BIOLOGY OF REPRODUCTION 55, (1996)

BIOLOGY OF REPRODUCTION 55, 386-392 (1996) Direct Actions of the Luteinizing Hormone-Releasing Hormone Agonist, Deslorelin, on Anterior Pituitary Contents of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH), LH and FSH Subunit Messenger Ribonucleic Acid, and Plasma Concentrations of LH and FSH in Castrated Male Cattle' William J. Aspden, 3 4 Alexandra Rao, 5 Paul T. Scott, 4 lain. Clarke, 5 Timothy E. Trigg, 6 John Walsh, 6 and Michael. D'Occhio 2, 3 CSIRO Division of Tropical Animal Production,I Tropical Beef Centre, Rockhampton Mail Centre, Rockhampton, Queensland 4702, Australia Department of Biology, 4 Central Queensland University, Rockhampton, Queensland 4702, Australia Prince Henry's Institute of Medical Research, s Clayton, Melbourne, Victoria 3168, Australia Peptech Animal Health Pty Ltd.,6 Dee Why, New South Wales 2099, Australia ABSTRACT The objective in this study was to characterize direct effects of the LHRH agonist, deslorelin, on anterior pituitary gland function in male cattle in the absence of gonadal feedback. Castrated bulls (steers), 30 mo old, were allocated to four groups: group 1, control, no treatment (n = 8); group 2, five deslorelin implants (- 250 Ig total deslorelin/day) for 42 days (n = 8); group 3, control + LHRH (50 I.g i.m.) at weekly intervals (n = 3); group 4, five deslorelin implants + LHRH as for group 3 (n = 3). Plasma LH was similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 4, and continued to decrease to Day 41 (group 1, 1.71 + 0.20 ng/ml [mean + SEM]; group 2, 0.38 0.03 ng/ml [p < 0.0011). Mean plasma concentrations of FSH were similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 7, and declined to Day 41 (group 1, 43.5 3.9 ng/ml; group 2, 17.5 + 1.5 ng/ml [p < 0.001]). Steers in group 3 showed increases in plasma LH after injection of LHRH on all occasions, while steers in group 4 did not show increases in plasma LH from Day 14 onward. Mean relative pituitary contents (arbitrary units) of LHB- and FSHI3- subunit mrnas were reduced on Day 42 in steers treated with deslorelin (LHP: groups 1 and 3, 1.56 ± 0.27; groups 2 and 4, 0.08 ± 0.01 [p < 0.001]; FSHP: groups 1 and 3, 1.01 0.08; groups 2 and 4, 0.34 ± 0.07 [p < 0.001]). However, a-subunit mrna was similar for control steers and steers treated with deslorelin (groups 1 and 3, 1.00 0.11; groups 2 and 4, 0.86 + 0.12 [p > 0.11). Pituitary content of LH, but not FSH, was reduced in steers treated with deslorelin. In summary, steers treated with deslorelin showed desensitization to natural LHRH, and this was associated with reduced pituitary contents of LH and FSH,(-subunit mrnas, a reduction in pituitary content of LH, and decreases in plasma concentrations of LH and FSH. This demonstrated, for the first time, a direct action of LHRH agonist on LH and FSH 3-subunit gene expression in cattle, independent of gonadal feedback. Also, there was a differential effect of treatment with deslorelin on gonadotropin a- and -subunit mrna contents in the anterior pituitary. Accepted March 26, 1996. Received January 3, 1996. 'This study was supported, in part, by a University Research Grant from Central Queensland University, Rockhampton, Queensland, Australia. 2Correspondence: Dr. Michael J. D'Occhio, CSIRO Division of Tropical Animal Production, Tropical Beef Centre, PO Box 5545, Rockhampton Mail Centre, Rockhampton, Queensland 4702, Australia. FAX: 61 79 361034; e-mail: m.docchio@roll.rock.tap.csiro.au 386 INTRODUCTION Treatment with agonists of LHRH results in suppression of endocrine activity of the pituitary-testicular axis and eventual azoospermia in rats [1, 2], dogs [3-6], rams [7, 8], pigs [9], baboons [10], rhesus monkeys [11, 12], and men [13, 14]. In contrast, the pituitary-testicular axis remains apparently unchanged in mice [ 15, 16] and marmoset monkeys [17, 18] treated with LHRH agonist, while in the red deer, plasma concentrations of both LH and testosterone are increased during agonist treatment [19]. In several studies in the bull, treatment with LHRH agonists was associated with typical mean plasma concentrations of LH and FSH and increased