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Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity
General and Comparative Endocrinology 149 (2006) 236–243 www.elsevier.com/locate/ygcen
Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity A.L. Schmidt a,e, D.A. Taggart b, P. Holz c, P.D. Temple-Smith d,e, A.J. Bradley a,¤ a
b
Department of Anatomical Sciences, School of Biomedical Sciences, The University of Queensland, St. Lucia, Qld 4072, Australia Royal Zoological Society of S.A., C/O School of Earth and Environmental Science, The University of Adelaide, Nth Terrace, Adelaide, SA 5005, Australia c Healesville Sanctuary, Healesville, Vic. 3777, Australia d Conservation Research Unit, Zoological Parks and Gardens Board, PO Box 74, Parkville, Vic. 3052, Australia e Department of Zoology, University of Melbourne, Parkville, Vic. 3052, Australia Received 15 February 2006; revised 28 May 2006; accepted 8 June 2006 Available online 1 August 2006
Abstract The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-oV is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inXammatory responses. This study examines the endocrine proWle of male brush-tailed phascogales (Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations. © 2006 Elsevier Inc. All rights reserved. Keywords: Albumin; Cortisol; Corticosteroid binding globulin; Dasyuridae; Die-oV; Phascogale; Reproduction; Semelparity
1. Introduction There are seven species of semelparous dasyurid marsupials, characterized by: (i) highly seasonal breeding, occurring over a restricted period of time; (ii) female monoestry; (iii) sexual maturity at approximately 11 months of age; and (vi) stress-induced die-oV of all males within a few weeks of the conclusion of the breeding season, while females may survive to a second season (Lee et al., 1982). Of the seven species, Wve belong to the genus Antechinus and two belong to the genus Phascogale (Lee et al., 1982). Studies of life-history and reproductive physiology (Cuttle,
*
Corresponding author. Fax: +61 7 3365 1299. E-mail address: [email protected] (A.J. Bradley).
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1978, 1982; Soderquist, 1993a,b, 1994, 1995; Soderquist and Ealey, 1994; Soderquist and Lill, 1995; Millis et al., 1999) have resulted in the classiWcation of the brush-tailed Phascogale, Phascogale tapoatafa as the largest of these species. Female brush-tailed (P. tapoatafa) and red-tailed (Phascogale calura) phascogales may survive to a second breeding season in natural situations (Kitchener, 1981; Lee et al., 1982; Soderquist, 1994) and often do so in captivity (Cuttle, 1978, 1982; Lee et al., 1982; Millis, 1995), although there are indications that fecundity declines with age (Halley, 1992; Soderquist, 1993a,b, 1994). Survival beyond the Wrst breeding season is less common in males, but has been recorded in captive colonies (Cuttle, 1978, 1982; Halley, 1992; Soderquist, 1994). In the captive population of P. tapoatafa maintained at Healesville Sanctuary (Healesville, Victoria) since 1990, all males generally
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survive and maintain good health well beyond breeding, until they are eventually euthanased for management purposes (J. Phelan and M. Halley, unpublished data). Autopsies conducted on euthanased males show none of the pathological symptoms described in dead males from natural populations (Millis, 1995). In particular, there is no evidence of gastric ulceration/gastrointestinal hemorrhage, which is the primary cause of death in wild populations (Soderquist, 1994; Bradley, 1987, 1997, 2003). Observations on captive male P. tapoatafa from the Healesville population indicate that these animals show a single, limited round of sperm production and undergo complete reproductive senescence at the end of the Wrst breeding season. Although they may survive up to three years, histological studies indicate that there is permanent cessation of spermatogenesis at the end of the Wrst season (Millis, 1995; Millis et al., 1999) and a lack of redevelopment of secondary sexual characteristics (e.g. sternal gland activity) in subsequent years (Cuttle, 1978, 1982). This is similar to what occurs in Antechinus, but in P. tapoatafa, testicular sperm production continues into the mating season (Millis, 1995; Millis et al., 1999) while in Antechinus, sperm production ceases prior to breeding (Taylor and Horner, 1970; Kerr and Hedger, 1983). Physiological studies of natural populations of Antechinus stuartii have shown that, immediately before the die-oV period, males (but not females) show increased plasma corticosteroid and liver glycogen levels, and adrenal gland weight, while body weight, and sodium levels decline (Barnett, 1973). It was suggested that, as they approach die-oV, males decrease their food intake and become hypoglycaemic, as a result of corticosteroid activation of gluconeogenesis (Barnett, 1973); indicating that the underlying cause of death is physiological stress. This is supported by observations that male Antechinus also exhibit suppression of immune and inXammatory responses and develop a range of symptoms, including haemolytic anaemia (Cheal et al., 1976; Barker et al., 1978), parasitaemia (Barker et al., 1978; Bradley et al., 1980; Bradley, 1990a) and gastric ulceration (Barker et al., 1978; Soderquist, 1994; Bradley et al., 1980, 1997). Endocrine changes associated with male die-oV in P. calura are similar to those observed in Antechinus, with total plasma androgen concentrations reaching peak levels just prior to die-oV (Bradley et al., 1980; Bradley, 1987, 1990a, 1997). In the Phascogale species, anaemia and parasitaemia are less prevalent (Bradley, 1990a, 1997; Soderquist, 1994), but the primary cause of death in both groups is gastrointestinal hemorrhage (Bradley, 1990a, 1997; Soderquist, 1994). The causal role of elevated glucocorticoid levels in male semelparity was established through treatment of Antechinus with exogenous corticosteroids. Bradley et al. (1975, 1980) removed male A. stuartii from the wild and treated groups with daily injections of exogenous cortisol and testosterone and found that mortality rates were signiWcantly higher in groups treated with exogenous steroid. Increasing doses also produced increasing mortality levels (Bradley et al., 1975, 1980). In a direct comparison of breeding-
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related endocrine changes in Antechinus swainsonii, A. Xavipes (both of which exhibit the mass die-oV of males) and Sminthopsis crassicaudata (a related species which does not exhibit male die-oV) McDonald et al. (1981) then found that the marked increase in plasma cortisol concentration does not occur in either males of non-die-oV species or females of die-oV species. These early studies led to the hypothesis that glucocorticoid feedback becomes impaired in males during the breeding season (Barnett, 1973; Bradley et al., 1980; McDonald et al., 1986). The role of the glucocorticoid feedback mechanism was consequently investigated in A. swainsonii and P. calura and it was demonstrated that, while maximum corticosteroid binding capacity (MCBC) is always much greater than total corticosteroid in females of the die-oV species and both males and females of non-die-oV species, in males of die-oV species, MCBC is much lower than total corticosteroid (Bradley et al., 1980; McDonald et al., 1986; Bradley, 1990b). This indicates that the adrenal cortex continues to produce glucocorticoid despite the high plasma concentrations and the male die-oV is associated with a failure of glucocorticoid feedback (Bradley et al., 1980; McDonald et al., 1986; Bradley, 1990b). The aim of this study was to investigate the endocrine proWle of male P. tapoatafa surviving beyond the breeding season in captivity, to determine whether they display similar endocrine changes to those observed in wild populations. 2. Materials and methods 2.1. Study group The study colony included 14 adult (seven males; seven females) P. tapoatafa housed at Healesville Sanctuary (Healesville, Victoria), monitored through the 1995 breeding season (mating between 5th and 31st May). One female, and three males were wild-caught individuals, while the remainder were captive-born.
