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research-article |
School of Biological, Earth and Environmental Sciences,4 University of New South Wales, Sydney, New South Wales 2052, Australia
AgResearch,5 Wallaceville Animal Research Centre, Upper Hutt 6001, New Zealand
Peptech Animal Health Pty. Limited,6 Macquarie Park, New South Wales 2113, Australia
ABSTRACT
The present study evaluated the effects of chronic GnRH agonist (deslorelin) treatment on sexual maturation in the male tammar wallaby. Slow-release deslorelin or placebo implants were administered to male pouch young (n = 10/group) when they were between 180 and 200 days old, to determine if disruption of the pituitary-testicular axis during development altered the timing of sexual maturation or had long-term effects on adult reproductive function. Deslorelin treatment caused retardation of testicular growth and reduced the serum FSH and testosterone concentrations between 12 and 24 mo of age. Maturation of the hypothalamic-pituitary-testicular axis was also delayed in treated animals at 13 and 19 mo of age. Despite these alterations in the pattern and timing of neuroendocrine development, sexual maturation was not permanently blocked in these animals and deslorelin-treated animals reached sexual maturity at the same age as treated animals, as evidenced by a fully functional pituitary-testicular axis and proven fertility at 25 mo of age. The ability of the treated animals to reach puberty at the same time as control animals, despite delayed maturation of the hypothalamic-pituitary-testicular axis, suggests that puberty in the male tammar wallaby is additionally regulated by other, gonadotropin-independent factors.
FSH, gonadotropin-releasing hormone, GnRH agonist, male reproductive tract, marsupial, mechanisms of hormone action, puberty, testosterone
In eutherian mammals, the neonatal period is a critical time for the development of the hypothalamic-pituitary-gonadal axis. The neonatal activity of this axis plays roles in the masculinization of the central nervous system, determination of Sertoli cell number, maturation of feedback systems, development of the immune system, and the development of social and sexual behaviors in many species [1]. As such, disruption of normal neuroendocrine events during infancy can alter the pattern and timing of sexual maturation and adult pituitary-testicular function.
Analogs of GnRH have been used to disrupt sexual maturation in a range of mammalian species. Continuous exposure to a GnRH agonist during infancy delays the onset of puberty in rhesus monkeys [2] and rams [3], and this has been associated with retarded testicular growth, reduced concentrations of testosterone, alterations to sexual behavior, and diminished growth [2–5]. In rhesus monkeys, these effects are reversible [2], although there is evidence that alterations to sexual motivational systems may be retained in adulthood [5]. Immunization against GnRH during the neonatal period in sheep caused permanent infertility in the majority of the treated animals, even when anti-GnRH titers were no longer detectable [6].
At birth, marsupial mammals are extremely altricial. Most sexual differentiation occurs postnatally over a longer time period than in eutherian mammals [7], which means that they may be particularly susceptible to manipulation of the developing hypothalamic-pituitary-gonadal axis. Alterations to the pattern and timing of sexual maturation in some marsupial taxa may be of practical value for the management of overabundant marsupial populations [8]. The present study aimed to test the effect of the long-term GnRH agonist deslorelin, given during the second half of pouch life, on male sexual development in a model marsupial species, the tammar wallaby (Macropus eugenii). We have previously reported that treatment of adult tammars with long-acting deslorelin implants inhibits fertility in females [9, 10] but has no effect on testosterone secretion in males [11]. The differential responses to chronic GnRH agonist treatment between the sexes have been reported for numerous species, including cattle [12] and marmoset monkeys [13], although the underlying causes for these differences between the sexes are not understood. Investigating the effects of GnRH agonist treatment on juvenile males of these species should determine whether there are differences in the effects on testicular maturation versus the maintenance of testicular function.