secretion of testosterone [20-24]. Although mean plasma concentrations of LH were typical in bulls treated with agonist, pulsatile secretion of LH was abolished [22]. The latter observation provided evidence that gonadotroph cells of bulls treated with LHRH agonist are insensitive to natural LHRH; this was supported by the failure of bulls receiving agonist to show an increase in plasma LH after injection of LHRH [21]. Consistent with these observations, bulls treated with agonist had reduced numbers of pituitary LHRH receptors and decreased pituitary LH content [22]. The changes at the anterior pituitary of bulls treated with LHRH agonist would appear to be inconsistent with maintenance of typical mean plasma concentrations of LH [20-24]. It is possible that an apparent lack of change in mean plasma LH under these circumstances may reflect an inability of RIAs to discriminate subtle differences in plasma LH between gonadal-intact bulls, which under normal conditions tend to have relatively low plasma concentrations of LH, and bulls treated with LHRH agonist. In men treated with LHRH agonist, RIA of LH overestimated relatively low plasma concentrations of LH in comparison to immunofluorometric analysis [25]. Castration results in an increase in plasma concentrations of LH and FSH in bulls [21]. In the present study, therefore, castrated bulls (steers) were used as a model to examine direct effects of LHRH agonists on pituitary gonadotroph cell function in male cattle in the absence of gonadal feedback. We argued that since steers have relatively high plasma concentrations of LH and FSH, any down-regulation of pituitary function induced by treatment with an LHRH agonist would be clearly reflected in decreases in plasma LH and FSH. Any changes in pituitary contents of LH and FSH subunit mrnas in response to agonist treatment should likewise be readily apparent in steers. Accordingly, longterm-castrated steers were treated with the LHRH agonist,

MOLECULAR AND ENDOCRINE RESPONSES OF STEERS TO LHRH AGONIST 387 deslorelin, and observed for changes in pituitary contents of LH and FSH, LH and FSH subunit mrnas, and plasma concentrations of LH and FSH. The findings would indicate whether or not LHRH agonists can directly down-regulate the anterior pituitary gland in male cattle. This information is considered of fundamental importance for an understanding of the basis for apparent differential responses of the pituitary-testicular axis in males of different species during treatment with LHRH agonists. MATERIALS AND METHODS LHRH Agonist, Bioimplant Formulation, and Implantation The LHRH agonist, deslorelin (D-Trp 6 -Pro 9 -des-gly 10 - LHRH ethylamide [26]), was formulated into bioimplants that were 2.5 x 3 mm and contained 2.14 mg of deslorelin [21]. When incubated in vitro, the implants have an initial release rate of 50-100 g/24 h for 2-3 days (determined by HPLC and absorbance at 278 nm) and the release rate then stabilizes at approximately 50 pjg/24 h (Peptech Animal Health Pty Ltd., Sydney, Australia). Implants were placed s.c. in the ear under aseptic conditions by means of a commercial implanting device. Experimental Design Steers used in the study were maintained under range conditions except when required for experimentation. Standard management practices were used. Twenty-two, 30-mo-old Brahman (Bos indicus) steers were randomized on liveweight into four groups: group 1 (n = 8), control, no treatment; group 2 (n = 8), five deslorelin implants; group 3 (n = 3), LHRH injection (50 jig i.m.) on Days 0, 7, 14, 21, 28, and 41; group 4 (n = 3), five deslorelin implants, as well as LHRH injections as for group 3. Blood samples were taken from steers in groups 1 and 2 at 0 and 24 h and on Days 2, 4, 7, 11, 14, 18, 21, 25, 28, and 41 of treatment. For steers in groups 3 and 4, blood samples were taken on Days 0, 7, 14, 21, 28, and 41 immediately before LHRH injection (Time 0) and then at 30, 60, and 120 min after injection. Increases in plasma LH (ALH) and plasma FSH (AFSH) after injection of LHRH were calculated by subtracting the concentrations at Time 0 from respective maximal concentrations