aminoglutethimide administration to T-implanted orchidectomized dogs caused an abrupt rise in plasma LH and indirectly concluded that central aromatization of T to E2 was a prerequisite step for feedback regulation (Winter et al, 1983). Our conclusions also were supported by other work in the dog using the same aromatase inhibitor (Worgu! et a!, 1981). However, our data and that of Worgu! et a! (1981) could be interpreted to indicate that aminoglutethimide stimulated LH secretion directly or inhibited the feedback action of E2 on its receptor. In the present study, we implanted orchidectomized dogs with E2 and administered aminoglu-
No. 4 REGULATION OF GONADOTROPIN SECRETION. Awoniyi et al 237 Treatment TABLE 1. Plasma Concentrations of E2 (p9/mi) in Dogs Treated with PDS-E2 (Group 1); Empty PDS + AG (Group 2); and PDS-E2 + AG (Group 3) Days Group 1 Group 2 Group 3 Pretreatment 0-4 9.9 ± O.9Bt 5.6 ± 03A,b 6.2 ± 07B,b PDS 5-8 90.6 ± 65B,a 6.0 ± 03A,b 90.9 ± 86A,a Castration 9-14 89.8 ± 6.2a 2.8 ± 0,3B,b 101.4 ± AG treatment (not group 1) PDS removal 15-24 20-24 84.9 ± 8.6 ± 36B,a PDS-E2 = Polydimethylsiloxane capsules filled with estradiol; AG = aminoglutethimide. 3.4 ± 08B,b 3.5 ± 1.3 94.6 ± 93A,a 3.4 ± 05B,a tsuperscripts represent significant differences at the 0.05 level. Uppercase superscripts compare the effect of treatments within each group (vertical comparison). Lowercase superscripts compare the effect of treatments between the groups (horizontal comparison). tethimide. No effect of aminoglutethimide was observed, since E2 was able to inhibit both LH and FSH secretion following orchidectomy. We have concluded that aminoglutethimide does not affect E2 action directly. It is important to note that the concentration of E2 achieved in this study following E2 implantation was approximately 10 times that found in intact dogs. This high level of E2 may have been the cause of the chronic suppression of both LH and FSH observed following E2 removal in groups 1 and 3 (Fig. 1). In our previous studies in dogs, removal of T in orchidectomized dogs results in a prompt increase in both gonadotropins (Vincent et a!, 1979). The most convincing data regarding E2 involvement in LH regulation in the male are those of Ellinwood et a!. (1984), who found that following implantation of male monkeys with an aromatase inhibitor, 1,4,6-androstatriene-3,17,dione (ATD), increases in both plasma LH and T were observed. If E2 was administered concurrently with ATD, the effect of ATD was abolished and neither plasma LH nor T were affected. ATD treatment was shown to reduce peripheral E2 levels to 30% and hypothalamic aromatase activity to 10 to 20% of control values. They concluded, as have we, that E2 formation plays an important role in the negative feedback regulation of LH secretion in males. Similar conclusions have been made in the T-treated orchidectomized ram using aminoglutethimide treatment (Schanbacher, 1984). In a previous study (Worgu! eta!, 1981), aminoglutethimide was administered to intact dogs also treated with hydrocortisone to suppress ACTH and further suppress adrenal androgen secretion. This regimen resulted in increased plasma concentrations of LH to castrate levels within seven days of treatment. Accompanying this increase of LH were increases in plasma concentrations oft. In constrast to T, plasma concentrations of E2 remained low. Since aminoglutethimide administration caused an increase in plasma LH in the presence of increasing plasma concentrations of T and constant levels of peripheral E2, these authors also concluded that central, rather than peripheral aromatization, was blocked by the aminoglutethimide. In contrast to the data obtained in dogs and monkeys, it has been reported (Marynick et a!, 1979) that aromatization is not essential for LH regulation. Men were pretreated with the aromatase inhibitor testolactone (Teslac, E.R. Squibb, Princeton, NJ) and then TABLE 2. Plasma Concentrations of LH (ng/ml) in Dogs Treated with PDS-E2 (Group 1); Empty PDS + AG (Group 2); and PDS-E2 + AG (Group 3) Treatment Days Group 1 Group 2 Group 3 Pretreatment 0-.4 1.1 ± 02AB a 1.5 ± O.4 1.8 ± PDS 5-8 0.1 ± 002B b 2.8 ± 10C a 0.3 ± 02B,a Castration 9-14 1.2±0.3 8.2±O.9Ba AG treatment (not group 1) PDS removal 15-24 20-24 0.8 ± 03AB b 0.4 ± 01AB b 13.8 ± l.1 13.9 ± 1#{149}3A 0.1 ± 0.04 0.5 ± Superscripts represent significant differences at the 0.05 level. Uppercase superscripts compare the effect of treatments within each group (vertical comparison). Lowercase superscripts compare the effect of treatments between the groups (horizontal comparison).