The processes of sexual differentiation and puberty in male tammar wallabies are reasonably well-documented. Testicular cord formation and androgen production occur around the time of birth, with testicular descent completed by 65–72 days postpartum [7]. By 180–200 days, male gonadal weight is about half of that observed at the time of final pouch exit at 8 mo [7]. By 13 mo of age, the seminiferous tubules are without a central lumen and contain two main cell types: 1) supporting cells, which are the precursors of Sertoli cells; and 2) prespermatogonia [14]. By 19 mo of age, the seminiferous tubules are lined with Sertoli cells, spermatogonia, and primary spermatocytes, mitotic activity is evident in the germinal epithelium, and there are clumps of mature Leydig cells in the interstitial tissue. Moreover, the hypothalamic-pituitary axis has been shown to be fully mature by 19 mo of age, while the testicular response to LH is delayed until 25 mo [14]. Male tammars are considered to be fully mature at 25 mo of age, with all stages of spermatogenesis being present.
The present study assessed the effects of chronic GnRH agonist treatment on male tammar wallaby pouch young. The aim was to determine whether treatment of male pouch young with implants that release deslorelin over a period of approximately 12 mo would result in either a delay in the onset of puberty or longer lasting effects on the fertility of the animals in adulthood, after deslorelin release had ceased. In particular, we investigated whether deslorelin treatment had effects on: 1) skeletal growth during pouch life; 2) body weight, testicular size, and FSH and testosterone secretion between 12 and 25 mo; 3) pituitary responses to GnRH challenge at 13, 19, and 25 mo of age; 4) fertility at 25 mo of age; and 5) male social rank at the time of expected puberty.
The tammar wallabies used in the present experiments were held in grassed outdoor yards at the Macquarie University Fauna Park facility in Sydney, Australia. All animals were of Kangaroo Island descent. The animals were fed specially formulated "kangaroo" cereal pellets (Gordon Specialty Stock Feed, Yanderra, NSW, Australia) with water available ad libitum. The Macquarie University Animal Ethics Committee approved all the experimental work (approval no. 2002/006), and animal handling and husbandry were conducted in accordance with National Health and Medical Research Council of Australia [15] guidelines.
A timeline of events is outlined in Figure 1. Male tammar wallaby pouch young (PY) received either one 5-mg deslorelin implant (n = 10) or one placebo implant (n = 10) when they were between 180 and 200 days old (mean 192 ± 2 days for both the treatment and control groups). This age was chosen for implant placement as the PY are large enough to administer with ease the implant, yet they are still at an early stage of the testicular maturation process. Age was determined by measuring the head lengths of the young and consulting tammar wallaby growth curves [16]. PY were treated in smaller groups between August and October 2002, when there were sufficient numbers of male PY within the age range of 180–200 days (female PY of the same age were used in a companion experiment). Each subset of animals included both control and treated animals.
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At the time of implant insertion, the PY received a microchip that allowed them to be identified permanently, and they were weighed and had their tail, head, and pes (foot) measured with vernier calipers. These measurements were made at monthly intervals until the PY were 9 mo old (Fig. 1). The injection site was examined 1 wk posttreatment, to ensure that there was no negative reaction to implant placement. The PY remained with their mothers until after they were fully weaned (at more than 10 mo of age), at which time they were held in three bachelor groups, with a group of female tammar wallabies in a yard adjacent to each bachelor group.
Blood samples were collected from the males at monthly intervals from 12 to 18 mo of age, then once every 2 wk from 18 to 24 mo, to determine the testosterone and FSH concentrations. For 4 wk in January 2004, when the animals were approximately 24 mo old, blood samples were collected at weekly intervals. This was designed to detect the reported early breeding season peak in testosterone [17]. To measure directly the effects of deslorelin on the maturation of the hypothalamic-pituitary axis, GnRH challenge tests were performed at 13, 19, and 25 mo of age, as described by Williamson et al. [14]. Animals were weighed using hanging scales (Salter, model 235 6S; max. = 10 kg, d = 0.05 kg) at the time of sample collection, and the length of the left testis (excluding the epididymis) and the combined width of both testes were measured using vernier calipers.