2.2. Blood sampling Regular blood sampling commenced on 29th March, prior to onset of breeding and continued through until 28th July, approximately two months after the end of the breeding period. Animals were collected from the nest-box and anaesthetized with isoXuorance (1-chloro-2,2,2-triXuoroethyl diXuoromethyl ether; VM Supplies, Melbroune) in oxygen, administered by mask. Blood samples were collected from the lateral caudal vein into EDTA-treated syringes Wtted with a 26-guage needle and centrifuged at 2500g for 5 min, to separate plasma, as described in Millis et al. (1999). To ensure that individual animals were not adversely aVected by repeated blood sampling, blood haematocrit values were regularly determined by veterinary staV. Females were bled twice weekly (to monitor oestrogen concentrations for a concurrent study). Males were bled twice weekly through May and June, but in July sampling frequency was reduced to once per week. Two males were euthanased in early July, before the Wnal bleeding, for use in a concurrent study (Millis et al., 1999), meaning the last Wve sampling dates involved only Wve animals. Morphological evidence (Millis et al., 1999) indicated that the breeding season occurred between 8 and 26th May, with the peak mating period occurring between 17 and 22 May.
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2.3. Albumin Plasma albumin concentration was determined on 10 L plasma samples using the bromocresol green spectrophotometric method of Rodkey (1965) and bovine serum albumin (BSA) (Commonwealth Serum Laboratories, Melbourne, Australia) standards. An albumin absorbance standard curve was constructed using BSA standards ranging from 2 to 8 g/dL. Serial dilutions of both P. tapoatafa and BSA standards showed that parallelism was acceptable and the dye binding characteristics of BSA closely approximates that of P. tapoatafa and P. calura (Bradley, unpublished data). The constant for the low aYnity binding of cortisol to P. tapoatafa plasma was determined at 36 °C in 1:5 diluted heat-treated plasma. To denature the high aYnity cortisol binding sites on the CBG, 1:5 diluted plasma was incubated at 60 °C for 30 min. The relationship between the plasma albumin concentration and the low aYnity binding of cortisol to heat-treated diluted plasma was determined and thereafter, the albumin concentration was used as an index of low aYnity binding.
2.4. Maximum corticosteroid binding capacity Plasma high aYnity corticosteroid binding capacity (MCBC) was measured by the gel Wltration method of Doe et al. (1964) and the dextrancoated charcoal method as described by McDonald et al. (1981).
2.5. Cortisol Plasma cortisol levels were determined by radioimmunoassay, as described in Millis et al. (1999). Standard curve construction and conversion of radiation counts to concentrations was performed using AssayZap software (Biosoft, Cambridge, United Kingdom).
2.6. Steroid partitioning Plasma samples were diluted with a phosphate buVer (0.05 M, pH 7.4) containing 0.01% mercury-[(O-carboxyphenyl)-thio]-ethyl (Thimerosal, Sigma Chemical Co., USA) as a preservative. Partitioning of cortisol and corticosterone into CBG bound, albumin bound and free components was calculated by the method of Tait and Burstein (1964) as described for the closely related P. calura (Bradley, 1987), with the high aYnity binding constant KT of 6.2 £ 107 M¡1 at 36 °C for P. tapoatafa (Bradley, 1982) used to calculate the partitioning of cortisol within blood plasma.
2.7. Statistical analysis All parameters were measured from duplicated samples where possible. Variation between duplicated samples was assessed through analysis of variance (ANOVA). No signiWcant variation was detected and duplicate samples were then combined and averaged prior to further analysis. All results are presented as mean § SE. Repeated measures ANOVA analyses of data were conducted using Jandel ScientiWc statistical software (Jandel Corporation, heilerSoftware, GmbH). As steroid partitioning was initially calculated in percentage form, data were arcsine transformed prior to analysis. Where the ANOVA showed statistically signiWcant diVerences, multiple comparisons were made using both Bonferroni’s and Student–Newman–Keuls tests. The null hypothesis was rejected at p < 0.05.
3. Results
detected at any point (Fig. 1a). Prior to onset of breeding (29th March), male plasma albumin concentration was 3.48 § 0.41 g/dL (Fig. 1b). At the beginning of the breeding season (3rd May), albumin concentration increased signiWcantly (p D 0.0100) to 5.60 § 0.22 g/dL. No further signiWcant changes were detected, and at the end of the sampling period, plasma albumin concentration was 4.82 § 0.22 g/dL. 3.2. Maximum corticosteroid binding capacity Prior to breeding, female MCBC was 11.80 § 0.55 g/dL. The only signiWcant (p D 0.0198) variation occurred on 2nd June (at the end of the mating season), when MCBC concentration dropped to 8.83 § 0.44 g/dL (Fig. 1a). Male MCBC ranged from 9.6 § 0.24 g/dL prior to breeding (19th April) to 7.8 § 0.67 g/dL during the breeding period (24th May) (Fig. 1b), although no signiWcant variations were detected. 3.3. Cortisol Prior to onset of breeding (29th March) female plasma cortisol concentration was 0.36 § 0.03 g/dL. No signiWcant variation occurred, however a slight decline (p D 0.0545) to 0.30 § 0.06 g/dL (Fig. 2a) was observed during the peak mating period. Male cortisol concentration showed no signiWcant variation during the sampling period (Fig. 2b). The peak concentration of 0.14 § 0.01 g/dL was recorded approximately three weeks after mating (14th June), but this was not a statistically signiWcant peak. The minimum concentration of 0.04 § 0.01 g/dL was recorded during the mating period (17th May). 3.4. Steroid partitioning Steroid partitioning remained constant in both sexes over the sampling period, with no signiWcant variation in the amount of plasma bound by either CBG or albumin, or in free steroid (Fig. 3a and b). Prior to onset of breeding (29th March), 6.37 § 0.83% of steroid in females was albumin bound, 89.10 § 0.67% was CBG bound and 4.53 § 0.22% was free (Fig. 3a). At the end of the sampling period (28th July; two months after breeding) 6.07 § 0.34% of steroid was albumin bound, 88.37 § 0.01% was CBG-bound and 5.53 § 0.38% was free. Prior to onset of breeding (29th March), 5.38 § 0.75% of steroid in males was albumin bound, 88.90 § 1.67% was CBG-bound and 5.75 § 0.10% was free (Fig. 3b). At the end of the sampling period, 6.95 § 0.22% of steroid in males was albumin bound, 87.30 § 0.20% was CBG-bound and 5.73 § 0.15% was free. In both sexes, MCBC was always greater than plasma cortisol levels.
3.1. Albumin 4. Discussion Female albumin concentration remained constant throughout the sampling period, ranging from 3.50 § 0.23 to 5.70 § 0.98 g/dL. No signiWcant Xuctuations were
Phascogale calura, A. stuartii and A. swainsonii show increases in plasma cortisol levels immediately prior to
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Fig. 1. Changes in concentration of plasma albumin and maximum corticosteroid binding capacity (MCBC) in captive brush-tailed phascogales (Phascogale tapoatafa) during the 1995 breeding season. (a) Changes in female MCBC and albumin with the breeding season. Female albumin concentration (n D 7) remained constant throughout the sampling period. MCBC however, showed some Xuctuation. Prior to breeding, MCBC concentration 11.80 § 0.55 g/dL (n D 7) (29th March). This declined (p D 0.0198) to 8.83 § 0.44 g/dL at the end of the mating season (2nd June). (b) Changes in male MCBC and albumin with the breeding season. Prior to onset of breeding (29th March), male plasma albumin concentration was 3.48 § 0.41 g/dL. This increased signiWcantly to 5.67 § 0.22 g/dL (p D 0.0100) at the beginning of the breeding season (3rd May), before declining to 4.82 § 0.46 g/dL by the end of the sampling period (28th July).
Fig. 2. Changes in plasma cortisol concentrations across the breeding season. (a) Changes in female MCBC and albumin with the breeding season. Female cortisol concentration showed no statistically signiWcant variation across the breeding season (n D 7). (b) Changes in male cortisol with the breeding season. Male cortisol concentration showed no statistically signiWcant variation during the sampling period (n D 7).
