A.J. TILBROOK, 2 ' 3 D.M. DE KRETSER, 4 and I.J. CLARKE 5 ABSTRACT

BIOLOGY OF REPRODUCTION 49, 779-788 (1993) Human Recombinant Inhibin A Suppresses Plasma Follicle-Stimulating Hormone to Intact Levels But Has No Effect on Luteinizing Hormone in Castrated Rams' A.J. TILBROOK, 2 ' 3 D.M. DE KRETSER, 4 and I.J. CLARKE 5 Victorian Institute of Animal Science, 3 Werribee, Victoria 3030, Australia Institute of Reproduction and Development, 4 Monash University, Clayton, Victoria 3168, Australia Prince Henry's Institute of Medical Research,s Monash Medical Centre, Clayton, Victoria 3168, Australia ABSTRACT This study tested the hypothesis that inhibin is a major negative feedback regulator of FSH secretion but has minimal effects on LH secretion in rams. In experiment 1, castrated rams (wethers) were given either vehicle or human recombinant inhibin A (hr-inhibin) as three s.c. or three i.v. 50-jig injections 6 h apart or as one 50-ltg i.v. injection followed by 100-jpg infusion over 12 h. Human recombinant inhibin suppressed plasma FSH while the vehicle had no effect. The greatest suppression in plasma FSH was achieved following i.v. administration of hr-inhibin given either by repeated injection or by infusion. In experiment 2, wethers were given vehicle or a 50-jig i.v. injection followed by 800-jg infusion of hr-inhibin over 12 h. Infusion of hr-inhibin suppressed plasma FSH with a maximal suppression of 53.3% occurring between 15 and 24 h after the start of treatment. During this period, the plasma concentrations of FSH and inhibin were in the range of values for intact rams. Human recombinant inhibin did not influence plasma LH in either experiment. This study demonstrated that physiological treatment with inhibin, in the absence of testosterone, has the capacity to suppress plasma concentrations of FSH in wethers to the levels found in intact rams. INTRODUCTION The rapid rise in plasma concentrations of the gonadotropins LH and FSH after castration of rams [1-7] indicates that secretion of the gonadotropins is under the negative feedback control of testicular hormones. While control of FSH and LH secretion by the negative feedback of testicular steroids has been studied extensively in rams, the importance of the testicular glycoprotein hormone inhibin as a feedback regulator is less well understood. Many studies have shown that both androgens and estrogens negatively regulate the secretion of FSH and LH [1-6, 8-15], although the doses of testicular steroids used have not always been in the physiological range. When a physiological dose of testosterone was administered to castrated rams for 7 days, the plasma concentrations of LH were reduced into the range of values found in intact rams but there was no significant reduction of plasma concentrations of FSH [16]. Furthermore, the effect of testosterone was mediated through a hypothalamic effect on GnRH secretion [16], whereas the effect of inhibin on FSH is exerted at the level of the pituitary gland [17]. These findings suggest that the negative feedback regulation of LH may be predominantly under the control of testicular steroids while the control of FSH is likely to involve other testicular products such as inhibin. That inhibin has a physiological role as a feedback regulator of FSH secretion in rams is suggested from studies in which there was an increase in the plasma concentrations of FSH in ram lambs immunized against partially purified Accepted May 25, 1993. Received January 4, 1993. 'Supported by the National Health and Medical Research Council of Australia. 2 Correspondence and current address: Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. FAX: 61-3-565-2547. inhibin derived from bovine follicular fluid [18] and in adult rams immunized against the a-subunit of human recombinant inhibin [19]. Furthermore, we recently showed that a single i.v. injection of human recombinant inhibin A (hrinhibin) to castrated rams suppressed plasma concentrations of FSH with a maximal suppression of 20% occurring 6-10 h after injection [17]. This time course of action and degree of suppression of FSH is similar to the effects in ovariectomized ewes of hr-inhibin [20] and purified native bovine inhibin of 31 kda [21]. While there is evidence that inhibin may play a role in the feedback regulation of FSH secretion in rams, there is a need to apply more physiological treatments for a proper assessment of the role of inhibin in the control of