In January and February 2004, when the males were approximately 24 mo of age, behavioral studies were conducted to determine the dominance hierarchy in two of the bachelor groups. In March 2004, after the 25-mo GnRH challenge, three control and three treated males were moved to individual yards and paired with females, to test in a direct manner their fertility.
The GnRH agonist deslorelin (D-Trp6-Pro9-des-gly10-GnRH ethylamide) was formulated into implants that contained 5 mg of deslorelin (Suprelorin; Peptech Animal Health Pty. Ltd., Macquarie Park, Australia), as previously described [18]. This dosage has previously been shown to be effective at suppressing reproduction in adult female tammar wallabies [9, 10]. In a real-time in vitro dissolution system, the release of deslorelin was approximately 1 µg/day for periods of approximately 1 yr [18]. Although the in vivo release rate in tammar wallabies is not known, reproduction by female tammar wallabies is suppressed for approximately 12 mo [10], which gives some indication of the duration of deslorelin release in vivo. The implants were prepackaged in a commercial implanting device that was sterilized by e-beam radiation, and were injected subcutaneously near the shoulder blades. The injection site was sealed with a veterinary tissue adhesive (Vetbond; 3M Animal Care Products, St. Paul, MN). The 5-mg deslorelin implant measures 2.3 mm in width and 12.5 mm in length.
Regular blood samples were collected from the lateral tail veins of conscious animals between 0730 h and 1000 h using a 21G winged infusion set (Surflo; Terumo Corp.) and a 5-ml syringe. Blood was transferred immediately into serum separation tubes (Vacuette, Greiner Labortechnik, Austria). The serum was separated by centrifugation and stored in duplicate aliquots at –20°C until assayed for FSH and testosterone.
Animals were placed in a hessian sack with their tails protruding from a hole at the base of the sack. A 22G indwelling catheter (Terumo) was fitted to the lateral tail vein and kept patent with heparinized saline (250 IU/ml in sterile 0.9% NaCl) between each sample. Blood samples (2 ml) were collected at –30, –15, 0, 15, 30, 60, and 90 min relative to the injection of GnRH (2 µg/kg i.v. in sterile 0.9% saline; Fertagyl; Intervet (Aust.) Pty. Ltd., Castle Hill, Australia).
All samples from individual animals were run in the same assay to reduce variability. Samples that exceeded the limits of the standard curve were diluted down to an optimum endpoint within the standard curve limits.
Testosterone assay.
Testosterone concentrations were determined using the automated Immulite 2000 immunoassay analyzer with a solid-phase competitive chemiluminescent enzyme immunoassay. Single 20-µl serum samples were assayed using the Immulite total testosterone kit (L2KTW2; Diagnostics Product Corp., Los Angeles, CA). The solid phase is made up of a polystyrene bead enclosed within the Immulite test unit, which is coated with a polyclonal rabbit antibody against testosterone. The antibody is highly specific for testosterone, with 0.6% cross-reactivity with androstenedione, 0.5% cross-reactivity with 5
-androstan-3ß, 17ß-diol, 2% cross-reactivity with 5-dihydro-testsoterone, 0.7% cross-reactivity with methyltestosterone, and 0.1% cross-reactivity with progesterone.
Serial dilutions of early breeding season male tammar wallaby serum in charcoal-stripped wallaby serum were found to be linear and parallel to the serial dilutions of human plasma. The assay sensitivity was 0.2 ng/ml. The intraassay coefficients of variation calculated for three quality control pools that contained 4.92 ± 0.14 ng/ml, 2.58 ± 0.26 ng/ml, and 1.41 ± 0.16 ng/ml (mean ± SD) were 2.8%, 10.1%, and 11.4%, respectively. The interassay coefficients of variation for three quality control pools that contained 5.84 ± 0.55 ng/ml, 2.86 ± 0.24 ng/ml, and 1.64 ± 0.17 ng/ml (mean ± SD) were 9.4%, 10.4%, and 8.3%, respectively.