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Fig. 3. No signiWcant variation in steroid partitions was recorded in either female or male Phascogale tapoatafa. (a) Tuan female steroid partitioning. CBG binding of steroid in females remained around 90% throughout the study period. Albumin binding and free steroid levels accounted equally for the remaining steroid (5–6% albumin bound, 4–5% free). (b) Tuan male steroid partitioning. As in females, approximately 90% of steroid in males was CBG bound at all times, while 5–6% was albumin bound and 4–5% remained free. By the end of the sampling period, »7% of steroid in males was albumin bound, »87% was CBG-bound and »6% was free.
die-oV in wild populations (Barnett, 1973; Bradley et al., 1975, 1976, 1980; McDonald et al., 1981, 1986; Bradley, 1987, 1990b, 1997), but no signiWcant increases in cortisol levels were detected in captive male P. tapoatafa surviving beyond the breeding season. The peak cortisol concentration observed in these animals (0.14 § 0.01 g/dL) occurred following mating (14th June), but was much lower than recorded in wild populations of P. calura, where concentrations range from 1 to 2 g/dL in females (Bradley, 1987) and 4–5 g/dL in males (Bradley, 1987). Maximum corticosteroid binding capacity (MCBC) also remained relatively constant (ranging from 7.8 to 9.6 g/dL in males and from 8.8 to 11.5 g/dL in females) and lies within a similar range to that observed in wild female P. calura (»6 to 8 g/dL). In wild males however, MCBC declines as low as 1.9 g/dL during the die-oV period (Bradley, 1987). The observation of no signiWcant increase in plasma cortisol, and no signiWcant decrease in MCBC, in captive P. tapoatafa surviving beyond the breeding season therefore suggests that the stress-related die-oV is a response to events or conditions unique to natural populations. A second notable diVerence between the captive population and wild populations of semelparous dasyurids lies in testosterone levels. In the captive population used in this study, testosterone concentration reached a maximum of 6.25 § 0.56 ng/mL during the breeding period (Millis et al., 1999). In wild P. calura, testosterone concentrations rise to almost twice this level (8–9 ng/mL) immediately prior to
die-oV (Bradley, 1987) and this contributes to the decline in CBG levels because of an inverse relationship between plasma testosterone concentration and MCBC (Bradley et al., 1980; Bradley, 1990b). The eVects of stress then cause a further decline in CBG concentrations (Bradley et al., 1980; Bradley, 1990b), which compounds this eVect. The lower levels of testosterone and cortisol in the captive population may therefore be critical and insuYcient to produce the same decrease in CBG observed in natural populations. One possible factor inXuencing the absence of the mating related androgen and cortisol surges, and subsequent decline in steroid-binding capacity, is social environment. In a study of the eVects of mating and agonistic experience on male A. stuartii, Scott (1987) found that plasma androgen and MCBC in males isolated from all conspeciWcs did not vary from that of males in physical contact with conspeciWc males, or in intermittent contact with oestrous females. However, by the end of the breeding season, males in daily contact with conspeciWcs showed greater plasma corticosteroid concentrations and higher mortality rates than isolated males. Communal nesting is common in Antechinus (LazenbyCohen and Cockburn, 1988) and has been observed in natural populations of P. tapoatafa, but this appears to represent a response to unusual thermoregulatory challenge and the species is usually solitary (Rhind, 2003). DiVerences in sociality between the two genera may be reXected in the prevalence of secondary causes of mortality such as
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parasite infection. While Antechinus species exhibit high levels of parasitic infection prior to die-oV (Barker et al., 1978), in Phascogale species, parasitic infection is less common (Bradley, 1990a; Soderquist, 1994). However, the high level of parasitic infection seen in some Antechinus populations is not duplicated in others (A. Bradley, unpublished data) and parasite-induced mortality cannot be used as a universal explanation of post-mating mortality of males. In both species, the primary cause of death is gastrointestinal hemorrhage following development of gastrointestinal ulcers (Bradley, 1990a, 1997; Soderquist, 1994), suggesting that there is some other, underlying process that drives male die-oV. Possible factors can be identiWed through examination of other life-history features. Immediately prior to die-oV (one week before disappearance), wild male P. calura and P. tapoatafa undergo a terminal dispersal; travelling large distances in a short period of time (Soderquist, 1994; Soderquist and Ealey, 1994; Bradley, 1997). The terminal dispersal occurs during winter, when nighttime temperatures are low and males trapped during this period are in poor condition, showing symptoms of thermoregulatory stress, and negative nitrogen balance (Bradley, 1997), indicative of homeostatic stress. Boonstra (1994) has proposed that progressive deterioration in endocrine feedback (along the hippocampal– hypothalamic–pituitary–adrenal axis) means older animals are particularly prone to homeostatic stress when faced with such conditions. However, male Phascogales are only 11 months old at the time of the terminal dispersal and age is unlikely to be a major factor. Bradley (1997) has suggested that the stress response of male Phascogale species is an adaptive mechanism, designed to mobilize energy resources during the later stages of mating and combat the thermoregulatory challenge associated with the terminal dispersal. This is supported by evidence that marsupials sometimes undergo stress-related endocrine changes to produce energy when required (Lee and Cockburn, 1985; Lee and McDonald, 1985). This “Adaptive Stress Senescence” hypothesis (Bradley, 1997; Boonstra, 2005), which may also explain similarities in the endocrine proWles of semelparous Wsh species (Stein-Behrens and Sapolsky, 1992), is based on the suggestion that exposure of the hippocampal glucocorticoid receptors to high concentrations of free cortisol results in damage to the hippocampal–hypothalamic–pituitary–adrenocortical axis and consequent impairment of glucocorticoid control (Sapolsky, 1992; Boonstra, 1994, 2005). Observations in wild populations of semelparous dasyurids have provided previous support for this hypothesis. The decreased immune function observed in natural populations is produced by a syngergisteric interaction between free cortisol and testosterone concentrations (Woollard, 1971; Barnett, 1973; Bradley et al., 1976, 1980; McDonald et al., 1981; Bradley, 1990a,b), accompanied by a decrease in corticosteroid binding globulin (Bradley et al., 1976; Bradley, 1990b), leading to an increase in the amount of
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free steroid within the system. During this period, the tissues of males are exposed to large amounts of glucocorticoid (Bradley, 1987). In P. calura, the tissues of males may be exposed to 25 times more free cortisol than the tissue of females (Bradley, 1990b). The absence of the stress response in captive males that survive beyond breeding provides further support. The nature of the captive-breeding program at Healesville Sanctuary is such that the inXuence of the social environment is limited. Females are maintained in permanent enclosures and individual males are rotated in a “round robin” fashion. In 1995, there were eight males and seven females, meaning each male spent only two days with a given female, before being transferred to a new cage with a diVerent female, or a solitary holding pen, for a further 2 days. The total period of exposure to females spanned only 2–3 weeks, limiting any eVect of aggression from pregnant females. Captive males were also fed a balanced, high-quality diet, and a variety of nesting materials (fresh Eucalypt and Acacia leaves and shredded newspaper) were continually available (Halley, 1992). Captive conditions also meant that these males could not disperse as they would in the last days of the wild breeding season. Removal of the key triggers for the stress response (i.e. agonistic interactions with other individuals and the terminal dispersal) may mean that individual males never enter a phase of negative nitrogen balance. A complimentary explanation for the absence of the stress response in captive populations may be provided by data indicating that semelparous dasyurids which experience intense competition produce low numbers of spermatozoa relative to other mammals (Woolley, 1966; Dewsbury, 1982; Kerr and Hedger, 1983; Taggart and Temple-Smith, 1990a,b). It has been suggested that this strategy allows males to conserve energy that can then be directed toward competition for mates (Taggart et al., 1997). In the semelparous dasyurids, increased androgen levels during the breeding period may be an adaptation to a highly competitive environment, allowing individuals to increase bulk, energy and aggression, and gain a competitive advantage, without negative eVects on sperm production because spermatogenesis ceases before mating (Taylor and Horner, 1970; Kerr and Hedger, 1983). If this were the case, the diVerence in population densities between Antechinus and Phascogale species (Soderquist, 1995; Bradley, 1990b, 1997) would also explain the persistence of sperm production into the breeding season in P. tapoatafa (Millis, 1995; Millis et al., 1999). Acknowledgments The authors gratefully acknowledge the generous support of the management and staV of Healesville Sanctuary and their provision of holding facilities and animals. In particular, we thank Merrill Halley, Jim Phelan and Justin Gamble for their assistance in establishing and monitoring experimental groups and Todd Soderquist for advice