238 Journal of Andrology. July/August 1986 Vol. 7 TABLE 3. Plasma Concentrations of FSH (ng/ml) in dogs treated with PDS-E2 (Group 1); Empty PDS + AG (Group 2); and PDS-E2 + AG (Group 3) Treatment Days Group 1 Group 2 Group 3 Pretreatment 0-4 177 ± 93B a, 173 ± 137C a 1% ± 101A a PDS 5-8 167 ± 66B a 177 ± 99C,a 181 ± Castration 9-14 173 ± 97B b 372 ± 239B,a 203 ± 134A b AG treatment (not group 1) PDS removal 15-24 20-24 157 ± 96B b 210 ± 172A t, 662 ± 366A a 740 ± 368A a 200 ± 106A b 203 ± 103A b Superscripts represent significant differences at the 0.05 level. Uppercase superscripts compare the effect of treatments within each group (vertical comparison). Lowercase superscripts compare the effect of treatments between the groups (horizontal comparison). infused with T. Whereas plasma T concentrations rose following Teslac treatment, plasma E2 concentrations remained unchanged. Since LH and FSH were reduced either under both the Teslac plus T regimen or when T was infused in the absence of Teslac, these authors concluded that T directly altered gonadotropin secretion. However, the possibility that the decrease in gonadotropin secretion was due to central aromatization of T to E2 was not excluded. Their experiments could be interpreted as a reduction in peripheral but not central aromatization. Other data obtained in the male rat (Krey et a!, 1982) contradict those reported for dogs and monkeys. Normally, T induces elevations in brain nuclear estrogen receptor levels. When blockage of central aromatization via ATD administration was accomplished, no rise in plasma LH was observed in T- implanted castrated male rats. These authors concluded that aromatization was not crucial to gonadotropin regulation in the male rat. The reasons for the different results reported in the dog (Worgul et al, 1981; Winter et al, 1983) and those obtained in the monkey (Ellinwood et al, 1984), as compared to those conducted in men (Marynick et al, 1979) and the rat (Krey et al, 1982), may be threefold: 1) there may be a species difference in steroid specificity to negative feedback; 2) the dog and monkey may have different susceptibilities to centrally inhibited aromatization as compared to men and rats; and 3) the different aromatase inhibitors used and/or their dosages may have been the cause of the different results. While further work is required in the male, it is clear from our studies in male dogs that aromatization is involved in LH secretion. References Block GJ, MaskenJ, Kragt CL, Ganong WF. Effect of testosterone on plasma LH in adult male rats of various ages. Endocrinology 1974; 94:947-951. DePalatis L, Moore J, Falvo RE. Plasma concentrations of testosterone and LH in the male dog. J Reprod Fertil 1978; 52:201-207. D Occhio MJ, Schanbacher BD, Kinder JE. Relationship between serum testosterone concentration and patterns of luteinizing hormone secretion in male sheep. Endocrinology 1982; 110:1547-1554. Ellinwood WE, Hess DL, Roselli CE, Spies HG, ReskoJA. Inhibition of aromatization stimulates luteinizing hormone and testosterone secretion in adult male rhesus monkeys. J Clin Endocrinol Metab 1984; 59:1088-1096. Falvo RE, Vincent DL, LathropJC, Toenjes A. Effects of testosterone and testosterone propionate administration on luteinizing hormone secretion in the male mongrel dog. Biol Reprod 1979; 21:807-812. Falvo RE, Vincent DL. Testosterone regulation of follicle stimulating hormone secretion in the dog. J Androl 1980; 1: 197-201. Falvo RE, Gerrity M, Pirmann J, Winter M, Miller J, Vincent DL. Testosterone pretreatment and the response of pituitary LH to gonadotropin releasing hormone (GnRH) in the male dog. Androl 1982; 3:193-198. Gamier DH, Terqui M, Pelletier J. Plasma concentrations of