FSH and LH assays. Plasma FSH and LH concentrations were determined using the heterologous RIAs developed by Moore et al. [19, 20] for use in brushtail possums (Trichosurus vulpecula). The ß subunits of LH and FSH have been cloned from another macropodid, the red kangaroo (Macropus rufus), and there is a high degree of deduced amino acid sequence identity (96% and 91%, respectively) with the ß subunits of LH and FSH in possums [21, 22]. The sequence similarity for kangaroo LH and FSH versus a variety of eutherian species generally ranges from 70% to 80% [21, 22]. The relatively high degree of sequence identity suggests that the heterologous RIAs developed by Moore et al. [19, 20] are adequate for measurements of FSH and LH in tammar wallaby plasma, and this was supported by the assay validation. The assays were validated by demonstrating parallelism between serial dilutions of tammar wallaby pituitary homogenates and plasma pools from castrated wallabies and the possum LH [11] and FSH (the present study) standard curves. Cross-reactivity between possum FSH and LH has not been observed in these assays [19, 20].
The FSH assay used rabbit antiserum raised against human FSH [23], which was kindly supplied by Dr. Alan McNeilly (University of Edinburgh, Scotland). The reference standards were made up with purified possum FSH in hypophysectomized sheep serum, and 125I was conjugated to the purified possum FSH (provided by Lloyd Moore, AgResearch, Wallaceville, New Zealand). All samples were assayed in duplicate. The sensitivity of the assay was 0.3 ng possum FSH/ml serum. The interassay coefficients of variation calculated for three quality control pools that contained 0.45 ± 0.06 ng/ml, 3.07 ± 0.25 ng/ml, and 5.31 ± 0.44 ng/ml (mean ± SD) were 15.0%, 8.3%, and 8.3%, respectively. The intraassay coefficients of variation for the same pools were 11.6%, 7.6%, and 7.9%, respectively.
The LH assay used rabbit antiserum raised against ovine LH (Wa-R oLH; AgResearch, Wallaceville, New Zealand). The reference standards were made up with purified possum LH in hypophysectomized sheep serum, and 125I was conjugated to the purified possum LH (provided by Lloyd Moore, AgResearch, Wallaceville, New Zealand). All samples were assayed in duplicate. The assay sensitivity was 0.15 ng possum LH/ml serum. The interassay coefficients of variation calculated for three quality control pools that contained 0.47 ± 0.07 ng/ml, 0.97 ± 0.11 ng/ml, and 4.85 ± 0.42 ng/ml (mean ± SD) were 15.6%, 11.4%, and 8.6%, respectively. The intraassay coefficients of variation for the same pools were 7.2%, 7.4%, and 8.1%, respectively.
Continuous scan sampling [24] was used to observe agonistic/dominance behavior in the two male bachelor groups. Group 1 contained four control and two treated individuals and group 2 had four control and three treated individuals. Observations were conducted in the late afternoon, when the animals were most active. Incidences of agonistic behavior (hostile and avoidance behaviors) between individuals were observed, and the ‘winners' and ‘losers' of each interaction were recorded. A dominance hierarchy was determined by the directionality of hostile and avoidance behaviors and a dominance index was calculated for each individual using the formula I = Na/(Na + Nb), where Na is the number of interactions won and Nb is the number of interactions lost [25]. Animals with the highest win rate were considered to be dominant.
Three control and three treated males were each paired with two females when they were 25 mo old. These females had a PY removed on February 16 2004 (Day 0), to stimulate reactivation of a dormant blastocyst, with birth and postpartum estrus occurring approximately 26 days later or estrus occurring approximately 29 days later in the event of a nonpregnant cycle [26, 27]. These females were examined on days 26, 28, and 31 for evidence of a copulatory plug. Any young that were born at this time (which were conceived from a previous mating) were removed on Day 31 (March 18), to allow any embryos conceived from the mating with the experimental males to proceed to term. The females were subsequently recaptured after an additional 28 and 40 days to look for a new PY.