after injection of LHRH. Liveweights (kg) were recorded on Days 0 and 41. Pituitary glands were excised after slaughter on Day 42. Anterior pituitary glands were hemisected midsagittally, plunged into liquid nitrogen, and then stored at -80 C until processed. Hormone Assays Plasma concentrations of LH and FSH were determined through use of double-antibody RIAs [27]. Intra- and interassay coefficients of variation for both assays were < 10% based on duplicate samples. Sensitivities of the assays were 0.2 ng USDA bovine (b)lh-b-5/ml and 4.0 ng USDA bfsh-bp3/ml. Pituitary LH and FSH Content Hemipituitaries were weighed and then homogenized in sucrose buffer (10 mm Tris, 0.1% BSA [w:v], NaN3 0.1% [w:v], sucrose 25 mm; ph 7.4; 4C [28]). Homogenates were centrifuged at 30 000 X g for 30 min at 4 C [28], and the resulting supernatants were diluted and assayed for concentrations of LH and FSH by means of the same assay procedures as specified above for plasma LH and FSH. Results are expressed as nanograms hormone/milligrams anterior pituitary tissue. Pituitary RNA Extraction Total cellular RNA was extracted as previously described [29]. Briefly, a hemipituitary from each animal was weighed and then homogenized in 9.5 ml of lysis buffer (5 M guanidine thiocyanate, 5% -mercaptoethanol, 18 mm N-lauroyl-sarcosine, 1.25 M cesium chloride, 10 mm Tris buffer [ph 7.4], 10 mm EDTA). Total cellular RNA was isolated by ultra-centrifugation of lysates through a 1.25 M/5.7 M cesium chloride density gradient at 80 000 g for 14 h at 20 C. RNA pellets were dissolved in 400 l of sterile distilled water; they were then precipitated by adding 40 Il of 3 M sodium acetate solution (ph 5.2) and 800 I of cold 100% ethanol and placement at -80 C for 2 h. After centrifugation in a microfuge for 15 min, pellets were washed in 70% ethanol, dried, and then dissolved in 100 I1 of sterile distilled water. Concentrations of RNA were estimated by spectrophotometry. Northern Blot Analysis Northern blot analysis was based on the procedure of Mercer and Clarke [30]. Aliquots (12.5 g) of total cellular RNA were run on a glyoxal denaturing agarose gel (1.2%) and then Northern blotted onto Hybond-N membrane (Amersham, Buckinghamshire, UK). Gonadotropin subunit cdna probes were used to assess relative pituitary mrna contents. The bovine LH13-(525 bp) and bovine FSH[-subunit (559 bp) cdna probes used were kindly provided by Dr. S. Chapel (Integrated Genetics, Framingham, MA); the rat a-subunit cdna probe (- 500 bp) [31] was kindly provided by Dr. W. Chin (Brigham and Women's Hospital, Boston, MA). The rat a-subunit cdna is 80% homologous to that of cattle [31, 32]. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe was used to eliminate differences due to loading [33]. Aliquots (25 ng) of cdna probes were random-prime labeled (Readyprime; Amersham) with [a- 32 P]-dCTP (Readyview; Amersham). The GAPDH riboprobe was labeled during RNA transcription by addition of RNA SP6 polymerase and [- 32 P]-dUTP [33]. In each hybridization, 1 x 106 cpm/ml of incorporated probe was used. Hybridization buffer consisted of 50% formamide, 50 mm phosphate buffer (ph 8.0), 5-strength SSC (0.75 M NaC1, 0.075 M sodium citrate; ph 7.0), 0.2% (w:v) BSA, 0.2% (w:v) polyvinylpyrrolidone-40, 0.2% (w:v) ficoll, Escherichia coli trna (40 RIg/ml), and herring sperm DNA (20 Kg/ml). The blot was sequentially probed for LH[3-, FSH[-, and a-subunit mrna and then for GAPDH mrna. The membrane was hybridized in 10 ml of hybridization buffer for 16 h at 42 C in a hybridization oven (Hybaid Micro-4; Hybaid Ltd, Middlesex, UK); it was then washed twice at room temperature for 20 min in double-strength SSC + 0.1% (w:v) SDS and exposed to Fuji RX100 (Fuji Film Co., Tokyo, Japan) x-ray film. Higher-stringency washes were undertaken where required to reduce background further. The membrane was stripped of probe (98 C, 0.1% [w:v] SDS + 0.1- strength SSC, for 10 min) and checked overnight on x-ray film before each new hybridization. Autoradiography results were quantitated by densitometry using an Olympus CUE-2 image analysis system (Olympus Corporation, Lake Success, NY).