FSH secretion. Physiological treatments of inhibin are also necessary to determine whether or not inhibin influences the LH secretion in rams. Although it is generally considered that the negative feedback regulation of LH is controlled predominantly by the testicular steroids, treatment of castrated rams with ovine [22] or bovine [23] follicular fluid, which contains some inhibinlike activity, was found to significantly suppress plasma LH. Also, inhibin was found to enhance GnRH-induced release of LH from sheep pituitary cells in vitro [24, 25] and to increase the number of GnRH receptors [26]. In contrast, administration of purified inhibin preparations to ewes [21] and heifers [27] did not affect the plasma concentrations of LH. To date there have been no physiological studies on the effect of purified inhibin on plasma LH in rams. Since the pattern of immunoactive inhibin in the plasma of rams is nonpulsatile [28], continuous treatment with purified inhibin is likely to provide more meaningful information about its role in the feedback control of gonadotropins than are single injections. Study of the direct effects 779

780 TILBROOK ET AL. of inhibin on plasma concentrations of FSH and LH has been limited by the lack of availability of sufficient quantities of purified inhibin preparations to conduct physiological studies. It is now possible to address this issue using hr-inhibin, which is bioactive in castrated rams [17]. The present study tested the hypothesis that inhibin is a major regulator of FSH secretion but has minimal effects on LH secretion in rams by determining the effectiveness of continuous treatment with hr-inhibin in suppressing plasma concentrations of FSH and LH in castrated rams. In an attempt to achieve an experimental paradigm that would give meaningful physiological data, we compared different modes of administration of hr-inhibin. Animals MATERIALS AND METHODS Adult Romney Marsh rams that had been castrated within the first 3 wk of birth (wethers) were used in experiments 1 and 2; for the duration of the experiments, the animals were penned individually in an animal house and offered a maintenance ration and water ad libitum. The mean (+ SE) live weight of the animals was 65.8 + 1.5 kg. These experiments were conducted at the Victorian Institute of Animal Science, Werribee, Victoria, Australia, during the nonbreeding season for this breed of sheep [29]. A group of 20 adult Romney Marsh rams were used to provide an indication of the plasma concentrations of FSH in intact rams during the nonbreeding season. The care and experimental use of the animals in these experiments conformed with the requirements of the Australian Prevention of Cruelty to Animals Act, 1986, and the National Health and Medical Research Council/Commonwealth Scientific and Industrial Research Organisation/Australian Agricultural Council Code of Practice for the Care and Use of Animals for Experimental Purposes. Experimental Procedure Plasma concentrations of FSH in intact rams. A blood sample was collected by venipuncture from each of the 20 intact rams at the same time that experiments 1 and 2 were conducted. Plasma was collected by centrifugation and stored at -15C for assay for FSH. Experiment 1 Twelve wethers were allocated to four groups (n = 3) and were treated with 1) one i.v. injection of vehicle followed by an infusion i.v. of vehicle for 12 h; 2) three s.c. injections of 50 Ixg of hr-inhibin 6 h apart; 3) three i.v. injections of 50 Rg of hr-inhibin 6 h apart; or 4) one i.v. injection of 50 g of hr-inhibin followed by an infusion i.v. of 100 plg of hr-inhibin for 12 h. Thus, all wethers in groups 2, 3, and 4 received 150 pxg of hr-inhibin over 12 h. The i.v. injections and infusions were given into the jugular vein via indwelling catheters (Dwellcath, Tuta Laboratories, Lane Cove, Australia); the s.c. injections were given under the skin of the foreleg. Vehicle or hr-inhibin was infused at a constant rate (2 ml/h) via a syringe infusion pump. Jugular venous samples were collected from all animals via indwelling catheters every 10 min for 3 h prior to treatment, throughout the treatment period, and for 6 h after the treatment period. Blood samples were then collected hourly over the next 6 h (from 18 to 24 h after the commencement of treatment) and every 10 min for 3 h 24 and 48 h after the commencement of treatment. Plasma was collected by centrifugation and stored at -15C for assay for