To determine whether the variation in age at the time of implant placement had an effect on physiological parameters, a repeated measures analysis of variance was conducted comparing age at treatment time (divided into four age groups: 180–185 days, 186–190 days, 191–195 days, and 196–200 days) to FSH concentration and testes width over time (separate analyses for the treated and control groups). There was no significant difference between age at implant time and FSH concentrations (P < 0.05) or testes width (P > 0.05) in the treated and control animals (nor was there a significant interaction between age and time [P > 0.05] for FSH and testes width). These results suggest that the variation in implant placement time did not influence the results. In addition, there was no correlation between age at treatment and peak LH or testosterone concentrations in response to GnRH throughout the study (Pearson Correlation, P > 0.05 for treated and control animals). As such, the data for all the male PY were pooled into treated and control groups for all subsequent analyses on the effects of deslorelin on sexual development.
The data for hormone concentrations, skeletal measurements, testis size, and animal weight over time (for the duration of the study) were analyzed by ANOVA using the General Linear Model (GLM) repeated measures procedure of SPSS [28], the model being: y = treatment, time, treatment x time, with time as the repeated subject. Testis size was not corrected for weight because the weight did not differ between the groups throughout the study. The mean rate of increase in hormone (FSH and testosterone) concentrations over time was determined by calculating the slope of the linear regression line for each group. The slopes for each group were then compared using a two-sample t-test, to determine if there was a significant difference in the rate of increase for the treated and control males. The criteria for determining whether an individual had a positive response to the GnRH challenge were based on the pattern of response and the magnitude of the peak relative to the starting concentrations [11]. A positive response was recorded if the peak value was greater than two-times the standard deviation of the three pre-GnRH samples (–30, –15, and 0 min) and if the peak occurred between 15 and 30 min or 90 min after GnRH injection for LH and testosterone, respectively, based on the pattern of response in control animals. Comparisons of the mean testosterone and LH concentrations for each group at a single time-point were analyzed using paired-sample t-tests for comparisons within groups or two-sample t-tests for comparisons between groups. Where necessary, the data were log-transformed before analysis, to attain homogeneity of variance. However, the results are reported as untransformed arithmetic means ± SEM. Because of the small number of animals in the fertility trial, these results were not analyzed statistically.
No negative reactions were observed in the control or treated animals in response to the administration of placebo or deslorelin implants.
There was no significant difference in the growth rate of treated and control animals in terms of the head, tail, or pes length (P > 0.05 for treatment and treatment x time interaction for all three measurements) of animals between 6 and 9 mo of age. Similarly, there was no significant difference in the weights of the control and treated animals between 6 and 25 mo of age (P > 0.05 for treatment and treatment x time interaction).
Combined testes width was the testicular size parameter that was most accurately measured from a young age and thus, it was chosen as the best measure of testicular development for the current experiment. Between 7 and 11 mo of age, there was no significant difference in the combined testes widths of the treated and control animals (P > 0.05 for treatment and treatment x time interaction; Fig. 2), with the combined testes width slowly increasing over time in both groups during this period (time, P < 0.05). From 12 to 25 mo of age, there was a significant treatment (P < 0.05) and treatment x time interaction (P < 0.001), as the testicular growth rate was significantly slower in treated males. By 25 mo of age, there was no significant difference between the treated and control animals (Control, 48.0 ± 1.1 mm; Treated, 45.3 ± 2.0 mm; P > 0.05), and testes size had reached a plateau in both groups.
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Serum FSH concentrations were significantly lower in the treated than in the control animals between 10 and 25 mo of age (P < 0.01 for treatment, time and treatment x time interaction; Fig. 3). The FSH concentrations were low in both groups between 10 and 14 mo (undetectable in treated animals). The FSH level then began to increase in both groups, albeit at a significantly faster rate in control animals (P < 0.005). By 25 mo of age, the FSH concentrations were still significantly lower in treated males (Control, 25.59 ± 2.96 ng/ml; Treated, 16.05 ± 2.65 ng/ml; P < 0.05).