luteinizing hormone and testosterone on castrated rams treated with testosterone and testosterone propionate. Reprod Fertil 1977; 49:359-361. Graves PE, Salhanick HA. Stereoselective inhibition of aromatase by enantiomers of aminoglutethimide. Endocrinology 1979; 105:52-57. Krey LC, MacLusky NJ, David PC, Lieberburg Dl, Roy EJ. Different intracellular mechanisms underlie testosterone s suppression of basal and stimulation of cyclic luteinizing hormone release in male and female rats. Endocrinology 1982; 110: 2159-2167. Marynick SF, Loriaux DL, Sherins RJ, Pita JC, Lipsett MB. Evidence that testosterone can suppress pituitary gonadotropin secretion independently of peripheral aromatization. J Clin Endocrinol Metab 1979; 49:396-398. Naftolin F, Ryan KJ. The metabolism of androgens in central neuroendocrine tissues. J Steroid Biochem 1975; 6:993-997. Plant TM, Hess DL, Hotchkiss J, Knobil E. 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No.4 REGULATION OF GONADOTROPIN SECRETION. Awoniyi et a! 239 115:944-950. Steiner RA, Schiller H, Barber J, Gale CC. LH regulation in monkeys (Macace nemestrina): Failure of testosterone and dihydrotestosterone to block the estradiol-induced gonadotropin surge. Biol Reprod 1978; 19:51-56. Swerdloff RS, Walsh PC. Testosterone and oestradiol suppression of LH and FSH in adult male rats: Duration of castration, duration of treatment and combined treatment. Acta Endocrinol 1973; 73:11-21. Swerdloff RS, Heber RE. Endocrine control of testicular function from birth to puberty. In: Burger H, de Kretser D, eds. The testis. New York: Raven Press, 1980; 107-115. Vincent DL, Kepic TA, LathropJC, Falvo RE. Testosterone regulation of luteinizing hormone secretion in the male dog. mt J Androl 1979; 2:241-249. Winter M, Pirmann J, Falvo RE, Schanbacher BD, MillerJ. Steroid control of gonadotropin secretion in the acutely orchidectomized dog. J Reprod Fertil 1982; 64:449-455. Winter M, Falvo RE, Schanbachem BD, Verholtz S. Regulation of gonadotropin secretion in the male dog: role of estradiol. Androl 1983; 4:319-323. Worgul Ti, Santen Ri, Samojlik E, Irwin C, Falvo RE. Evidence that brain aromatization regulates LH secretion in the male dog. Am J Physiol 1981; 241:E246-250. Call for Abstracts for the XIIIth Annual Meeting of the International Embryo Transfer Society The International Embryo Transfer Sociwety will hold its 13th annual meeting from January 25-27, 1987, at the Burlington Hotel, Dublin, Ireland. Free communications at this meeting will be presented as posters, of which abstracts will be published in the January, 1987, issue of Theriogenology. Abstracts submitted for consideration must be in English, and must be prepared according to Theriogenology s guidelines on special forms for direct offset reproduction, which are obtainable from the lets office. The abstract must report original data that has not been published previously, and must contain a statement of objectives, experimental methods, results, and conclusions. Students may enter their submissions in a competition, and may also be eligible to compete for a travel grant to attend the meeting. Potential competitors should request further details of eligibility requ irements, judging procedures, and prizes from the lets office before submitting their abstracts. The deadline for receipt of abstracts is August 15, 1986. Abstracts will not be considered if received after the due date. They should be mailed to: Dr. Maurice Boland Department of Agriculture University Lyons Newcastle College Estate P.O. Co. Dublin, Ireland For information on the main program contact: Dr. Sarah M. Seidel Executive Secretary International Embryo Transfer Society 3101 Arrowhead Road, LaPorte, CO 80535 Tel: (303) 482-1088