FSH, LH, and inhibin. Separate catheters were used to infuse hr-inhibin and to take blood samples. Experiment 2 In this experiment, 8 wethers were allocated to two groups (n = 4) and were given either 1) an iv. injection and 12- h infusion of vehicle or 2) an i.v. injection of 50 ftg hrinhibin followed by an i.v. infusion of 800,ug hr-inhibin over 12 h. Blood samples were collected via indwelling jugular catheters every 15 min for 2 h prior to treatment, during treatment, and for 12 h after the end of treatment (i.e., 24 h after the commencement of treatment), then hourly until 60 h after the commencement of treatment. Further blood samples were collected every 12 h until 6 days (144 h) after the commencement of treatment. The plasma from these samples was processed as above and stored at -15 C until assayed for FSH, LH, and inhibin. Separate catheters were used to infuse hr-inhibin and to collect blood samples. Preparation of Hr-Inhibin The hr-inhibin, which had been purified from a recombinant mammalian cell line and characterized as described by Tierney et al. [30], was obtained from Biotech Australia Pty. Ltd. (Sydney, Australia). The preparation was originally stored in -35% acetonitrile/0.1% trifluoroacetic acid at -70 0 C. Each sample was thawed; then BSA (0.1% final concentration) was added, the acetonitrile removed by evaporation under nitrogen, and the remaining sample gel-filtered (Sephadex G25, PD 10 columns; Pharmacia, Uppsala, Sweden) in Dulbecco's phosphate buffer, ph 7.2 [31]. Aliquots of the inhibin fraction were stored at -70 0 C. For use in these experiments, each hr-inhibin sample was diluted in sterile saline to a concentration of either 25 or 50 VLg/ mil. The vehicle consisted of volumes of the phosphate buffer and saline identical to the volumes of hr-inhibin and saline used in each case. Radioimmunoassays The RIAs for FSH were conducted as described by Bremner et al. [32] using NIADDK-oFSH-RP1 as the standard. In twenty assays, the mean ( SE) assay sensitivity was 0.3 +-

EFFECT OF INHIBIN ON GONADOTROPINS IN RAMS 781 TABLE 1. Mean (± SE) plasma concentrations of FSH (ng/ml) in the wethers in experiment 1 treated with vehicle (veh.) or hr-inhibin given as 3 50-1g (s.c. or i.v.) injections 6 h apart or as a 50-ipg i.v. injection and 100-1.g infusion over 12 h (inf.). The stages of the experiment shown are pre-treatment (pre-treat) and hours relative to the start of treatment. The times 0-6 h and 6-12 h refer to the first and second half of the treatment period, respectively.* Hours relative to the start of treatment Treatment Pre-treat. 0-6 6-12 12-18 24-27 48-51 Veh. 10.7 0.2 11.0-0.1 10.9 ± 0.2 11.1 ± 0.1 11.3 ± 0.2 11.2-0.2 s.c. 14.02 ± 0.3 13.4 ± 0.2 12.7 ± 0.2* 12.1 ± 0.1' 11.6 ± 0.3* 13.3 ± 0.3 i.v. 23.2 ± 1.2 22.2 ± 0.8 16.8 ± 0.3* 14.4 ± 0.7** 14.8 ± 0.9* 20.3 ± 1.0 inf. 13.1 ± 0.4 12.7 ± 0.3** 9.0-0.2** 7.03-0.2** 10.1 ± 0.3* 13.0 ± 0.4 *Within rows means significantly different from pre-treatment values are indicated by *p < 0.05 and **p < 0.01. 0.03 ng/ml, and the range in sensitivities was 0.2-0.4 ng/ ml with a maximum precision of 2.6-6.6% at 6.3-9.1 ng/ ml. The intraassay coefficient of variation of < 10% was between 0.7-1.6 and 27.9-61.9 ng/ml, and the interassay coefficient of variation was 12.8% at 5 ng/ml and 10.23% at 10 ng/ml. Plasma concentrations of LH were measured by RIA as described by Lee et al. [33], with NIH LH S18 used as the standard. Eight assays for LH were conducted; mean (+ SE) assay sensitivity was 0.2 0.02 ng/ml, and range in sensitivity was 0.2-0.3 ng/ml. The maximum point of precision was 3.7-6-6% at 4.4-8.2 ng/ml. The intraassay coefficient of variation of < 10% was between 0.5-1.2 and 26.9-57.7 ng/ml, and the interassay coefficient of variation was 6.4% at 14 ng/ml. Immunoactive inhibin in the plasma was measured by use of a double-antibody RIA based on an antiserum (1989) raised against bovine 31-kDa inhibin and using iodinated 31-kDa bovine inhibin as tracer [34]. This assay has been validated for measurement of ovine inhibin [35] and crossreacts 288% with pro-ac, a product of the a-subunit of inhibin [36]. There is no significant cross-reactivity with activin A, transforming growth factor 13, and Mfillerian-inhibiting substance. For five assays of inhibin, the mean ( SE) assay sensitivity was 0.06 + 0.002 ng/ml with a range of 0.05-0.06 ng/ml. The maximum point of precision was 3.3-6.7% at 0.5-0.6 ng/ml. The intraassay coefficient