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Serum testosterone concentrations were low in the treated and control animals between 10 and 16 mo of age (undetectable in treated animals). The subsequent increase in testosterone was significantly delayed in the treated animals (P < 0.01 for time, and P < 0.05 for treatment and treatment x time; Fig. 4), although there was no significant difference in the rate of increase (P > 0.05). Testosterone concentrations peaked in both groups between 23 and 25 mo of age, which coincided with the initiation of the 2004 breeding season. Despite the significant difference in testosterone concentrations between 10 and 25 mo of age, by the end of the sampling period, there was no significant difference between the treated and control animals (Control, 2.64 ± 0.60 ng/ml; Treated, 3.41 ± 0.67 ng/ml; P > 0.05).
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At 13 mo of age, the basal LH and testosterone concentrations were below the detection limit of the assay in most of the males. All the control males (n = 9) had a positive response to GnRH challenge, as observed by an increase in the circulating LH concentrations between 15 and 30 min postinjection (peak of 1.93 ± 0.31 ng/ml at 30 min; Fig. 5a), which was significantly higher than the peak observed in treated males at this age (peak of 0.60 ± 0.32 ng/ml at 15 min; P < 0.01 for treatment and treatment x time interaction; Fig. 5a). This small increase in LH in treated males was primarily accounted for by significant increases in 2/7 animals. Despite the significant difference in the LH response to GnRH at 13 mo of age, there was no significant difference between the treated and control animals in terms of their testosterone responses to GnRH at this age (P > 0.05 for treatment and treatment x time; Fig. 6a). A small but significant increase in testosterone concentration (P < 0.01) was observed in most of the control males at 90 min postinjection (peak of 0.60 ± 0.20 ng/ml). In the treated males, there was a significant increase in testosterone concentration 90 minu postinjection (peak of 0.39 ± 0.13 ng/ml), but again this small increase was primarily accounted for by significant increases in the same two animals.
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By 19 mo of age, there were significant increases in both LH and testosterone in all the animals in response to GnRH (P < 0.01 for time for both hormones; Figs. 5b and 6b), although the pattern of response was significantly different between the two groups (P < 0.05 for treatment x time interaction for LH and testosterone). The peak LH response was significantly lower in treated animals at 30 min (Control, 3.28 ± 0.43 ng/ml; Treated, 1.65 ± 0.58; P < 0.05), as was the peak testosterone response at 90 min (Control, 4.24 ± 0.65 ng/ml; Treated, 1.63 ± 0.52 ng/ml; P < 0.01). By 25 mo of age, there was no significant difference in the magnitude or timing of response to GnRH between the treated and control animals, with LH concentrations peaking at 30 min (Control, 3.40 ± 0.38 ng/ml; Treated, 3.77 ± 0.74 ng/ml; P > 0.05; Fig. 5c) and testosterone peaking at 90 min (Control, 4.88 ± 1.03 ng/ml; Treated, 5.42 ± 0.86 ng/ml; P > 0.05; Fig. 6c).
Table 1 illustrates the dominance rank order of the males based on the direction of agonistic interactions among the males. In both groups, control animals held the top two dominance positions. The highest rank achieved by a treated male was third.
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All six males (three control and three treated males) successfully sired young when they were individually paired with females at 25 mo of age (Table 2).
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Treatment of male tammar wallaby pouch young with slow-release deslorelin implants, commencing at between 180 and 200 days of age, resulted in a significant retardation of testicular growth, reduced levels of circulating FSH and testosterone, and delayed maturation of the hypothalamic-pituitary-testicular axis. However, this delay did not permanently block sexual maturation. By 25 mo of age, the expected time of puberty, there was no significant difference in testis size, serum testosterone concentration, and pituitary-testicular response to GnRH challenge between the treated and control males, although the FSH concentrations remained significantly lower in the treated animals. Despite the altered timing of neruoendocrine events, treated animals were capable of siring offspring at 25 mo of age.