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and discussion. All experimental procedures were reviewed and approved by The Monash University Animal Experimentation Ethics panel prior to commencement of the study and blood samples were collected by Healesville Sanctuary’s veterinary staV (P. Holz and J. Phelan). References Barker, I.K., Beveridge, I., Bradley, A.J., Lee, A.K., 1978. Observations on spontaneous stress-related mortality among males of the dasyurid marsupial Antechinus stuartii Macleay. J. Zool. 26, 435–447. Barnett, J.L., 1973. A stress response in Antechinus stuartii (Macleay). Aust. J. Zool 21, 501–503. Boonstra, R., 1994. Population cycles in microtines: the senescence hypothesis. Evol. Ecol. 8, 196–219. Boonstra, R., 2005. Equipped for life: the adaptive role of the stress axis in male mammals. J. Mamm. 86, 236–247. Bradley, A.J., McDonald, I.R., Lee, A.K., 1975. EVects of exogenous cortisol on mortality of a dasyurid marsupial. J. Endocrinol. 66, 281–282. Bradley, A.J., McDonald, I.R., Lee, A.K., 1976. Corticosteroid binding globulin and mortality in a dasyurid marsupial. J. Endocrinol. 70, 323–324. Bradley, A.J., McDonald, I.R., Lee, A.K., 1980. Stress and mortality in a small marsupial (Antechinus stuartii, Macleay). Gen. Comp. Endocrinol. 40, 188–200. Bradley, A.J., 1982. Steroid binding proteins in the plasma of dasyurid marsupials. In: Archer, M. (Ed.), “Carnivorous Marsupials”. Surrey Beatty and Sons, Sydney, pp. 651–657. Bradley, A.J., 1987. Stress and mortality in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). Gen. Comp. Endocrinol. 67, 85–100. Bradley, A.J., 1990a. Seasonal eVects on the haematology and blood chemistry in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). Aust. J. Zool. 37, 533–543. Bradley, A.J., 1990b. Failure of glucocorticoid feedback during breeding in the male red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). J. Steroid. Biochem. 37, 155–163. Bradley, A.J., 1997. Reproduction and life history in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae): the adaptive-stress senescence hypothesis. J. Zool. 241, 739–755. Bradley, A.J., 2003. Stress, hormones and mortality in small dasyurid marsupials. In: Jones, M., Dickman, C., Archer, M. (Eds.), Predators with Pouches: The Biology of Carnivorous Marsupials. CSIRO Press, Sydney, pp. 250–263. Bradley, A.J., McDonald, I.R., Lee, A.K., 1980. Stress and mortaility in a small marsupial (Antechinus stuartii, Macleay). Gen. Comp. Endocrinol. 40, 188–200. Cheal, P.D., Lee, A.K., Barnett, J.L., 1976. Changes in the haematology of Antechinus stuartii (Marsupialia) and their association with male mortality. Aust. J. Zool. 24, 299–311. Cuttle, P. 1978. The behavior in captivity of Phascogale tapoatafa (Meyer). MSc Thesis, Department of Zoology, Monash University, Clayton, Victoria, Australia. Cuttle, P., 1982. Life history strategy of the dasyurid marsupial Phascogale tapoatafa. In: Archer, M. (Ed.), ‘Carnivorous Marsupials’. Royal Zoological Society of New South Wales, Sydney. Dewsbury, D.A., 1982. Ejaculate cost and male choice. Am. Nat. 119, 601–610. Doe, R.P., Fernandez, R., Seal, U.S., 1964. Measurement of corticosteroid-binding globulin in man. J. Clin. Endocrinol. 24, 1029–1039. Halley, M., 1992. Maintenance and captive breeding of the brush-tailed phascogale Phascogale tapoatafa. Int. Zool. 31, 71–78. Kerr, J.B., Hedger, M.P., 1983. Spontaneous spermatogenic failure in the marsupial mouse Antechinus stuartii Macleay (Dasyuridae: Marsupialia). Aust. J. Zool. 31, 445–466.
Kitchener, D.J., 1981. Breeding, diet and habitat preference of Phascogale calura (Marsupialia: Dasyuridae) in the southern wheat belt, Western Australia. Rec. West. Aust. Mus. 9, 173–186. Lazenby-Cohen, K.A., Cockburn, A., 1988. Lek promiscuity in a semelparous mammal, Antechinus stuartii (Marsupialia: Dasyuridae)? Behav. Ecol. Sociobiol. 22, 195–202. Lee, A.K., Cockburn, A., 1985. Evolutionary Ecology of Marsupials. Cambridge University Press, Cambridge, UK. Lee, A.K., McDonald, I.R., 1985. Stress and population regulation in small mammals. Oxford Rev. Reprod. B 7, 261–304. Lee, A.K., Woolley, P., Braithwaite, R.W., 1982. Life history strategies of dasyurid marsupials. In: Archer, M. (Ed.), ‘Carnivorous Marsupials’. Royal Zoological Society of New South Wales, Sydney. McDonald, I.R., Lee, A.K., Bradley, A.J., Than, K.A., 1981. Endocrine changes in dasyurid marsupials with diVering mortality patterns. Gen. Comp. Endocrinol. 44, 292–301. McDonald, I.R., Lee, A.K., Than, K.A., Martin, R.W., 1986. Failure of glucocorticoid feedback in males of a population of small marsupials (Antechinus swainsonii) during the period of mating. J. Endocrinol. 108, 63–68. Millis, A.L. 1995. Reproductive and genetic studies of the brush-tailed phascogale, Phascogale tapoatafa (Marsupialia: Dasyuridae). BSc Honours Thesis, Department of Anatomy, Monash University, Clayton, Victoria, Australia. Millis, A.L., Taggart, D.A., Bradley, A.J., Phelan, J., Temple-Smith, P.D., 1999. Reproductive biology of the brush-tailed phascogale, Phascogale tapoatafa (Marsupialia: Dasyuridae). J. Zool. 248, 325–335. Rhind, S.G., 2003. Communal nesting in the usually solitary marsupial, Phascogale tapoatafa. J. Zool. 261, 345–351. Rodkey, F.L., 1965. Direct spectrophotometric determination of albumin in human serum. Clin. Chem. 11, 478–487. Sapolsky, R., 1992. Neuroendocrinology of the stress response. In: Becker, J.B., Breedlove, S.M., Crews, D. (Eds.), Behavioural Endocrinology. MIT Press, Cambridge Massachusetts, USA, pp. 287–324. Scott, M.P., 1987. The eVect of mating and agonistic experience on adrenal function and mortality of male Antechinus stuartii (Marsupialia). J. Mamm. 68, 479–486. Soderquist, T.R., 1993a. Maternal strategies of Phascogale tapoatafa (Marsupialia: Dasyuridae). I. Breeding seasonality and maternal investment. Aust. J. Zool. 41, 549–566. Soderquist, T.R., 1993b. Maternal strategies of Phascogale tapoatafa (Marsupialia: Dasyuridae). II. Juvenile thermoregulation and maternal attendance. Aust. J. Zool. 41, 567–576. Soderquist, T.R. 1994. Reproductive strategies of Phascogale tapoatafa (Marsupialia: Dasyuridae). PhD Thesis, Department of Ecology and Evolutionary Biology, Monash University, Clayton, Victoria. Soderquist, T.R., 1995. Spatial organization of the arboreal carnivorous marsupial, Phascogale tapoatafa. J. Zool. 237, 385–398. Soderquist, T.R., Ealey, L., 1994. Social interactions and mating strategies of a solitary carnivorous marsupial, Phascogale tapoatafa, in the wild. Wildlife Res. 21, 527–542. Soderquist, T.R., Lill, A., 1995. Natal dispersal and philopatry in the carnivorous marsupial Phascogale tapoatafa (Dasyuridae). Ethology 99, 297–312. Stein-Behrens, B.A., Sapolsky, R.M., 1992. Stress, Glucocorticoids and Aging. Aging Clin. Exp. Res. 4, 197–210. Taggart, D.A., Temple-Smith, P.D., 1990a. The eVects of breeding season and mating on total number and relative distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii. J. Reprod. Fertil. 88, 81–91. Taggart, D.A., Temple-Smith, P.D., 1990b. An unusual mode of progressive motility in spermatozoa from the dasyurid marsupial, Antechinus stuartii. Reprod. Fert. Develop. 2, 107–114. Taggart, D.A., Selwood, L., Temple-Smith, P.D., 1997. Sperm production, storage, and the synchronization of male and female reproductive cycles in the iteroparous, stripe-faced dunnart (Sminthopsis macroura; Marsupialia): relationship to reproductive strategies within the Dasyuridae. J. Zool. 243, 725–736.