of variation of < 10% was between 0.1-0.2 and 1.9-2.2 ng/ml, and the interassay coefficient of variation was 7.2% at 2.0 ng/ ml and 1.8% at 0.9 ng/ml. Statistical Analyses In both experiments, plasma concentrations of FSH and LH, amplitude of LH pulses, and number of LH pulses per hour were compared at various stages of the experiment through use of analysis of variance. Paired comparisons were made using least significant differences. In experiment 1, for each of the treatment groups, comparisons were made between pretreatment and 0-6 h, 6-12 h, 12-18 h, 24-27 h, and 48-51 h after the start of treatment. The 0-6 h and 6-12 h after the start of treatment represented the first and second 6 h of the treatment period, respectively. For plasma concentrations of FSH and LH, the mean of the concentration over each period was compared. For the amplitude TABLE 2. Mean (± SE) a) plasma concentrations of LH (ng/ml), b) amplitude of LH pulses (ng/ml), and c) number of LH pulses per hour in the wethers in experiment 1 treated with vehicle (veh.) or hr-inhibin given as 3 50-pg (s.c. or i.v) injections 6 h apart or as a 50-pg i.v. injection and 100-pRg infusion over 12 h (inf.). The stages of the experiment shown are pre-treatment (pre-treat) and hours relative to the start of treatment. The times 0-6 h and 6-12 h refer to the first and second half of the treatment period, respectively.* Hours relative to the start of treatment Treatment Pre-treat. 0-6 6-12 12-18 24-27 48-51 a) Concentration Veh. 6.4 + 0.3 6.7 ± 0.3 7.4 ± 0.3 6.9 ± 0.2 7.2 ±+ 0.3 6.5 ± 0.2 s.c. 6.9 + 0.2 6.3 ± 0.2 6.7 ± 0.2 7.0-0.2 7.2 ± 0.2 6.3 ± 0.2 i.v. 10.04 + 0.5 9.6-0.4 10.7 ± 0.5 10.8 ± 0.5 11.7 ± 0.6 10.9 ± 0.6 inf. 6.4 + 0.3 6.8 + 0.2 7.2 ± 0.2 6.4 ± 0.2 7.6 ± 0.3 8.2-0.4 b) Pulse amplitude Veh. 3.04 + 0.8 3.3 ± 1.1 3.8 ± 1.1 1.9 ± 0.2 2.4 ± 0.4 2.3 ± 0.5 s.c. 2.7 ± 0.1 2.7 ± 0.1 2.3 ± 0.1 2.4 ± 0.2 2.4 + 0.2 2.5 ± 0.2 i.v. 2.6 ± 0.7 2.7-0.7 2.9 ± 0.6 2.9 ± 1.1 3.9 ± 0.8 4.3 ± 2.0 inf. 2.8 ± 0.5 3.4 ± 0.6 3.2 + 0.7 2.5 + 0.2 4.4 + 0.9 4.3 ± 1.4 c) Number of pulses per hour Veh. 1.4 0.1 1.4 ± 0.1 1.3 + 0.1 1.5 0.1 1.6 0.1 1.6 + 0.1 s.c. 1.3 0.2 1.4 ± 0.2 1.3 0.2 1.1 0.2 1.4 0.1 1.2 0.1 i.v. 1.1 0.2 1.5 ± 0 1.3 + 0.2 1.1 0.2 1.3 0.2 1.3 ± 0.2 inf. 1.2 0.1 1.3 ± 0.2 1.3-0.1 1.3 0.1 1.3 0.2 1.6 + 0.2 *There are no significant differences between stages of the experiment for any of the parameters of plasma LH.

782 la r0 E 5- o 4 c "-3-5- E 4- r' c 3- Z I 1 z 0-2- - 1 Ii- - n 7 - /u 60 - \ 50 - c 40 - z30 - a b c TILBROOK ET AL. i.v. injection L 11 i.v. vehicle infusion.a ^ A s.c. inhibin injection-0,6&12h K i.v. inhibin injection-0,6&12h &. A I? r\ z - Z nl zs A ~ E 20 c 15 z 10 I 5 ẕ 0-3 : Ad ;n :i -n - 4t ln 0 3 6 9 12 15 18 HOURS FIG. 1. Plasma concentrations of immunoactive inhibin (ng/ml) in each of the wethers treated for 12 h with vehicle or hr-inhibin in experiment 1. The wethers were treated with a) an i.v. injection and 12-h infusion of vehicle; b) 3 s.c. injections of 50 Lg hr-inhibin 6 h apart; c) 3 i.v. injections of 50 Ig hr-inhibin 6 h apart; or d) an i.v injection of 50 Lg hr-inhibin followed by a 12-h infusion of 100 pg hr-inhibin. and frequency of LH pulses, the number of pulses in each period was calculated. In experiment 2, plasma concentrations of FSH for the wethers infused with saline and hrinhibin were compared between pretreatment and 0-6 h, 6-12 h, 15-21 h, 18-24 h, 30-36 h, 42-48 h, 72 h, 96 h, 120 h, and 144 h after the start of treatment. For LH, the plasma concentrations, the amplitude of pulses, and the number of pulses per hour were compared over the first 24 h of the experiment when frequent samples were collected (i.e., pretreatment and 0-6 h, 6-12 h, 12-18 h, and 18-24 h after the start of treatment). Pulses of LH were defined according to Karsch et al. [37] as abrupt increases that were greater than assay sensitivity, that exceeded the previous value by at least three times the standard deviation of the previous value, and that were followed by a progressive decline at a rate consistent with the reported half-life for LH of 29 min [38]. The frequency of LH pulses was expressed as the number of pulses per hour; the amplitude of pulses of LH was calculated as the difference between the peak and the preceding nadir. RESULTS Plasma Concentrations of FSH in Intact Rams The mean (+ SE) plasma concentration of FSH in the intact rams was 5.1 + 0.9 ng/ml with a range in values from 0.4 to 12.2 ng/ml.