The observation that deslorelin treatment retards testicular growth in juvenile male tammar wallabies is consistent with the hypothesis that growth and maturation of the testis are acutely dependent on stimulation from pituitary gonadotropins. In both the treated and control tammars, there was a significant positive correlation between serum FSH concentrations and testis size (data not shown), which suggests that FSH is an important regulator of testicular size in this species. The recent development of laboratory mice strains with genetic disruption of both the FSH ß subunit and its receptor has provided further evidence that FSH signaling is essential for maintaining normal testicular size, seminiferous tubule diameter, as well as sperm number and motility in eutherian mammals [29]. In humans and rats, it has been reported that an adequate FSH concentration is necessary in the early stages of puberty for the proliferation and differentiation of Sertoli cells [30, 31]. Given that seminiferous tubules constitute the bulk of testicular tissue in many species [3, 30], it is likely that the reduced testicular size in treated tammar wallabies reflects a reduced rate of Sertoli cell differentiation during deslorelin treatment, resulting in a smaller volume of seminiferous tubule tissue than in control animals.
Although FSH is clearly important for the development of the testis, our results indicate that testis growth up to 11 mo of age is gonadotropin (specifically FSH)-independent. Thereafter, the growth of the testis is reduced, but not totally halted, in treated males coincident with deslorelin-induced FSH suppression up to 16–17 mo. As the effects of deslorelin begin to wane at this time-point, the FSH concentrations slowly increase and testis growth increases in treated males, with treated males catching up to the control males by 21 mo of age. The fact that testicular development was not totally inhibited in treated tammars between 12 and 24 mo, but merely showed a slower rate of increase, demonstrates that the degree of pituitary suppression was not sufficient to abolish totally gonadotropin secretion. Similar observations have been reported after GnRH agonist treatment of prepubertal rams [3]. This is supported by the observation in the current study that FSH concentrations were detectable, albeit at lower levels, from 14 mo onwards. By the time of expected puberty, the testis size in treated males had increased and was not significantly different from that of the control animals, which suggests that deslorelin treatment does not irreversibly affect Sertoli cell differentiation.
The reduced testosterone secretion in treated males is presumably related to a reduction in FSH and LH secretion from the pituitary in response to deslorelin treatment. Androgen production by the testis is dependent on the stimulation of Leydig cells by LH. In turn, Leydig cell growth and activity are indirectly influenced by FSH through its effects on Sertoli cells [32]. The basal FSH concentrations were significantly lower in the treated than in the control tammars, even at the conclusion of the study when the treated animals were known to be fertile. Basal LH concentrations in tammar wallabies are typically below the sensitivity thresholds of available RIA techniques and could not be measured in the present study. However, there was evidence of pituitary desensitization, as the treated males could not respond to GnRH challenge with a surge of LH at 13 mo.
The in vivo release rate of deslorelin from this implant in tammar wallabies has not been determined, although deslorelin is expected to be released for up to 1 yr [18], which corresponds to the approximate contraceptive duration when adult female tammar wallabies are treated with these implants [10]. The endocrine data presented in the present paper clearly demonstrate that treated animals had desensitized pituitaries at 13 mo of age but that the effects of the implants were waning by 19 mo of age (or 13 mo postinsertion of implant), at which time the implants had presumably stopped releasing deslorelin. When the wallabies reached 25 mo of age, there was no evidence of pituitary desensitization, with the exception of lower basal FSH concentrations relative to control animals. When prepubertal bulls underwent chronic deslorelin treatment from 3 mo of age, the associated retardation of testicular growth was correlated with a delay in the onset of puberty [33]. The finding that treated tammars are able to catch up to control animals in terms of testicular development and fertility, despite evidence of significant delays in pituitary and testicular maturation as well as significantly reduced FSH concentrations at 24 mo, was surprising. This suggests that other gonadotropin-independent factors influence testicular maturation, e.g., body weight.