A.L. Schmidt et al. / General and Comparative Endocrinology 149 (2006) 236–243 Tait, J.F., Burstein, S., 1964. In vivo studies of steroid dynamics in man. In: Pincus, C., Thiman, K.V., Astwood, E.B. (Eds.), The Hormones, vol. V. Academic Press, New York, pp. 441–557. Taylor, J.M., Horner, B.E., 1970. Gonadal activity in the marsupial mouse, Antechinus bellus, with notes on other species of the genus. J. Mamm. 51, 659–668.
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Woollard, P., 1971. DiVerential mortality of Antechinus stuartii (Macleay): nitrogen balance and somatic changes. Aust. J. Zool. 19, 347–353. Woolley, P., 1966. Reproduction in Antechinus species and other dasyurid marsupials. Symp. Zool. Soc. Lond. 15, 281–294.
Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity A.L. Schmidt a,e, D.A. Taggart b, P. Holz c, P.D. Temple-Smith d,e, A.J. Bradley a,¤ a
b
Department of Anatomical Sciences, School of Biomedical Sciences, The University of Queensland, St. Lucia, Qld 4072, Australia Royal Zoological Society of S.A., C/O School of Earth and Environmental Science, The University of Adelaide, Nth Terrace, Adelaide, SA 5005, Australia c Healesville Sanctuary, Healesville, Vic. 3777, Australia d Conservation Research Unit, Zoological Parks and Gardens Board, PO Box 74, Parkville, Vic. 3052, Australia e Department of Zoology, University of Melbourne, Parkville, Vic. 3052, Australia Received 15 February 2006; revised 28 May 2006; accepted 8 June 2006 Available online 1 August 2006
Abstract The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-oV is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inXammatory responses. This study examines the endocrine proWle of male brush-tailed phascogales (Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations. © 2006 Elsevier Inc. All rights reserved. Keywords: Albumin; Cortisol; Corticosteroid binding globulin; Dasyuridae; Die-oV; Phascogale; Reproduction; Semelparity
1. Introduction There are seven species of semelparous dasyurid marsupials, characterized by: (i) highly seasonal breeding, occurring over a restricted period of time; (ii) female monoestry; (iii) sexual maturity at approximately 11 months of age; and (vi) stress-induced die-oV of all males within a few weeks of the conclusion of the breeding season, while females may survive to a second season (Lee et al., 1982). Of the seven species, Wve belong to the genus Antechinus and two belong to the genus Phascogale (Lee et al., 1982). Studies of life-history and reproductive physiology (Cuttle,
*
Corresponding author. Fax: +61 7 3365 1299. E-mail address: [email protected] (A.J. Bradley).
0016-6480/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2006.06.003
1978, 1982; Soderquist, 1993a,b, 1994, 1995; Soderquist and Ealey, 1994; Soderquist and Lill, 1995; Millis et al., 1999) have resulted in the classiWcation of the brush-tailed Phascogale, Phascogale tapoatafa as the largest of these species. Female brush-tailed (P. tapoatafa) and red-tailed (Phascogale calura) phascogales may survive to a second breeding season in natural situations (Kitchener, 1981; Lee et al., 1982; Soderquist, 1994) and often do so in captivity (Cuttle, 1978, 1982; Lee et al., 1982; Millis, 1995), although there are indications that fecundity declines with age (Halley, 1992; Soderquist, 1993a,b, 1994). Survival beyond the Wrst breeding season is less common in males, but has been recorded in captive colonies (Cuttle, 1978, 1982; Halley, 1992; Soderquist, 1994). In the captive population of P. tapoatafa maintained at Healesville Sanctuary (Healesville, Victoria) since 1990, all males generally
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survive and maintain good health well beyond breeding, until they are eventually euthanased for management purposes (J. Phelan and M. Halley, unpublished data). Autopsies conducted on euthanased males show none of the pathological symptoms described in dead males from natural populations (Millis, 1995). In particular, there is no evidence of gastric ulceration/gastrointestinal hemorrhage, which is the primary cause of death in wild populations (Soderquist, 1994; Bradley, 1987, 1997, 2003). Observations on captive male P. tapoatafa from the Healesville population indicate that these animals show a single, limited round of sperm production and undergo complete reproductive senescence at the end of the Wrst breeding season. Although they may survive up to three years, histological studies indicate that there is permanent cessation of spermatogenesis at the end of the Wrst season (Millis, 1995; Millis et al., 1999) and a lack of redevelopment of secondary sexual characteristics (e.g. sternal gland activity) in subsequent years (Cuttle, 1978, 1982). This is similar to what occurs in Antechinus, but in P. tapoatafa, testicular sperm production continues into the mating season (Millis, 1995; Millis et al., 1999) while in Antechinus, sperm production ceases prior to breeding (Taylor and Horner, 1970; Kerr and Hedger, 1983). Physiological studies of natural populations of Antechinus stuartii have shown that, immediately before the die-oV period, males (but not females) show increased plasma corticosteroid and liver glycogen levels, and adrenal gland weight, while body weight, and sodium levels decline (Barnett, 1973). It was suggested that, as they approach die-oV, males decrease their food intake and become hypoglycaemic, as a result of corticosteroid activation of gluconeogenesis (Barnett, 1973); indicating that the underlying cause of death is physiological stress. This is supported by observations that male Antechinus also exhibit suppression of immune and inXammatory responses and develop a range of symptoms, including haemolytic anaemia (Cheal et al., 1976; Barker et al., 1978), parasitaemia (Barker et al., 1978; Bradley et al., 1980; Bradley, 1990a) and gastric ulceration (Barker et al., 1978; Soderquist, 1994; Bradley et al., 1980, 1997). Endocrine changes associated with male die-oV in P. calura are similar to those observed in Antechinus, with total plasma androgen concentrations reaching peak levels just prior to die-oV (Bradley et al., 1980; Bradley, 1987, 1990a, 1997). In the Phascogale species, anaemia and parasitaemia are less prevalent (Bradley, 1990a, 1997; Soderquist, 1994), but the primary cause of death in both groups is gastrointestinal hemorrhage (Bradley, 1990a, 1997; Soderquist, 1994). The causal role of elevated glucocorticoid levels in male semelparity was established through treatment of Antechinus with exogenous corticosteroids. Bradley et al. (1975, 1980) removed male A. stuartii from the wild and treated groups with daily injections of exogenous cortisol and testosterone and found that mortality rates were signiWcantly higher in groups treated with exogenous steroid. Increasing doses also produced increasing mortality levels (Bradley et al., 1975, 1980). In a direct comparison of breeding-
237