EFFECT OF INHIBIN ON GONADOTROPINS IN RAMS 783 TABLE 3. Mean (+ SE) plasma concentrations (ng/ml) of FSH in the wethers of experiment 2 treated with an i.v. injection and 12-h infusion of vehicle (veh.) or a 50-pg i.v. injection and 12-h infusion of 800 lpg of hr-inhibin over 12 h. The stages of the experiment shown are pretreatment (pre-treat) and hours relative to the start of treatment. The times 0-6 h and 6-12 h refer to the first and second half of the treatment period, respectively.* Hours relative Treatment to start of treatment Vehicle Hr-inhibin Pre-treat 19.1 + 4.1 11.7 + 1.7 0-6 19.7 + 4.1 11.1 + 1.4 6-12 19.1 + 4.3 7.9 + 1.2* 15-21 18.2 + 4.6 5.5 +- 0.8* 18-24 18.7 ± 4.6 5.5 + 0.9** 30-36 19.8 + 4.7 8.4 + 1.5 42-48 20.9 + 4.6 11.3 + 1.8 72 22.0 + 4.3 11.6 + 1.6 96 23.02 ±+ 5.0 11.2 + 0.6 120 23.01 + 5.0 14.01 + 2.3 144 23.01 ±+ 5.0 13.7-1.9 *Within columns, means significantly different (p < 0.01) from pre-treat are indicated by **. Experiment 1 Plasma FSH. Plasma concentrations of FSH were suppressed in the wethers administered hr-inhibin, while the vehicle had no effect (Table 1). Plasma concentrations of FSH were not significantly decreased in the first 6 h of treatment, but significant decreases from pretreatment levels were evident 6-12 h after the start of treatment in the wethers given s.c. or i.v. injections (p < 0.05) of hr-inhibin and in those infused with hr-inhibin (p < 0.01). In all groups, plasma concentrations of FSH remained significantly (p < 0.05) lower than pretreatment concentrations for at least 24-27 h after the start of treatment, but were similar to pretreatment values 48-51 h after the start of treatment (Table 1). The largest decreases in plasma FSH occurred in the wethers given hr-inhibin as i.v. injections or as an i.v. injection followed by infusion; for both these modes of administration, the maximal suppression in plasma concentrations of FSH was observed 12-18 h after the start of treat- 40 n E 30 c 20 r* I L 10 0 / I I I 1 0 6 12 18 24 30 36 42 48 72 96 120 144 HOURS -f I ZD 20 E r C L) 15 10 5 sp-b'"/"/v7 ;pp9a V O- ki"p4~~~~~8::: 0 0 6 12 18 24 30 36 42 48 HOURS j' ~,. 72 96 120 144 FIG. 2. Plasma concentrations of FSH (ng/ml) in wethers treated for 12 h with vehicle or hr-inhibin in experiment 2. The wethers were treated with a) an i.v. injection and 12-h i.v. infusion of vehicle or b) an i.v, injection of 50 jig hr-inhibin followed by an infusion of 800 llg hr-inhibin over 12 h.