388 ASPDEN ET AL. E 11- M a E 0 a @2 E N c U- C E a Q. 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 L 0 Ou an 50 40 30 20 10 o- Time 0 to the time of peak response in the LHRH tests within each treatment, the aired-sample t-test was used. Where necessary, data were logl0 transformed before analysis to improve homogeneity of variance. Results for plasma concentrations of LH, FSH, and testosterone (ng/ml), pituitary LH and FSH content (nanograms hormone/milligrams anterior pituitary tissue), mrna content (mean relative units), and liveweight (kg) are means + SEM. RESULTS Changes in Liveweights Liveweight (kg) means at assignment to treatment groups were for group 1, 453 + 11; for group 2, 465 I I I I I I 13; for group 3, 462 + 21; and for group 4, 452 15 kg; 7 14 21 28 35 42 (p > 0.05). There were no significant (p > 0.05) differences in liveweight gains over the treatment period between the Day of treatment four groups of steers (group 1, 11.8 + 2.4; group 2, 5.5 2.3; group 3, 10.7 + 1.8; group 4, 16.0 + 5.3, kg). FIG. 1. Plasma conc entrations (ng/ml) of LH and FSH in controls (group 1, open circles) and in steers treated with deslorelin (group 2, solid circles). Results are me. Plasma LH and FSH Mean plasma concentrations of LH for steers in groups 1 and 2 are shown in Figure 1. There was no difference (p > 0.05) in mean plasma concentrations of LH on Day 0 between steers in group 1 (1.39 ± 0.13 ng/ml) and steers in group 2 (1.36 + 0.12 ng/ml). Mean plasma LH was first significantly (p < 0.05) reduced in steers treated with deslorelin as compared with controls on Day 4 (group 1, 1.69 ± 0.12 ng/ml; group 2, 1.20 0.11 ng/ml) and further declined until on Day 41 the plasma concentrations of LH were for group 1, 1.71 ± 0.20 ng/ml and for group 2, 0.38 + 0.03 ng/ml (p < 0.001). Analysis from Day I to Day 41 showed treatment, time, and treatment x time all to be I I I I I, significant (p < 0.001), as shown in Figure 1 by the decline 0 7 14 21 28 35 42 in plasma LH over time in treated steers relative to controls. The plasma ALH responses of steers in groups 3 and 4 Day of treatment after injection of LHRH are shown in Table 1. Groups 3 and 4 did not differ (p > 0.05) in the ALH response to LHRH injection on Day 0, but differed (p < 0.01) on all other days. Within group 3, the LH response to LHRH injection was lower on Day 21 than in four of the other tests, the reason not being known. For steers in group 3, all plasma LH responses were significant (p < 0.05) when paired Statistical Analysses comparisons were undertaken between the LH concentratime points were examined by ANOVA tion at Time 0 and the maximum concentration after injec- Data at single procedures using the General Linear Models (GLM) pro- tion of LHRH. For steers in group 4, increases in plasma cedure of SAS/S;TAT [34]. Data analyses over time were undertaken by re t peated measures analysis using SAS/STAT procedure MIXE D with REML estimation, and of type autoregressive(l) [: 35]. The model was y = treatment, time, treatment X time, with animal as the repeated subject. The CONTRAST startement of SAS/STAT procedure of GLM LH after injection of LHRH were significant (p < 0.05) on Days 0 and 7, and not significant (p > 0.05) on Days 14, 21, 28, and 41. The mean plasma concentrations of FSH for steers in groups 1 and 2 are shown in Figure 1. There was no dif- ference (p > 0.05) in mean plasma concentrations of FSH was used for comiparisons of group means. For comparisons on Day 0 between steers in group 1 (38.03 2.01 ng/ml) of increases in pilasma LH (ALH) and FSH (AFSH) from and steers in group 2 (46.98 5.21 ng/ml). Plasma FSH TABLE 1. Increases in plasma concentrations of LH (ALH; ng/ml) after i.m. injection of 50 plg natural LHRH in control steers (group 3) and steers treated with deslorelin (group 4) (means + SEM, n = 3). Day 0 7 14 21 28 41 Control 8.86a - 0.97 8.90 x t 0.60 9.47 a x + 1.11 4.1 5 y + 0.25 14.05 X t+ 5.32 8.03= a y + 2.50 Deslorelin 6.31 + 0.96 1.03'Y + 0.11 0. 44 bz + 0.10 0.1 2 b z + 0.12 0.1 6 bz 0.14 0.08 b, 0.07 ab Means in columns without a common superscript differ (p < 0.05). xy, Means in rows without a common superscript differ (p < 0.05).