related endocrine changes in Antechinus swainsonii, A. Xavipes (both of which exhibit the mass die-oV of males) and Sminthopsis crassicaudata (a related species which does not exhibit male die-oV) McDonald et al. (1981) then found that the marked increase in plasma cortisol concentration does not occur in either males of non-die-oV species or females of die-oV species. These early studies led to the hypothesis that glucocorticoid feedback becomes impaired in males during the breeding season (Barnett, 1973; Bradley et al., 1980; McDonald et al., 1986). The role of the glucocorticoid feedback mechanism was consequently investigated in A. swainsonii and P. calura and it was demonstrated that, while maximum corticosteroid binding capacity (MCBC) is always much greater than total corticosteroid in females of the die-oV species and both males and females of non-die-oV species, in males of die-oV species, MCBC is much lower than total corticosteroid (Bradley et al., 1980; McDonald et al., 1986; Bradley, 1990b). This indicates that the adrenal cortex continues to produce glucocorticoid despite the high plasma concentrations and the male die-oV is associated with a failure of glucocorticoid feedback (Bradley et al., 1980; McDonald et al., 1986; Bradley, 1990b). The aim of this study was to investigate the endocrine proWle of male P. tapoatafa surviving beyond the breeding season in captivity, to determine whether they display similar endocrine changes to those observed in wild populations. 2. Materials and methods 2.1. Study group The study colony included 14 adult (seven males; seven females) P. tapoatafa housed at Healesville Sanctuary (Healesville, Victoria), monitored through the 1995 breeding season (mating between 5th and 31st May). One female, and three males were wild-caught individuals, while the remainder were captive-born.
2.2. Blood sampling Regular blood sampling commenced on 29th March, prior to onset of breeding and continued through until 28th July, approximately two months after the end of the breeding period. Animals were collected from the nest-box and anaesthetized with isoXuorance (1-chloro-2,2,2-triXuoroethyl diXuoromethyl ether; VM Supplies, Melbroune) in oxygen, administered by mask. Blood samples were collected from the lateral caudal vein into EDTA-treated syringes Wtted with a 26-guage needle and centrifuged at 2500g for 5 min, to separate plasma, as described in Millis et al. (1999). To ensure that individual animals were not adversely aVected by repeated blood sampling, blood haematocrit values were regularly determined by veterinary staV. Females were bled twice weekly (to monitor oestrogen concentrations for a concurrent study). Males were bled twice weekly through May and June, but in July sampling frequency was reduced to once per week. Two males were euthanased in early July, before the Wnal bleeding, for use in a concurrent study (Millis et al., 1999), meaning the last Wve sampling dates involved only Wve animals. Morphological evidence (Millis et al., 1999) indicated that the breeding season occurred between 8 and 26th May, with the peak mating period occurring between 17 and 22 May.
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2.3. Albumin Plasma albumin concentration was determined on 10 L plasma samples using the bromocresol green spectrophotometric method of Rodkey (1965) and bovine serum albumin (BSA) (Commonwealth Serum Laboratories, Melbourne, Australia) standards. An albumin absorbance standard curve was constructed using BSA standards ranging from 2 to 8 g/dL. Serial dilutions of both P. tapoatafa and BSA standards showed that parallelism was acceptable and the dye binding characteristics of BSA closely approximates that of P. tapoatafa and P. calura (Bradley, unpublished data). The constant for the low aYnity binding of cortisol to P. tapoatafa plasma was determined at 36 °C in 1:5 diluted heat-treated plasma. To denature the high aYnity cortisol binding sites on the CBG, 1:5 diluted plasma was incubated at 60 °C for 30 min. The relationship between the plasma albumin concentration and the low aYnity binding of cortisol to heat-treated diluted plasma was determined and thereafter, the albumin concentration was used as an index of low aYnity binding.
2.4. Maximum corticosteroid binding capacity Plasma high aYnity corticosteroid binding capacity (MCBC) was measured by the gel Wltration method of Doe et al. (1964) and the dextrancoated charcoal method as described by McDonald et al. (1981).
2.5. Cortisol Plasma cortisol levels were determined by radioimmunoassay, as described in Millis et al. (1999). Standard curve construction and conversion of radiation counts to concentrations was performed using AssayZap software (Biosoft, Cambridge, United Kingdom).
2.6. Steroid partitioning Plasma samples were diluted with a phosphate buVer (0.05 M, pH 7.4) containing 0.01% mercury-[(O-carboxyphenyl)-thio]-ethyl (Thimerosal, Sigma Chemical Co., USA) as a preservative. Partitioning of cortisol and corticosterone into CBG bound, albumin bound and free components was calculated by the method of Tait and Burstein (1964) as described for the closely related P. calura (Bradley, 1987), with the high aYnity binding constant KT of 6.2 £ 107 M¡1 at 36 °C for P. tapoatafa (Bradley, 1982) used to calculate the partitioning of cortisol within blood plasma.
2.7. Statistical analysis All parameters were measured from duplicated samples where possible. Variation between duplicated samples was assessed through analysis of variance (ANOVA). No signiWcant variation was detected and duplicate samples were then combined and averaged prior to further analysis. All results are presented as mean § SE. Repeated measures ANOVA analyses of data were conducted using Jandel ScientiWc statistical software (Jandel Corporation, heilerSoftware, GmbH). As steroid partitioning was initially calculated in percentage form, data were arcsine transformed prior to analysis. Where the ANOVA showed statistically signiWcant diVerences, multiple comparisons were made using both Bonferroni’s and Student–Newman–Keuls tests. The null hypothesis was rejected at p < 0.05.
3. Results
detected at any point (Fig. 1a). Prior to onset of breeding (29th March), male plasma albumin concentration was 3.48 § 0.41 g/dL (Fig. 1b). At the beginning of the breeding season (3rd May), albumin concentration increased signiWcantly (p D 0.0100) to 5.60 § 0.22 g/dL. No further signiWcant changes were detected, and at the end of the sampling period, plasma albumin concentration was 4.82 § 0.22 g/dL. 3.2. Maximum corticosteroid binding capacity Prior to breeding, female MCBC was 11.80 § 0.55 g/dL. The only signiWcant (p D 0.0198) variation occurred on 2nd June (at the end of the mating season), when MCBC concentration dropped to 8.83 § 0.44 g/dL (Fig. 1a). Male MCBC ranged from 9.6 § 0.24 g/dL prior to breeding (19th April) to 7.8 § 0.67 g/dL during the breeding period (24th May) (Fig. 1b), although no signiWcant variations were detected. 3.3. Cortisol Prior to onset of breeding (29th March) female plasma cortisol concentration was 0.36 § 0.03 g/dL. No signiWcant variation occurred, however a slight decline (p D 0.0545) to 0.30 § 0.06 g/dL (Fig. 2a) was observed during the peak mating period. Male cortisol concentration showed no signiWcant variation during the sampling period (Fig. 2b). The peak concentration of 0.14 § 0.01 g/dL was recorded approximately three weeks after mating (14th June), but this was not a statistically signiWcant peak. The minimum concentration of 0.04 § 0.01 g/dL was recorded during the mating period (17th May). 3.4. Steroid partitioning Steroid partitioning remained constant in both sexes over the sampling period, with no signiWcant variation in the amount of plasma bound by either CBG or albumin, or in free steroid (Fig. 3a and b). Prior to onset of breeding (29th March), 6.37 § 0.83% of steroid in females was albumin bound, 89.10 § 0.67% was CBG bound and 4.53 § 0.22% was free (Fig. 3a). At the end of the sampling period (28th July; two months after breeding) 6.07 § 0.34% of steroid was albumin bound, 88.37 § 0.01% was CBG-bound and 5.53 § 0.38% was free. Prior to onset of breeding (29th March), 5.38 § 0.75% of steroid in males was albumin bound, 88.90 § 1.67% was CBG-bound and 5.75 § 0.10% was free (Fig. 3b). At the end of the sampling period, 6.95 § 0.22% of steroid in males was albumin bound, 87.30 § 0.20% was CBG-bound and 5.73 § 0.15% was free. In both sexes, MCBC was always greater than plasma cortisol levels.