784 TILBROOK ET AL. TABLE 4. Mean (- SE) a) plasma concentrations of LH (ng/ml), b) amplitude of LH pulses (ng/ml), and c) number of LH pulses per hour over the first 24 h in the wethers treated with an i.v. injection and 12-h infusion of vehicle or a 50-,Ig i.v. injection and 12-h infusion of 800 pg of hr-inhibin over 12 h in experiment 2. The stages of the experiment are pre-treatment (pre-treat) and hours relative to the start of treatment. The times 0-6 and 6-12 refer to the first and second half of the treatment period, respectively.* Hours relative to the start of treatment LH parameter Pre-treat 0-6 6-12 12-18 18-24 a) Concentration vehicle 5.0 + 0.8 4.8 + 0.5 7.8-1.4 7.4 + 1.7 7.0 + 1.1 hr-inhibin 3.6 ±+ 0.3 3.9 ± 0.3 4.6 ± 0.5 4.1 ±+ 0.5 4.2 + 0.4 b) Amplitude vehicle 2.4 ± 1.1 1.9 + 0.2 3.1-0.7 2.3 ±+ 0.4 2.6 ± 0.4 hr-inhibin 1.6 -+ 0.6 1.3 + 0.2 1.2 + 0.2 1.2 ±+ 0.1 1.8 + 0.4 d) Pulses/hour vehicle 0.8 ± 0.3 1.2 + 0.2 1.2 ± 0.2 1.0 ± 0.1 1.7 +0.4 hr-inhibin 1.3 ± 0.3 0.9 + 0.3 1.1 ± 0.2 0.8-0.1 1.04 + 0.2 *Within treatments, there are no significant differences between stages of the experiment. ment (i.e., in the 6 h following the end of treatment). For the wethers given i.v. injections of hr-inhibin, plasma concentrations of FSH were suppressed by 27.7% (p < 0.05) between 6 and 12 h after the start of treatment and by 38.1% (p < 0.01) between 12 and 18 h after the start of treatment; they were still 36.4% (p < 0.05) lower than pretreatment concentrations 24-27 h after the start of treatment (Table 1). Plasma concentrations of FSH were 31.7% (p < 0.01) lower than pretreatment concentrations 6-12 h after the start of hr-inhibin infusion; maximal suppression in plasma FSH, of 46.3% (p < 0.01), occurred 12-18 h after the start of hr-inhibin infusion. Concentrations were still 22.6% (p < 0.05) lower than pretreatment levels 24-27 h after the start of the infusion treatment. For the wethers given s.c. injections of hr-inhibin, plasma concentrations of FSH were 13.4% lower than pretreatment concentrations 12-18 h af- 8 6 E c z m I z 4 2 0 0 12 24 48 72 96 120 144 168 HOURS FIG. 3. Plasma concentrations of immunoactive inhibin (ng/ml) in wethers treated with a) an i.v. injection and infusion of vehicle for 12 h or b) an i.v. injection of 50 ig hr-inhibin followed an infusion of 800 Ig hr-inhibin over 12 h.

EFFECT OF INHIBIN ON GONADOTROPINS IN RAMS 785 ter the start of treatment (p < 0.05) and 17% lower 24-27 h after the start of treatment (p < 0.05). Plasma LH. None of the treatments with hr-inhibin or vehicle significantly affected mean ( SE) plasma concentrations of LH, amplitude of LH pulses, or number of LH pulses per hour (Table 2). For each treatment group, there were no significant differences between stages of the experiment. Plasma inhibin. Figure 1 shows the plasma concentrations of immunoactive inhibin for the wethers in each treatment group in experiment 1. The plasma concentrations (ng/ml) of immunoactive inhibin were mostly undetectable in the wethers treated with vehicle and in those receiving s.c. injections of hr-inhibin (Fig. 1). Among wethers given s.c. injections of hr-inhibin, only 6 samples measured above the sensitivity of the assay. Over the 12-h treatment period, the mean ( SE) plasma concentration of immunoactive inhibin was 1.3 + 0.3 ng/ml in wethers given i.v. injections of hr-inhibin and was 0.5 ± 0.1 ng/ml in those infused with hr-inhibin. These concentrations were significantly (p < 0.01) different. Experiment 2 Plasma FSH. Infusion of 800 plg of hr-inhibin for 12 h after an i.v. injection of 50 jig hr-inhibin significantly (p < 0.01) suppressed plasma concentrations of FSH in the wethers in experiment 2; vehicle had no effect (Table 3; Fig. 2). Plasma concentrations of FSH in wethers infused with hr-inhibin were significantly (p < 0.01) lower than pretreatment values 6-12 h after the start of treatment, with mean ( SE) concentrations reduced from 11.7 + 1.7 to 7.9 + 1.2 ng/ml (Table 3). The maximal suppression occurred between 15 and 24 h after the start of treatment; at this time the mean plasma concentrations fell to 5.5 ng/ml with a range of values from 4.4 to 8.0 ng/ml, representing a 53.3% (p < 0.01) suppression from pretreatment values (Table 3). These values were similar to those observed in the intact rams (see above). Although the plasma concentrations of FSH were approximately 28% lower than pretreatment levels 30-36 h after the start of treatment, this difference did not reach statistical significance. Plasma concentrations of FSH were similar to pretreatment values for the remainder of the experiment. Plasma LH. The mean (+ SE) plasma concentrations of LH, amplitudes of LH pulses, and numbers of LH pulses per hour are shown in Table 4. For both groups, there were no significant changes in any of the parameters of plasma LH during the experiment. Plasma inhibin. Figure 3 shows the plasma concentrations of immunoactive inhibin in wethers infused with vehicle or given an i.v. injection of 50 jig in combination with 12-h infusion of 800,g hr-inhibin in experiment 2. The mean (+ SE) plasma concentration of immunoactive inhibin over the 12-h infusion with hr-inhibin was 3.8 + 0.8 ng/ml. We found that the mean ( SE) plasma concentration of immunoactive inhibin in untreated rams of this breed during the nonbreeding season is 3.7 ± 0.2 ng/ ml [28]. DISCUSSION The experiments reported here provide convincing evidence that inhibin plays