MOLECULAR AND ENDOCRINE RESPONSES OF STEERS TO LHRH AGONIST 389 TABLE 2. Increases in plasma concentrations of FSH (AFSH; ng/ml) after i.m. injection of 50 I.g natural LHRH in control steers (group 3) and steers treated with deslorelin (group 4) (means SEM, n = 3). 0 7 14 21 28 41 Control Deslorelin 0.49 x ±+ 2.43 1.78' ax 2.09 12.45-3.82 4.23 " X - 1.95 16.01W a 1. 21 b X, 1.78 1.52 11.71W Y + 0.53 2.5 3 b x + 1.81 16.79ay + 3.72 2.11 bx ± 0.97 7.08axy - 3.63 2.91 ax - 1.91 a,b Means in columns without a common superscript differ (p < 0.05). -'Y Means in rows without a common superscript differ (p < 0.05). Day was first significantly reduced (p < 0.05) in steers treated with deslorelin as compared with control steers on Day 7 (group 1, 38.37 + 2.82 ng/ml; group 2, 27.28 3.14 ng/ml) and further declined until on Day 41 the plasma concentrations of FSH were for group 1, 43.47 3.94 ng/ml and for group 2, 17.49 + 1.52 ng/ml (p < 0.001). Analysis from Day 1 to Day 41 showed that treatment, time, and treatment x time were all significant (p < 0.001), as shown in Figure 1 by the decline in plasma FSH in treated steers relative to controls. The plasma AFSH responses of steers in groups 3 and 4 after injection of LHRH are shown in Table 2. In contrast to findings for LH, there was no consistent increase in plasma FSH for steers in group 3 after injection of LHRH. This may have been related to the relatively small number of steers used to assess responses to LHRH. Steers in group 4 did not (p > 0.05) show an increase in plasma FSH on any occasion after injection of LHRH, including the LHRH injection given on Day 0 before implantation with deslorelin. Pituitary LH and FSH Contents Pituitary contents of LH and FSH from controls and from steers treated with deslorelin are shown in Table 3. Pituitary content of LH is presented separately for the four treatment groups. Pituitary content of FSH was pooled for groups 1 and 3 and for groups 2 and 4, respectively, as there were no differences (p > 0.05) in pituitary FSH content. Pituitary content of LH was reduced (p < 0.05) in control steers periodically treated with GnRH as compared with control steers not treated with GnRH. Steers treated with deslorelin had significantly (p < 0.001) reduced pituitary content of LH compared to steers not treated with deslorelin. Pituitary content of FSH did not differ (p = 0.56) between steers treated with deslorelin and controls. Pituitary LH and FSH mrnas There was no significant difference (p > 0.05) in yield of total cellular RNA between the four groups (group 1, 0.84 + 0.12; group 2, 0.81 0.10; group 3, 0.94 0.10; group 4, 1.05 + 0.05 pxg/mg tissue). Northern blot autoradiography results of probing for steer pituitary LH3-, FSH3-, and at-subunit mrna and GAPDH mrna are shown in Figure 2. The mrna content for each gonadotropin subunit did not differ (p > 0.05) for groups 1 and 3 or for groups 2 and 4, respectively, and the respective data were pooled (n = 11). Relative pituitary contents of the gonadotropin subunit mrnas between control steers and steers treated with deslorelin are shown in Figure 3. There was a significant decrease (p < 0.001) in relative pituitary content of LHP3-subunit mrna in steers treated with deslorelin (0.08 + 0.01) as compared with control steers (1.56 0.27). Relative pituitary content of FSHI-subunit was reduced (p < 0.001) in steers treated with deslorelin (0.34 ± 0.07) compared with controls (1.01 + 0.08). However a-subunit mrna relative content was similar (p = 0.39) in steers treated with deslorelin (0.86 + 0.12) and controls (1.00 + 0.11). DISCUSSION The present findings demonstrated, for the first time, a direct action of LHRH agonist in suppressing anterior pituitary gland contents of LH and FSH -subunit mrnas in male cattle. This was similar to the recently reported observation of reduced pituitary contents of LH and FSH -subunit mrnas in castrated male rats treated with LHRH agonist [36]. In steers, the relative decrease was greater for the LH -subunit than for the FSH -subunit, and this was associated with a decrease in pituitary content of LH but TABLE 3. Pituitary LH and FSH content in control steers and steers treated with deslorelin (means + SEM). LH FSH Treatment n (ng/mg pituitary) n (ng/mg pituitary) Control 8 227.9 + 25.6' 11 1515 + 168a Control + GnRH 3 144.2 + 28.1 b Deslorelin 8 25.6 + 1.9' 11 1390 + 127a Deslorelin + GnRH 3 38.1-5.5 c a.bc Means within columns without a common superscript differ (a from b, p < 0.05; and b from, p < 0.001). FIG. 2. Northern blot autoradiography results of probing for pituitary LHP-, FSHP-, and a-subunit mrna and GAPDH mrna in steers. Group 1 (control, lanes 1-8), group 3 (control + LHRH tests, lanes 9-11), group 2 (deslorelin, lanes 12-19), and group 4 (deslorelin + LHRH tests, lanes 20-22).