The treated males successfully sired offspring when individually paired with females, thus demonstrating their fertility. However the question remains as to whether they would have successfully sired offspring if they had to compete with nontreated males. Observations of the dominance hierarchies amongst treated and control animals in the present study suggest that prepubertal deslorelin treatment influences male social rank in adulthood. In rhesus monkeys, prolonged neonatal exposure to a GnRH agonist altered the systems that regulate sexual motivation after puberty [5]. However, prepubertal GnRH agonist treatment and the resultant reduction in androgen concentrations are unlikely to have long-lasting effects on male-type sexual behavior in tammar wallabies, as there is no indication that testosterone has any long-term organizational effects on the neural pathways in this species [34].
Previously, we have reported that treatment with slow-release deslorelin implants has no effect on testis size or on the LH and testosterone concentrations in adult male tammar wallabies, even when they are treated with four-times the dose that is effective in females [11]. Although the fertility of these animals was not tested, the maintenance of testicular size is probably indicative of ongoing spermatogenesis. This disparity in the effects of deslorelin treatment on adult and juvenile male wallabies suggests that there are differences in the degree of gonadotropic support required for sexual maturation versus maintenance of testicular function.
In addition to documenting the effects of chronic GnRH agonist treatment on the prepubertal male tammar wallaby, the present study provides additional information on the maturation of the hypothalamic-pituitary-gonadal axis in this species. Previous studies have suggested that the hypothalamic-pituitary axis is fully mature at 19 mo of age but that the pituitary-testicular axis takes longer to mature [14]. This was based on GnRH challenge and testicular histology results. Our findings from GnRH challenges of control animals support the notion that the hypothalamic-pituitary axis is mature by 19 mo of age, but in contrast to the previous study, we found that the testosterone responses to GnRH were not significantly different at 19 and 25 mo of age, which suggests that the pituitary-testicular axis was also mature at this age. In addition, combined testes width and FSH concentrations began to plateau at this age. The reasons for the discrepancies between the two studies are most likely related to variations between individual animals and the small sample size in the previous study (n = 4 for the GnRH challenge). Williamson et al. [14] found that one out of three 19-mo-old males had all stages of spermatogenesis present and had higher concentrations of testosterone in response to GnRH injection, demonstrating that some males are indeed sexually mature at this age. Although we did not investigate testicular histology in the current study, our results suggest that it is not uncommon for male tammars to be sexually mature by 19 mo of age.
In conclusion, chronic treatment with the GnRH agonist deslorelin commencing at 6 mo of age results in retardation of testicular growth and reduced FSH and testosterone concentrations between 12 and 24 mo of age. These effects are most likely related to GnRH agonist-induced pituitary desensitization. Despite this retardation of the maturation of the pituitary-testicular axis, treated males still reach puberty (i.e., have a fully functional hypothalamic-pituitary-testicular axis and are fertile) at the same age as control males. Thus, chronic GnRH agonist treatment during the second half of pouch life cannot be used as a means of delaying puberty in this species. While it is possible that earlier application of deslorelin implants may have had a longer-lasting effect, recent studies in prepubertal male cattle have not found this to be the case [33], and the administration of implants to smaller wallabies (<200 g) is problematic.
ACKNOWLEDGMENTS
We thank Ron Claassens, Anne Mouland, and James Cook for care of the animals and assistance with handling and sampling, Kim Whillock for conducting the behavioral observations, Dr. Janet Crawford, AgResearch for assistance with the LH and FSH assays, Dr. Margaret Wilkinson, Cameron Wood, and Mark Ridgwell of Royal North Shore Hospital, Sydney for assistance with the testosterone assay, and two anonymous referees for constructive comments on the manuscript.
FOOTNOTES
3Current address: School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand. 
1Supported by the Australian Research Council Linkage Grant Scheme (grant no. LP0219459 to D.W.C.).
Correspondence: 2FAX: 61 2 9385 1558; e-mail: cathherbert{at}unsw.edu.au
Received: 3 January 2007.
First decision: 21 January 2007.
Accepted: 27 February 2007.
REFERENCES
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