a major role in the negative feedback regulation of FSH in rams but has little effect on LH secretion. In both experiments, hr-inhibin in the absence of testosterone significantly suppressed plasma concentrations of FSH in castrated rams; but plasma concentrations, pulse frequency, and pulse amplitude of LH were not affected by hr-inhibin. These results corroborate our recent findings that hr-inhibin is bioactive in suppressing plasma FSH in wethers [17] and show that infusion of 800 g of hr-inhibin over 12 h can suppress plasma FSH to concentrations into the range of values found in intact rams. Human recombinant inhibin has also been found to be bioactive in ovariectomized ewes [20]. Although all modes of administration of hr-inhibin in experiment 1 suppressed plasma concentrations of FSH, administration directly into the jugular vein (either three i.v. injections 6 h apart or one iv. injection followed by a 12-h infusion) was more effective than s.c. injections. Plasma concentrations of inhibin during the treatment period were higher in the wethers given three i.v. injections of hr-inhibin than in those given one i.v. injection followed by a 12-h infusion; but this comparison can be misleading because of the manner in which inhibin was presented. The higher concentrations in the wethers given i.v. injections would be expected because an injection was given at the end of the treatment period, resulting in maximal concentrations immediately after injection. In these experiments we attempted to achieve an experimental regimen that would yield physiological information, and in this regard the infusion of hr-inhibin gave a more realistic pattern of plasma inhibin because the secretion of inhibin is nonpulsatile in intact rams [28]. Nevertheless, the plasma concentrations achieved in wethers infused with 100 jig of hr-inhibin over 12 h in experiment 1 were about eightfold lower than those found in intact rams [28], so a higher dose of hr-inhibin was infused in experiment 2. This produced plasma concentrations similar to those found in intact rams of this breed [28]. Importantly, this physiological treatment with hr-inhibin resulted in plasma concentrations of FSH during the period of maximal suppression that were similar to those measured in intact rams; this confirmed that inhibin has major negative feedback effects on FSH in rams, at least during the nonbreeding season. In contrast to the effects on FSH, the lack of effect of hrinhibin on plasma LH indicates that inhibin does not contribute to the negative feedback control of LH secretion in rams. Studies in which castrated rams were treated with crude preparations of inhibin consisting of follicular fluid

786 TILBROOK ET AL. [22, 23], or in which the effects of inhibin on pituitary cells from sheep [24-26] were investigated in vitro, have suggested that the secretion of LH may be affected by inhibin. However, we have now studied in vivo the effects of inhibin on LH secretion by administering a physiological treatment of purified inhibin to wethers. The lack of effect of hr-inhibin on plasma LH in wethers is similar to findings in ewes [21] and heifers [27], in which purified preparations of inhibin did not influence plasma LH. Our data suggest that inhibin selectively inhibits the secretion of FSH in rams, and this is in keeping with the accepted definition of inhibin as a glycoprotein hormone that preferentially inhibits the production and/or secretion of FSH [39]. Treatment of wethers with a physiological dose of testosterone for a week was reported to reduce plasma concentrations of LH to levels found in intact rams [16]; this suggests that the secretion of LH is principally under the negative feedback control of the testicular steroids. Although the results from experiment 2 demonstrate that inhibin plays a major role in the negative feedback control of FSH secretion in rams, testosterone is also a negative feedback regulator of this gonadotropin at the level of the pituitary gland. In a study during the nonbreeding season using wethers with hypothalamo-pituitary disconnection (HPD) [40] and replacement with GnRH, we administered hr-inhibin and testosterone alone and in combination and found that both hormones had actions directly on the pituitary to suppress plasma FSH [17]. Furthermore, it appeared that testosterone synergizes with inhibin in the negative feedback regulation of FSH, because the suppressive effects of hr-inhibin were significantly greater when the wethers were being treated with testosterone than when they were not receiving any steroid treatment [17]. An interaction between inhibin and steroids in the control of FSH secretion has also been suggested in ewes. The administration of low doses of estradiol and follicular fluid together produced a greater suppression in the plasma concentrations of FSH than did treatment with either hormone alone [41], and immunization against both hormones caused a greater rise in plasma concentrations of FSH than did immunization against either hormone alone [42]. It is difficult to assess the relative roles of inhibin and testosterone in the control of FSH secretion from our recent investigations with HPD wethers [17] because, although the dose of testosterone administered was physiological, the hr-inhibin treatment was a single i.v. injection. The relative roles of inhibin and testosterone in the negative feedback regulation of FSH require further investigation with