390 ASPDEN ET AL. W2.4 J $3 @3 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 <.oo001 Control LHp FSHp a FIG. 3. Pituitary LHI3-, FSHI3-, and a-subunit mrna relative contents (means + SEM) in controls (n = 11) and in steers treated with deslorelin (n = 11). I. not FSH. In contrast to the latter observation, it was reported by Melson et al. [22] that gonadal-intact bulls showed decreases in pituitary contents of both LH and FSH after 15 days of treatment with an LHRH agonist. Circulating concentrations of LH and FSH were decreased in steers receiving deslorelin, with the relative decrease again being greater for LH. A significant decline in circulating concentrations of LH and FSH after commencement of treatment occurred over 4-7 days and was likely due to a combination of down-regulation of gonadotroph cell LHRH receptors and desensitization of second messenger systems. The relative contributions of these two processes to the initial responses of gonadotroph cells to agonist treatment in cattle remains to be determined. Collectively, the findings in the present study established that cattle conform to the general phenomenon of pituitary down-regulation during treatment with LHRH agonist in males. The results also suggested that LHRH agonists can have a differential effect on LH and FSH synthesis, storage, and release in castrated male cattle. It is not apparent why the decreases in plasma concentrations of LH and FSH observed in steers in the present study are not also observed, albeit to a lesser degree, in gonadal-intact bulls treated with deslorelin [20-24]. Furthermore, in gonadal-intact bulls, typical mean plasma concentrations of LH during agonist treatment are associated with increased testosterone secretion [20-24]. Lunn et al. [18] found that intact male and female marmoset monkeys treated with LHRH agonist had typical plasma concentrations of LH and gonadal steroids, but that ovariectomized marmosets showed a decrease in plasma LH during agonist treatment. It was suggested that in intact marmosets, gonadal factors may serve to maintain typical secretion of LH during treatment with LHRH agonist [18]. Although the mechanism(s) whereby in some species gonadal factors may counter the effects of LHRH agonists on LH secretion has not been explained, it is possible that a similar response may occur in the bull. The red deer stag is also of particular interest in this regard, since this species shows increased LH and testosterone secretion during treatment with LHRH agonist [19]. It is possible that pituitary down-regulation is fundamentally a common response in males of all species and that differential responses in the pituitary-testicular axis are due to secondary mechanisms involving interactions between the pituitary and testes, which may differ between species. The possibility of direct actions of LHRH agonists at the testes, with differences between species, cannot be excluded. We recently reported that gonadal-intact bulls treated with deslorelin failed to show an increase in plasma LH after injection of natural LHRH [21]. In the same study, bulls treated with deslorelin had not shown an increase in plasma LH within 14 days after castration and, at 14 days after castration, did not show an LH response to LHRH injection [21]. In this earlier study using acutely castrated bulls, it remained possible that the responses observed may have been due to longer-term effects of increased plasma concentrations of testosterone during treatment with deslorelin before castration. In the present study, long-termcastrated steers became insensitive to natural LHRH after 1 wk of treatment with LHRH agonist, as demonstrated by the absence of an acute increase in plasma LH after injection of LHRH. Down-regulation of the anterior pituitary in cattle, therefore, does not require testicular feedback. Controls did not show a consistent response in FSH after injection of LHRH. The control of FSH release from the pituitary in cattle involves complex interactions that include inhibin, activin, and follistatin [37]; this may explain the lack of a consistent FSH response to injection of LHRH. Because of the lack of a consistent FSH response to LHRH injection in controls, it was not possible to draw a conclusion on the effect of deslorelin treatment on the FSH response to LHRH. Pituitary contents of LH, and LH and FSH -subunit mrnas, remained detectable in steers treated with deslorelin. Plasma concentrations of LH and FSH also remained above respective assay sensitivities. It would appear, therefore, that expression of LH and FSH 3-subunit mrna, and synthesis and release of LH and FSH, can continue in steers despite a down-regulated pituitary, although at reduced levels. Most transcriptional systems, including the a-subunit promoter, have both hormone-induced and basal components, and it is likely that the latter could support the maintenance of mrna levels in a down-regulated and desensitized pituitary. Treatment with deslorelin had no effect on pituitary content of a-subunit mrna in steers. Similar findings were recently reported for castrated rats treated with LHRH agonist [36]. It would