3.1. Albumin 4. Discussion Female albumin concentration remained constant throughout the sampling period, ranging from 3.50 § 0.23 to 5.70 § 0.98 g/dL. No signiWcant Xuctuations were
Phascogale calura, A. stuartii and A. swainsonii show increases in plasma cortisol levels immediately prior to
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239
Fig. 1. Changes in concentration of plasma albumin and maximum corticosteroid binding capacity (MCBC) in captive brush-tailed phascogales (Phascogale tapoatafa) during the 1995 breeding season. (a) Changes in female MCBC and albumin with the breeding season. Female albumin concentration (n D 7) remained constant throughout the sampling period. MCBC however, showed some Xuctuation. Prior to breeding, MCBC concentration 11.80 § 0.55 g/dL (n D 7) (29th March). This declined (p D 0.0198) to 8.83 § 0.44 g/dL at the end of the mating season (2nd June). (b) Changes in male MCBC and albumin with the breeding season. Prior to onset of breeding (29th March), male plasma albumin concentration was 3.48 § 0.41 g/dL. This increased signiWcantly to 5.67 § 0.22 g/dL (p D 0.0100) at the beginning of the breeding season (3rd May), before declining to 4.82 § 0.46 g/dL by the end of the sampling period (28th July).
Fig. 2. Changes in plasma cortisol concentrations across the breeding season. (a) Changes in female MCBC and albumin with the breeding season. Female cortisol concentration showed no statistically signiWcant variation across the breeding season (n D 7). (b) Changes in male cortisol with the breeding season. Male cortisol concentration showed no statistically signiWcant variation during the sampling period (n D 7).
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Fig. 3. No signiWcant variation in steroid partitions was recorded in either female or male Phascogale tapoatafa. (a) Tuan female steroid partitioning. CBG binding of steroid in females remained around 90% throughout the study period. Albumin binding and free steroid levels accounted equally for the remaining steroid (5–6% albumin bound, 4–5% free). (b) Tuan male steroid partitioning. As in females, approximately 90% of steroid in males was CBG bound at all times, while 5–6% was albumin bound and 4–5% remained free. By the end of the sampling period, »7% of steroid in males was albumin bound, »87% was CBG-bound and »6% was free.
die-oV in wild populations (Barnett, 1973; Bradley et al., 1975, 1976, 1980; McDonald et al., 1981, 1986; Bradley, 1987, 1990b, 1997), but no signiWcant increases in cortisol levels were detected in captive male P. tapoatafa surviving beyond the breeding season. The peak cortisol concentration observed in these animals (0.14 § 0.01 g/dL) occurred following mating (14th June), but was much lower than recorded in wild populations of P. calura, where concentrations range from 1 to 2 g/dL in females (Bradley, 1987) and 4–5 g/dL in males (Bradley, 1987). Maximum corticosteroid binding capacity (MCBC) also remained relatively constant (ranging from 7.8 to 9.6 g/dL in males and from 8.8 to 11.5 g/dL in females) and lies within a similar range to that observed in wild female P. calura (»6 to 8 g/dL). In wild males however, MCBC declines as low as 1.9 g/dL during the die-oV period (Bradley, 1987). The observation of no signiWcant increase in plasma cortisol, and no signiWcant decrease in MCBC, in captive P. tapoatafa surviving beyond the breeding season therefore suggests that the stress-related die-oV is a response to events or conditions unique to natural populations. A second notable diVerence between the captive population and wild populations of semelparous dasyurids lies in testosterone levels. In the captive population used in this study, testosterone concentration reached a maximum of 6.25 § 0.56 ng/mL during the breeding period (Millis et al., 1999). In wild P. calura, testosterone concentrations rise to almost twice this level (8–9 ng/mL) immediately prior to
die-oV (Bradley, 1987) and this contributes to the decline in CBG levels because of an inverse relationship between plasma testosterone concentration and MCBC (Bradley et al., 1980; Bradley, 1990b). The eVects of stress then cause a further decline in CBG concentrations (Bradley et al., 1980; Bradley, 1990b), which compounds this eVect. The lower levels of testosterone and cortisol in the captive population may therefore be critical and insuYcient to produce the same decrease in CBG observed in natural populations. One possible factor inXuencing the absence of the mating related androgen and cortisol surges, and subsequent decline in steroid-binding capacity, is social environment. In a study of the eVects of mating and agonistic experience on male A. stuartii, Scott (1987) found that plasma androgen and MCBC in males isolated from all conspeciWcs did not vary from that of males in physical contact with conspeciWc males, or in intermittent contact with oestrous females. However, by the end of the breeding season, males in daily contact with conspeciWcs showed greater plasma corticosteroid concentrations and higher mortality rates than isolated males. Communal nesting is common in Antechinus (LazenbyCohen and Cockburn, 1988) and has been observed in natural populations of P. tapoatafa, but this appears to represent a response to unusual thermoregulatory challenge and the species is usually solitary (Rhind, 2003). DiVerences in sociality between the two genera may be reXected in the prevalence of secondary causes of mortality such as
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parasite infection. While Antechinus species exhibit high levels of parasitic infection prior to die-oV (Barker et al., 1978), in Phascogale species, parasitic infection is less common (Bradley, 1990a; Soderquist, 1994). However, the high level of parasitic infection seen in some Antechinus populations is not duplicated in others (A. Bradley, unpublished data) and parasite-induced mortality cannot be used as a universal explanation of post-mating mortality of males. In both species, the primary cause of death is gastrointestinal hemorrhage following development of gastrointestinal ulcers (Bradley, 1990a, 1997; Soderquist, 1994), suggesting that there is some other, underlying process that drives male die-oV. Possible factors can be identiWed through examination of other life-history features. Immediately prior to die-oV (one week before disappearance), wild male P. calura and P. tapoatafa undergo a terminal dispersal; travelling large distances in a short period of time (Soderquist, 1994; Soderquist and Ealey, 1994; Bradley, 1997). The terminal dispersal occurs during winter, when nighttime temperatures are low and males trapped during this period are in poor condition, showing symptoms of thermoregulatory stress, and negative nitrogen balance (Bradley, 1997), indicative of homeostatic stress. Boonstra (1994) has proposed that progressive deterioration in endocrine feedback (along the hippocampal– hypothalamic–pituitary–adrenal axis) means older animals are particularly prone to homeostatic stress when faced with such conditions. However, male Phascogales are only 11 months old at the time of the terminal dispersal and age is unlikely to be a major factor. Bradley (1997) has suggested that the stress response of male Phascogale species is an adaptive mechanism, designed to mobilize energy resources during the later stages of mating and combat the thermoregulatory challenge associated with the terminal dispersal. This is supported by evidence that marsupials sometimes undergo stress-related endocrine changes to produce energy when required (Lee and Cockburn, 1985; Lee and McDonald, 1985). This “Adaptive Stress Senescence” hypothesis (Bradley, 1997; Boonstra, 2005), which may also explain similarities in the endocrine proWles of semelparous Wsh species (Stein-Behrens and Sapolsky, 1992), is based on the suggestion that exposure of the hippocampal glucocorticoid receptors to high concentrations of free cortisol results in damage to the hippocampal–hypothalamic–pituitary–adrenocortical axis and consequent impairment of glucocorticoid control (Sapolsky, 1992; Boonstra, 1994, 2005). Observations in wild populations of semelparous dasyurids have provided previous support for this hypothesis. The decreased immune function observed in natural populations is produced by a syngergisteric interaction between free cortisol and testosterone concentrations (Woollard, 1971; Barnett, 1973; Bradley et al., 1976, 1980; McDonald et al., 1981; Bradley, 1990a,b), accompanied by a decrease in corticosteroid binding globulin (Bradley et al., 1976; Bradley, 1990b), leading to an increase in the amount of
