CSIRO PUBLISHING
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Reproduction, Fertility and Development, 2007, 19, 891–898
Pituitary and testicular endocrine responses to exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone in male dogs treated with GnRH agonist implants A. JunaidiA,B,E,F , P. E. WilliamsonA , G. B. MartinB , P. G. StantonC , M. A. BlackberryB , J. M. CumminsA and T. E. TriggD A Division
of Veterinary and Biomedical Sciences, Murdoch University, Perth, Murdoch, WA 6150, Australia. B School of Animal Biology, The University of Western Australia, Crawley, WA 6009, Australia. C Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia. D Peptech Animal Health Pty Limited, 19–24 Khartoum Road, Macquarie Park, NSW 2133, Australia. E Present address: Faculty of Veterinary Medicine, Gadjah Mada University, J1. Olahraga - Karangmalang, Yogyakarta 55281, Indonesia. F Corresponding author. Email:
[email protected]
Abstract. The present study tested whether exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) can stimulate LH and testosterone secretion in dogs chronically treated with a GnRH superagonist. Twenty male adult dogs were assigned to a completely randomised design comprising five groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other four groups received a 6-mg implant containing a slow-release formulation of deslorelin (d-Trp6 -Pro9 -des-Gly10 –LH-releasing hormone ethylamide). The same four control dogs were used for all hormonal challenges, whereas a different deslorelin-implanted group was used for each challenge. Native GnRH (5 µg kg−1 bodyweight, i.v.) was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 µg kg−1 bodyweight, i.v.) was injected on Days 16, 26, 41 and 101. On all occasions after Day 25–26 postimplantation, exogenous GnRH and LH elicited higher plasma concentrations of LH and testosterone in control than deslorelin-treated animals (P < 0.05). It was concluded that, in male dogs, implantation of a GnRH superagonist desensitised the pituitary gonadotrophs to GnRH and also led to a desensitisation of the Leydig cells to LH. This explains, at least in part, the profound reduction in the production of androgen and spermatozoa in deslorelin-treated male dogs. Additional keywords: desensitisation, deslorelin, testosterone.
Introduction Long-term treatment with a gonadotrophin-releasing hormone (GnRH) agonist suppresses the activity of the pituitary–gonadal axis in males of many species, including rodents, sheep and primates (Labrie et al. 1978; Vickery and McRae 1980; Akhtar et al. 1983; Mann et al. 1984; Schurmeyer et al. 1984; Lincoln et al. 1986; Roger et al. 1986; Ward et al. 1989; Caraty et al. 1990; Martin et al. 1996). In canines also, chronic treatment with a superagonist strongly reduces luteinising hormone (LH) secretion, leading to a marked decline in the production of testosterone, as well as involution of the testes and accessory sex glands (Labrie et al. 1980; Vickery 1982; Vickery et al. 1985; Cavitte et al. 1988; Lacoste et al. 1989a, 1989b; Trigg et al. 2001; Junaidi et al. 2003). In contrast with these species, red deer stags treated with the agonist buserelin (1.5 mg subcutaneous rod, 0.5 cm long) showed increases in both LH and testosterone secretion (Lincoln 1987); however, in marmoset monkeys, the same agonist caused a © CSIRO 2007