physiological treatments of both hormones. The negative feedback control of FSH in rams is further complicated by a possible difference in the roles of inhibin and testosterone with respect to the stage of the breeding season. Despite the significant effect of testosterone on plasma FSH in HPD wethers during the nonbreeding season [17], a similar treatment of HPD wethers during the breeding season did not influence the plasma concentrations of FSH; this showed that the direct pituitary actions of testosterone during the breeding season are minimal [16]. Also, administration of a physiological dose of testosterone to hypothalamo-pituitary-intact wethers during the breeding season was unable to reduce plasma concentrations of FSH to intact levels [16]. These differences suggest that during the nonbreeding season, both inhibin and testosterone are important in the negative feedback control of FSH in rams, whereas during the breeding season, testosterone has minimal effects. Presumably, inhibin is the predominant feedback regulator of FSH secretion in rams during the breeding season, and this hypothesis needs to be tested. When the breeding cycle of Soay rams was artificially manipulated by lighting treatments or administration of melatonin, it was found that changes in plasma concentrations of inhibin occurred in parallel with the cycle of the diameter of the testes-with concentrations of inhibin, FSH, LH, and testosterone rising in unison with activation of the reproductive axis [43]. There was a positive correlation between the plasma concentrations of inhibin and FSH during the developing and regressing stages of the testicular cycle and a negative correlation during the active stage [43]. It was suggested that FSH stimulates inhibin secretion during the developing and regressing phases of the testicular cycle whereas during the active phase the negative feedback effects of inhibin predominate [43]. Despite these longitudinal investigations into the patterns of plasma inhibin, testosterone, and the gonadotropins in rams, the relative roles of inhibin and testosterone in the negative feedback control of FSH secretion at the various stages of the testicular cycle have not been ascertained. Nevertheless, the results of the present experiments and of our recent study with HPD wethers [17] collectively indicate that both inhibin and testosterone are major negative feedback regulators of FSH secretion in rams during the nonbreeding season. Although the mechanism of action of inhibin in suppressing FSH has not been studied in rams, it has been conclusively demonstrated in ovariectomized ewes [20,44] and heifers [45] that inhibin causes a rapid reduction in the levels of mrna for FSH3 in the pituitary gland. In ovariectomized ewes, transcription rates for the FSHi gene were reduced by 50% six hours after treatment and FSHPi mrna levels were virtually eliminated [20]. Also, hr-inhibin suppressed FSH3 mrna in vitro without any effect on transcription rate, and it was suggested that inhibin acts to reduce mrna for FSHP3 through one or more mechanisms additional to a reduction in transcription rate [20]. It is feasible that inhibin may have similar actions in rams, since the actions of hr-inhibin in wethers ([17], present study) and ovariectomized ewes [20] are similar. Nevertheless, it is notable that the recovery of plasma concentrations of FSH is slow after continuous treatment with hr-inhibin (30-36 h), but rapid after a single injection (12 h) [17, 20]. Thus earlier experiments using a single injection to study the ac-

EFFECT OF INHIBIN ON GONADOTROPINS IN RAMS 787 tion of inhibin may be misleading; continuous infusion, which closely approximates the physiological output of inhibin, may be more appropriate. In conclusion, this study demonstrated that administration of a physiological treatment of inhibin to wethers, in the absence of testosterone, has the capacity to suppress plasma concentrations of FSH to the intact range. Human recombinant inhibin was most effective in suppressing FSH when administered i.v., and an infusion of 800 g hr-inhibin over 12 h produced concentrations of immunoactive inhibin and FSH in the range measured in intact rams. This demonstrates that inhibin clearly plays a major role in the negative feedback control of FSH secretion in rams during the nonbreeding season. In contrast, none of the treatments with hr-inhibin affected plasma LH. Whereas our present findings and those from a recent study [17] collectively suggest that the negative feedback control of FSH secretion involves both inhibin and testosterone, it appears that the negative feedback regulation of LH secretion is predominantly under the influence of the testicular steroids. ACKNOWLEDGMENTS We thank A. Skinner, J. Muir, M. Purdon, T. Simpson, and T. Pisano for technical assistance. We are grateful to Biotech Australia Pty Ltd for the generous donation of hr-inhibin and to Dr. D.M. Robertson of Prince Henry's Institute of Medical Research for preparation of the hr-inhibin. We also thank NIDDK for RIA reagents. REFERENCES 1. Riggs BL, Malven PV. Spontaneous patterns of LH release in castrate male sheep and the effects of exogenous estradiol. J Anim Sci 1974; 38:1239-1244. 2. Schanbacher BD, Ford D. Gonadotropin secretion in cryptorchid and castrate rams and the acute effects of exogenous steroid treatment. Endocrinology 1977; 100:387-393. 3. 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