appear, therefore, that LHRH agonists can have differential effects on regulation of gene expression of the specific 3-subunits of LH and FSH as well as the common a-subunit of pituitary glycoprotein hormones. Also, thyrotroph cells, which presumably are not influenced by LHRH agonists, synthesize a-subunit as well. In contrast to the findings in castrated males, a-subunit mrna content was increased in intact male rats treated with LHRH agonist [36,38]; this was also reported for intact female sheep [39]. The apparent discrepancies in a-subunit mrna responses to LHRH agonist treatment between intact and castrated individuals have not been explained, but may involve pituitary-gonadal interactions in intact animals treated with agonist. In summary, treatment of steers with deslorelin resulted in decreased anterior pituitary content of LH, and LH and FSH 3-subunit mrnas, reduced plasma concentrations of LH and FSH, and pituitary insensitivity to natural LHRH. The anterior pituitary gland in male cattle, therefore, undergoes classical down-regulation and desensitization during treatment with the LHRH agonist deslorelin. Anterior pituitary content of (-subunit mrna was not influenced

MOLECULAR AND ENDOCRINE RESPONSES OF STEERS TO LHRH AGONIST 391 by treatment with deslorelin, suggesting a differential regulation of expression of gonadotropin -subunits as compared to the common a-subunit of anterior pituitary glycoprotein hormones. ACKNOWLEDGMENTS The authors thank Mr. G. Halford, Mr. J. Quilty, and Mr. G. Weldon ("Belmont" National Cattle Breeding Station) for their assistance in field activities and management of animals; Dr. D.L. Hamernik (Department of Physiology, University of Arizona, Tucson, AZ) for providing advice on RNA methodologies; Dr. D.J. Bolt (USDA Animal Hormone Program, Beltsville, MD) for the generous supply of LH and FSH assay reagents; and the staff of Teys Brothers Abattoir (Biloela, Queensland, Australia) for cooperation in obtaining pituitary glands. Support from the Livestock Improvement Program, CSIRO Division of Tropical Animal Production, is gratefully acknowledged. REFERENCES 1. Labrie F, Auclair C, Cusan I, Kelly PA, Pelletier G, Ferland L. Inhibitory effect of LHRH and its agonists on testicular gonadotropin receptors and spermatogenesis in the rat. Int J Androl Suppl 1978; 2: 303-318. 2. Ward JA, Purr BJA, Valaccia B, Curry B, Bardin CW, Gunsalus GL, Morris ID. Prolonged suppression of rat testis function by depot formulation of Zoladex, a GnRH agonist. J Androl 1989; 10:478-486. 3. Cavitte J-Ch, Lahlou N, Mialot JP, Mondain-Monval M, Mialot M, Nahoul K, Morel C, Roger M, Schally A-V. Reversible effects of long-term treatment with D-Trp 6 -LH-RH microcapsules on pituitarygonadal axis, spermatogenesis and prostate morphology in adolescent and adult dogs. Andrologia 1988; 20:249-263. 4. Lacoste D, Dube D, Trudel C, Belanger A, Labrie E Normal gonadal functions and fertility after 23 months of treatment of prepubertal male and female dogs with the GnRH agonist [D-Trp 6, des-gly- NH' 2]GnRH ethylamide. J Androl 1989; 10:456-465. 5. Lacoste D, Labrie F, Dube D, Belanger A, Tice T, Gilley RM, Pledger KL. Reversible inhibition of testicular androgen secretion by 3-, 5- and 6-month controlled release microsphere formulations of the LH-RH agonist [D-Trp 6, des-gly-nh 1 2]LH-RH ethylamide in the dog. J Steroid Biochem 1989; 33:1007-1011. 6. Vickery BH, McRae GI, Briones WV, Roberts BB, Worden AC, Schanbacher BD, Falvo RE. Dose-response studies on male reproductive parameters in dogs with nafarelin acetate, a potent LHRH agonist. J Androl 1985; 6:53-60. 7. Lincoln GA, Fraser, HM, Abbott MP Blockade of pulsatile LH, FSH and testosterone secretion in rams by constant infusion of an LHRH agonist. J Reprod Fertil 1986; 77:587-597. 8. Caraty A, Locatelli A, Delaleu B, Spitz IM, Schatz B, Bouchard P. Gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists do not alter endogenous GnRH secretion in short-term castrated rams. Endocrinology 1990; 127:2523-2529. 9. Xue JL, Dial GD, Bartsh S, Kerkaert B, Squires EJ, Marsh WE, Ferre G. Influence of a gonadotropin-releasing hormone agonist on circulating concentrations of luteinizing hormone and testosterone and tissue concentrations of compounds associated with boar taint. J Anim Sci 1994; 72:1290-1298. 10. Vickery BH, McRae GI. Effects of continuous treatment of male baboons with super agonists of LHRH. Int J Fertil 1980; 25:179-184. 11. Bint Akhtar F, Marshall GR, Wickings EJ, Nieschlag E. Reversible induction of azoospermia in rhesus monkeys by constant infusion of a gonadotropin-releasing hormone agonist using osmotic minipumps. J Clin Endocrinol & Metab 1983; 56:534-540. 12. Mann DR, Gould KG, Collins DC. Influence of continuous gonadotropin-releasing hormone (GnRH) agonist treatment on luteinizing hormone and testosterone secretion, the response to GnRH, and the testicular response to human chorionic gonadotropin in male rhesus monkeys. J Clin Endocrinol & Metab 1984; 58:262-267. 