transient rise in LH concentrations, after which the concentrations of both LH and testosterone remained at normal values over an extended period (Lunn et al. 1990, 1992). In sheep, treatment with leuprolide (subcutaneous injection of 0.1 mg kg−1 ) led to persistently high secretion of both LH and testosterone (Martin et al. 1996), in contrast with bulls, where treatment with leuprolide, buserelin or nafarelin led to elevated testosterone concentrations despite normal plasma LH concentrations (Melson et al. 1986; Rechenberg et al. 1986; Ronayne et al. 1993). Despite this variation in the endocrine outcome among species and among GnRH superagonists, these treatments are generally all thought to work the same way: by downregulating GnRH receptors and, thus, desensitising the pituitary gonadotrophs to endogenous GnRH. The reduced pituitary sensitivity to GnRH is evidenced by loss in responsiveness to a challenge with native GnRH, as has been demonstrated in male sheep, primates and cattle (Akhtar et al. 1983; Mann et al. 1984; Lincoln et al. 1986; Lunn et al. 1992; D’Occhio and Aspden 10.1071/RD07088
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1996; Martin et al. 1996; Aspden et al. 1998). Nevertheless, it is clear that this needs to be verified if we are to correctly interpret the mechanism of action of treatment for all combinations of species and superagonist chemistries (Martin et al. 1996). In contrast with the extensive literature focused on the effect of GnRH superagonists at the level of the gonadotroph, little is known about changes in the function of Leydig cells during long-term treatment. Changes in testosterone concentration may simply reflect passive responses to changes in LH secretion or they may be caused by changes in the responsiveness of the Leydig cells to LH. This has only been studied in rams treated with buserelin. The picture is not clear because daily injections desensitised both the gonadotrophs and the Leydig cells in one study (Fraser and Lincoln 1980), whereas a constant infusion clearly affected pituitary, but not testicular, responsiveness in another (Lincoln et al. 1986). Again, if we are to correctly interpret the mechanism of action of a treatment, responses to challenges with LH, as well as GnRH, are needed for all combinations of species and superagonist chemistries. These issues have led to the present study. We have been developing a very effective method for fertility control in dogs based on a subcutaneous implant containing the GnRH superagonist deslorelin (Trigg et al. 2001; Junaidi et al. 2003). To determine the mechanisms of action and the endocrine outcomes of this treatment for male dogs, we have studied the responses of the reproductive endocrine axis to challenges with exogenous native GnRH and bovine LH over a 100-day period after implantation. Our hypothesis was that chronic deslorelin treatment would reduce pituitary responsiveness to GnRH (indicated by LH secretion), but not testicular responsiveness to LH (indicated by testosterone secretion). Materials and methods All procedures complied with the National Health and Medical Research Council of Australia Code of Animal of Practice and were approved by the Animal Ethics and Experimentation Committee of Murdoch University. Animals, experimental design and treatments We used 20 mature male dogs (age 2–5 years, body mass 15–22 kg) of mixed breeding that were housed indoors at night and allowed outdoors every day for 2–6 h in large, shaded, sandy runs. All dogs had access to water ad libitum and were fed with biscuits (approximately 600 g per dog daily; Pedigree PAL; Uncle Ben’s of Australia, Wodonga, Vic., Australia) and canned meat (approximately 400 g per dog, three times per week; Pedigree PAL; Uncle Ben’s of Australia). Dogs were assigned to a completely random design comprising five groups of four animals each. Each dog in the control group received a blank implant and each dog in all other groups (Deslorelin-I, Deslorelin-II, Deslorelin-III and Deslorelin-IV) received a 6-mg deslorelin implant (Peptech Animal Health, Macquarie Park, NSW, Australia). The implants were cylindrical (0.23 × 15.2 mm) and contained 6 mg deslorelin (d-Trp6 -Pro9 -des-Gly10 –LH-releasing hormone (LHRH) ethylamide). The implants were prepackaged in a purpose-developed disposable implanter incorporating a sterile 13G needle and