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free steroid within the system. During this period, the tissues of males are exposed to large amounts of glucocorticoid (Bradley, 1987). In P. calura, the tissues of males may be exposed to 25 times more free cortisol than the tissue of females (Bradley, 1990b). The absence of the stress response in captive males that survive beyond breeding provides further support. The nature of the captive-breeding program at Healesville Sanctuary is such that the inXuence of the social environment is limited. Females are maintained in permanent enclosures and individual males are rotated in a “round robin” fashion. In 1995, there were eight males and seven females, meaning each male spent only two days with a given female, before being transferred to a new cage with a diVerent female, or a solitary holding pen, for a further 2 days. The total period of exposure to females spanned only 2–3 weeks, limiting any eVect of aggression from pregnant females. Captive males were also fed a balanced, high-quality diet, and a variety of nesting materials (fresh Eucalypt and Acacia leaves and shredded newspaper) were continually available (Halley, 1992). Captive conditions also meant that these males could not disperse as they would in the last days of the wild breeding season. Removal of the key triggers for the stress response (i.e. agonistic interactions with other individuals and the terminal dispersal) may mean that individual males never enter a phase of negative nitrogen balance. A complimentary explanation for the absence of the stress response in captive populations may be provided by data indicating that semelparous dasyurids which experience intense competition produce low numbers of spermatozoa relative to other mammals (Woolley, 1966; Dewsbury, 1982; Kerr and Hedger, 1983; Taggart and Temple-Smith, 1990a,b). It has been suggested that this strategy allows males to conserve energy that can then be directed toward competition for mates (Taggart et al., 1997). In the semelparous dasyurids, increased androgen levels during the breeding period may be an adaptation to a highly competitive environment, allowing individuals to increase bulk, energy and aggression, and gain a competitive advantage, without negative eVects on sperm production because spermatogenesis ceases before mating (Taylor and Horner, 1970; Kerr and Hedger, 1983). If this were the case, the diVerence in population densities between Antechinus and Phascogale species (Soderquist, 1995; Bradley, 1990b, 1997) would also explain the persistence of sperm production into the breeding season in P. tapoatafa (Millis, 1995; Millis et al., 1999). Acknowledgments The authors gratefully acknowledge the generous support of the management and staV of Healesville Sanctuary and their provision of holding facilities and animals. In particular, we thank Merrill Halley, Jim Phelan and Justin Gamble for their assistance in establishing and monitoring experimental groups and Todd Soderquist for advice
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and discussion. All experimental procedures were reviewed and approved by The Monash University Animal Experimentation Ethics panel prior to commencement of the study and blood samples were collected by Healesville Sanctuary’s veterinary staV (P. Holz and J. Phelan). References Barker, I.K., Beveridge, I., Bradley, A.J., Lee, A.K., 1978. Observations on spontaneous stress-related mortality among males of the dasyurid marsupial Antechinus stuartii Macleay. J. Zool. 26, 435–447. Barnett, J.L., 1973. A stress response in Antechinus stuartii (Macleay). Aust. J. Zool 21, 501–503. Boonstra, R., 1994. Population cycles in microtines: the senescence hypothesis. Evol. Ecol. 8, 196–219. Boonstra, R., 2005. Equipped for life: the adaptive role of the stress axis in male mammals. J. Mamm. 86, 236–247. Bradley, A.J., McDonald, I.R., Lee, A.K., 1975. EVects of exogenous cortisol on mortality of a dasyurid marsupial. J. Endocrinol. 66, 281–282. Bradley, A.J., McDonald, I.R., Lee, A.K., 1976. Corticosteroid binding globulin and mortality in a dasyurid marsupial. J. Endocrinol. 70, 323–324. Bradley, A.J., McDonald, I.R., Lee, A.K., 1980. Stress and mortality in a small marsupial (Antechinus stuartii, Macleay). Gen. Comp. Endocrinol. 40, 188–200. Bradley, A.J., 1982. Steroid binding proteins in the plasma of dasyurid marsupials. In: Archer, M. (Ed.), “Carnivorous Marsupials”. Surrey Beatty and Sons, Sydney, pp. 651–657. Bradley, A.J., 1987. Stress and mortality in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). Gen. Comp. Endocrinol. 67, 85–100. Bradley, A.J., 1990a. Seasonal eVects on the haematology and blood chemistry in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). Aust. J. Zool. 37, 533–543. Bradley, A.J., 1990b. Failure of glucocorticoid feedback during breeding in the male red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae). J. Steroid. Biochem. 37, 155–163. Bradley, A.J., 1997. Reproduction and life history in the red-tailed phascogale, Phascogale calura (Marsupialia: Dasyuridae): the adaptive-stress senescence hypothesis. J. Zool. 241, 739–755. Bradley, A.J., 2003. Stress, hormones and mortality in small dasyurid marsupials. In: Jones, M., Dickman, C., Archer, M. (Eds.), Predators with Pouches: The Biology of Carnivorous Marsupials. CSIRO Press, Sydney, pp. 250–263. Bradley, A.J., McDonald, I.R., Lee, A.K., 1980. Stress and mortaility in a small marsupial (Antechinus stuartii, Macleay). Gen. Comp. Endocrinol. 40, 188–200. Cheal, P.D., Lee, A.K., Barnett, J.L., 1976. Changes in the haematology of Antechinus stuartii (Marsupialia) and their association with male mortality. Aust. J. Zool. 24, 299–311. Cuttle, P. 1978. The behavior in captivity of Phascogale tapoatafa (Meyer). MSc Thesis, Department of Zoology, Monash University, Clayton, Victoria, Australia. Cuttle, P., 1982. Life history strategy of the dasyurid marsupial Phascogale tapoatafa. In: Archer, M. (Ed.), ‘Carnivorous Marsupials’. Royal Zoological Society of New South Wales, Sydney. Dewsbury, D.A., 1982. Ejaculate cost and male choice. Am. Nat. 119, 601–610. Doe, R.P., Fernandez, R., Seal, U.S., 1964. Measurement of corticosteroid-binding globulin in man. J. Clin. Endocrinol. 24, 1029–1039. Halley, M., 1992. Maintenance and captive breeding of the brush-tailed phascogale Phascogale tapoatafa. Int. Zool. 31, 71–78. Kerr, J.B., Hedger, M.P., 1983. Spontaneous spermatogenic failure in the marsupial mouse Antechinus stuartii Macleay (Dasyuridae: Marsupialia). Aust. J. Zool. 31, 445–466.
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