13. Roger M, Chaussain J-L, Berlier P. Bost M, Canlorbe P, Colle M, Francois R, Garandeau P, Lahlou N, Morel Y, Schally AV. Long-term treatment of male and female precocious puberty by periodic administration of a long-acting preparation of D-Trp 6 -luteinizing hormonereleasing hormone microcapsules. J Clin Endocrinol & Metab 1986; 62:670-677. 14. Schurmeyer T, Knuth UA, Freuschem CW, Sandow J, Akhtar FB, Nieschlag E. Suppression of pituitary and testicular function in normal men by constant gonadotropin-releasing hormone agonist infusion. J Clin Endocrinol & Metab 1984; 58:19-24. 15. Bex FJ, Corbin A, France E. Resistance of the mouse to the antifertility effects of LHRH agonists. Life Sci 1982; 30:1263-1269. 16. Wang NG, Sudaram K, Pavlou S, Rivier S, Vale W, Bardin CW. Mice are insensitive to the antitesticular effects of luteinizing hormone-releasing hormone agonists. Endocrinology 1983; 112:331-335. 17. Lunn SE Dixson AF Sandow J, Fraser HM. Pituitary-testicular function is suppressed by an LHRH antagonist but not by an LHRH agonist in the marmoset monkey. J Endocrinol 1990; 125: 233-239. 18. Lunn SF, Cowen GM, Morris KD, Fraser HM. Influence of the gonad on the degree of suppression induced by an LHRH agonist in the marmoset monkey. J Endocrinol 1992; 132:217-224. 19. Lincoln GA. Long-term stimulatory effects of a continuous infusion of LHRH agonist on testicular function in male red deer (Cervus elaphus). J Reprod Fertil 1987; 80:257-261. 20. Bergfeld EGM, D'Occhio MJ, Kinder JE. Continued desensitization of the pituitary gland in young bulls after treatment with luteinizing hormone-releasing hormone agonist deslorelin. Biol Reprod 1996; 54: 769-775. 21. D'Occhio MJ, Aspden WJ. Characteristics of luteinizing hormone (LH) and testosterone secretion, pituitary responses to LH-releasing hormone (LHRH), and reproductive function in young bulls receiving the LHRH agonist deslorelin: effect of castration on LH responses to LHRH. Biol Reprod 1996; 54:45-52. 22. Melson BE, Brown JL, Schoenemann HM, Tarnavasky GK, Reeves JJ. Elevation of serum testosterone during chronic LHRH agonist treatment in the bull. J Anim Sci 1986; 62:199-207. 23. Rechenberg Wv, Sandow J, Klatt P. Effect of long-term infusion of an LH-releasing hormone agonist on testicular function in bulls. J Endocrinol 1986; 109:R9-RII. 24. Ronayne E, Enright WJ, Roche JE Effects of continuous administration of gonadotropin-releasing hormone (GnRH) or a potent GnRH analogue on blood luteinizing hormone and testosterone concentrations in prepubertal bulls. Domest Anim Endocrinol 1993; 10:179-189. 25. Huhtaniemi I, Venho P, Jacobi G, Rannikko S. Responses of circulating gonadotropin levels to GnRH agonist treatment in prostatic cancer. J Androl 1991; 12:46-53. 26. Karten MJ, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function studies towards the development of agonists and antagonists: rationale and perspectives. Endocr Rev 1986; 7:44-66. 27. D'Occhio MJ, Gifford DR, Weatherly T, Setchell BP. Evidence of gonadal steroid-independent changes in activity of the central LH-releasing hormone pulse generator in developing bull calves. J Endocrinol 1986; 111:67-73. 28. Brown JL, Reeves JJ. Absence of specific luteinizing hormone releasing hormone receptors in ovine, bovine and porcine ovaries. Biol Reprod 1983; 29:1179-1182. 29. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18:5294-5299. 30. Mercer JE, Clarke IJ. Regulation of anterior pituitary gonadotropin subunit mrna levels by gonadotropin-releasing hormone in the ewe. J Neuroendocrinol 1989; 1:327-331. 31. Godine JE, Chin WW, Habener JE ca Subunit of rat pituitary glycoprotein hormones. J Biol Chem 1982; 257:8368-8371. 32. Erwin CR, Croyle ML, Donelson JE, Maurer RA. Nucleotide sequence of cloned complementary deoxyribonucleic acid for the a-subunit of bovine pituitary glycoprotein hormones. Biochemistry 1983; 22:4856-4860. 33. Tso JY, Sun X-H, Kao T-H, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cdnas: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 1985; 13:2485-2502. 34. SAS. SAS/STAT User's Guide, Volume 2, GLM-VARCOMP, Version 6, Fourth Edition. Cary, NC: Statistical Analysis System Institute Inc.; 1990: 891-996. 35. SAS. SAS/STAT Technical Report P-229, SAS/STAT Software: Changes and Enhancements, Release 6.07. Cary, NC: Statistical Analysis System Institute Inc.; 1992: 287-368. 36. Lerrant Y, Kottler M-L, Bergametti F, Moumni M, Blumberg-Tick J, Counis R. Expression of gonadotropin-releasing hormone (GnRH) re-

392 ASPDEN ET AL. ceptor gene is altered by GnRH agonist desensitization in a manner similar to that of gonadotropin -subunit genes in normal and castrated rat pituitary. Endocrinology 1995; 136:2803-2808. 37. Kogawa K, Nakamura T, Sugino K, Takio K, Titani K, Sugino H. Activin-binding protein is present in pituitary. Endocrinology 1991; 128:1434-1440. 38. Lalloz MRA, Detta A, Clayton RN. Gonadotropin-releasing hormone desensitization preferentially inhibits expression of the luteinizing hormone P-subunit gene in vivo. Endocrinology 1988; 122:1689-1694. 39. McNeilly JR, Brown P, Clark AJ, McNeilly AS. Gonadotropin releasing hormone modulation of gonadotropins in the ewe: evidence for differential effects on gene expression and hormone secretion. J Mol Endocrinol 1991; 7:35-43.