A. Junaidi et al.
were injected subcutaneously between the shoulder blades under aseptic conditions. Endocrine studies Using an indwelling intravenous cannula, 4 mL blood was sampled every 20 min for 2 h before the insertion of the deslorelin implant and, after implantation, every 20 min for 4 h, hourly for 6 h and then daily for 5 days. All dogs were used for these observations. Thereafter, blood was sampled twice weekly by venepuncture for the duration of the experiment, with the number of deslorelin-treated dogs decreasing as groups of four were killed for tissue collection. Exogenous GnRH and LH were administered on four occasions. Native GnRH decapeptide (Fertagyl; Intervet (Aust) Pty Ltd, Castle Hill, NSW, Australia), 5 µg kg−1 bodyweight, i.v., was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 µg kg−1 bodyweight, i.v.) was injected on Days 16, 26, 41 and 101 after implantation. The bovine LH was prepared as described by Stanton and Hearn (1987) and the doses used were chosen on the basis of preliminary experiments and on data from a previous study (Knol et al. 1993). Each GnRH–LH challenge involved the same group of four control dogs, but a different deslorelin-implanted group.After each challenge, the deslorelin-treated dogs were killed with an overdose of i.v. barbiturate for the recovery of tissues for another study. To measure the responses to exogenous GnRH and LH, jugular blood was sampled via an indwelling cannula at −40, −20, −10 and 0 min before injection, then every 10 min for 90 min and every 20 min to 150 min after injection. The blood was mixed with lithium heparin and centrifuged (1500g for 10 min at room temperature) immediately so that the plasma could be separated and stored at −20◦ C until assay. Hormone assays The plasma concentrations of LH were determined with a double-antibody radioimmunoassay system using canine reagents supplied by Dr A. F. Parlow (Director, Pituitary Hormones and Antisera Center, Harbor-UCLA, Medical Center, Torrance, CA, USA) and validated by Boyns et al. (1972). The assay was based on a polyclonal antiserum (AFP 8311890) to canine LH (cLH) that had been raised in a rabbit; cLH was also used for radioiodination (AFP-5214B) and reference (AFP-5216B). The limit of detection of the standard curve was 0.23 ± 0.12 ng per tube and the non-specific binding was always less than 6%. Each assay included six replicates of three pooled plasma samples containing LH at concentrations of 0.25 ± 0.30, 0.48 ± 1.40 and 0.70 ± 0.50 ng mL−1 . These samples were used to estimate variation within and between assays.The within-assay coefficients of variation (CV) were 11.7 ± 4.2%, 6.9 ± 2.3% and 13.6 ± 1.8%, respectively, whereas the between-assay CV were 11.8%, 13.5% and 9.4%, respectively. Plasma testosterone was measured using a non-extraction radioimmunoassay developed in our laboratory (Hötzel et al. 1995) using an antiserum (R3) raised against testosterone-3carboxymethyloxime–human serum albumin. The preparation 4-androsten-17β-ol-3-one (10 µg L−1 ; Sigma-Aldrich, St Louis, MO, USA) was used as the reference and 1,2,6,7-3 H-testosterone
GnRH–LH challenges in deslorelin-treated dogs
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Fig. 1. Plasma concentrations of luteinising hormone (LH; AFP-5216B; open circles) and testosterone (closed circles) in male dogs following the implantation of a blank implant (a) or an implant containing 6 mg deslorelin (b). The time of implantation was Minute 0 on Day 0. The data for the controls (blank implant) were derived from measurements on the same four dogs throughout the study. For Days 0–16, all deslorelintreated dogs (n = 16) are included in the data. A group of four deslorelin-treated dogs was removed after each challenge and only one group remained for Days 42–101. All points represent the mean ± s.e.m. (n = 4–16). For deslorelin-treated dogs, LH was undetectable after Day 13 and testosterone was undetectable after Day 30.
(specific activity = 3.33 TBq mm−1 ; Amersham, Sydney, NSW, Australia) was used as the tracer. Cross-reactions were 100% with testosterone, 70% with dihydrotestosterone, 3.7% with androstenedione and less than 0.05% with progesterone, 17-β-oestradiol, oestrone and oestriol. The limit of detection was 0.2 ± 0.1 ng mL−1 . All samples were measured in one assay. Statistical analyses For the outcomes to challenges with GnRH and LH, we defined three variables to describe the secretory pattern for LH and testosterone. For each dog, the pretreatment ‘baseline’ concentration was defined as the mean of values for samples taken before injection of GnRH or LH. The ‘response’ was defined as the mean concentration in samples taken every 10 min until 80 min after injection of GnRH or LH. The ‘recovery’ baseline was defined as the mean of values observed every 20 min from 90 to 150 min after the injection of GnRH or LH. We used analysis of variance for repeated-measures with Tukey’s multiple comparison test to assess the effect of treatment over time on plasma
concentrations of testosterone and LH using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± s.e.m. Results Effect of deslorelin implantation on the secretion of LH and testosterone In control dogs, there were no significant changes in plasma concentrations of LH or testosterone at any time during the study (Fig. 1a). In contrast, following insertion of the deslorelin implants, the LH concentration increased rapidly within 20 min and peak values were reached at approximately 40 min. Concentrations then started to decline, were very low (0.06 ± 0.03 ng mL−1 ) by the second week after implantation and, thereafter, LH became undetectable (
