Contextual modulation of androgen effects on agonistic interactions.

Seasonal changes in steroid hormones are known to have a major impact on social behavior, but often are quite sensitive to environmental context. In the bi-directionally sex changing fish, Lythrypnus dalli, stable haremic groups exhibit baseline levels of interaction. Status instability follows immediately after male removal, causing transiently elevated agonistic interactions and increase in brain and systemic levels of a potent fish androgen, 11-ketotestosterone (KT). Coupling KT implants with a socially inhibitory environment for protogynous sex change induces rapid transition to male morphology, but no significant change in social behavior and status, which could result from systemically administered steroids not effectively penetrating into brain or other tissues. Here, we first determined the degree to which exogenously administered steroids affect the steroid load within tissues. Second, we examined whether coupling a social environment permissive to sex change would influence KT effects on agonistic behavior. We implanted cholesterol (Chol, control) or KT in the dominant individual (alpha) undergoing sex change (on d0) and determined the effects on behavior and the degree to which administered steroids altered the steroid load within tissues. During the period of social instability, there were rapid (within 2 h), but transient effects of KT on agonistic behavior in alphas, and secondary effects on betas. On d3 and d5, all KT, but no Chol, treated females had male typical genital papillae. Despite elevated brain and systemic KT 5 days after implant, overall rates of aggressive behavior remained unaffected. These data highlight the importance of social context in mediating complex hormone-behavior relationships.

Levels of corticotropin-releasing hormone messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus and proopiomelanocortin mRNA in the anterior pituitary during late gestation in fetal sheep.

CRH regulates POMC gene expression and subsequent ACTH biosynthesis and release. In sheep, the preterm rise in fetal plasma ACTH commences at approximately 125 days gestation (dGA; 147 dGA = term), preceding the initiation of adrenocortical steroidogenesis. We hypothesized that an increase in CRH expression in the hypothalamic paraventricular nucleus (PVN) and POMC expression in the anterior pituitary in the late gestation sheep fetus may precede adrenal cortex maturation. Fetal sheep were obtained at 105-107 (n = 4), 128-130 (n = 5), and 138-140 (n = 4) dGA. Hypothalami were cryosectioned and subjected to in situ hybridization for ovine CRH mRNA. In all dGA groups, expression of CRH mRNA was observed throughout the rostrocaudal extent of the fetal PVN. The midrostral region of the fetal PVN where the dorsal and ventral divisions of the rostral PVN merge to form a single structure was selected for quantification. The number of copies of CRH probe hybridized per micron 3 were determined to estimate the quantity of hybridized CRH mRNA; the mean estimated CRH mRNA copy number per micron 3 midrostral PVN were 0.064 +/- 0.012 (105-107 dGA), 0.237 +/- 0.048 (128-130 dGA), and 0.108 +/- 0.034 (138-140 dGA; mean +/- SEM copies per micron 3 PVN). CRH mRNA signal significantly increased between 105-107 and 128-130 dGA (P < or = 0.05); 138-140 dGA levels of mRNA were not different from either 105-107 or 128-140 dGA levels. Regional variation in CRH mRNA levels were observed within the midrostral PVN between groups; at 138-140 dGA, a population of lateral midrostral PVN neurons maintain CRH mRNA levels greater than 105-107 dGA (P < 0.05), similar to those at 128-130 dGA. Fetal anterior pituitary RNA was subjected to Northern analysis for POMC mRNA. POMC mRNA levels in fetal anterior pituitaries were 14.1 +/- 2.2 (105-107 dGA), 28.9 +/- 10.9 (128-130 dGA), and 43.2 +/- 6 (138-140 dGA; mean +/- SEM arbitrary units). A significant increase (P < or = 0.05) was observed at 138-140 dGA compared to levels at 105-107 dGA. We conclude CRH mRNA levels in the fetal PVN increase coincident with increased POMC gene expression and the late gestation rise in fetal plasma ACTH. We speculate that a neuroendocrine stimulus at the fetal PVN may precipitate increased levels of CRH mRNA, initiating the maturation of the fetal hypothalamic-hypophyseal-adrenal axis, thus inducing the events of labor and delivery in sheep.

Serial adult sex change involves rapid and reversible changes in forebrain neurochemistry.

Species with multiple sexual phenotypes provide novel insights into neuroendocrine mechanisms underlying vertebrate sexuality. Many fish species are capable of socially mediated sex reversal: the marine goby (Trimma okinawae) is one of only four species known to change sex more than once and in either direction. We have demonstrated that socially mediated serial sex change involves significant and reversible changes in the size of arginine vasotocin-producing forebrain cells. Sex-specific mating and parental behavior also change during sex reversal: both are regulated by the vasotocin/vasopressin peptide family in vertebrates. These neural and behavioral changes are correlated with modifications in gonad form and function, the form of the sexually dimorphic genital papilla, and the presence of an active male accessory gonadal structure. The speed, reversibility, and extent of these changes in adult sexual phenotype is remarkable relative to the rigid nature of these characteristics in many vertebrates.

The nervus terminalis ganglion in Anguilla rostrata: an immunocytochemical and HRP histochemical analysis.

Immunocytochemistry and retrograde horseradish peroxidase (HRP) transport were used to study the ganglion of the nervus terminalis in the American eel, Anguilla rostrata. Luteinizing hormone releasing hormone (LHRH) like immunoreactivity was found in large, ganglion-like cells located ventromedially at the junction of the telencephalon and olfactory bulb and in fibers within the retina and olfactory epithelium. HRP transport from the retina demonstrated direct connections with both the ipsi- and contralateral populations of these ganglion-like cells. Given the well-documented role of both olfaction and vision during migratory and reproductive phases of the life cycle of eels, the robust nature of a nervus terminalis system in these fish may present a unique opportunity to study the behavioral correlates of structure-function organization in a discrete population of ganglion-like cells.

Post-fight levels of plasma lactate and corticosterone in male copperheads, Agkistrodon contortrix (Serpentes, Viperidae): differences between winners and losers.

During the mating seasons (late summer and spring), male copperheads (Agkistrodon contortrix; Serpentes, Viperidae) engage in aggressive physical interactions for priority of access to females. These fights generally involve two individuals and are characterized by prominent vertical displays, a high degree of physical contact, and the absence of biting. Ritualized aggression does not occur in females. Although intrasexual aggression in conspecifics has obvious energetic costs (e.g., lactate accumulation) that can affect subsequent behavior, few studies have addressed these costs in reptiles, and no studies have examined snakes. Moreover, recent studies suggest psychoneuroendocrine (catecholamines, glucocorticoids) regulation of metabolism during and following aggressive episodes. There were three main questions addressed in this study. Do winners and losers of staged, pair-wise encounters show differences in post-fight (60-min) levels of plasma lactate and corticosterone (CORT)? Are levels of plasma lactate correlated with levels of plasma CORT? Is fight duration correlated with levels of plasma lactate and CORT? Two different control groups (cage and arena) were used. Body length, body mass, duration of fighting, and season of testing were not correlated with levels of plasma lactate and CORT. At 60-min post-fight, losers had significantly higher levels of mean plasma lactate and CORT when compared to levels in winners and controls, and there were no significant differences between winners and controls. From our results, we suggest the following conclusions. First, elevated levels of CORT in losers, but not winners, result from psychoneuroendocrine factors rather than simple exercise. Second, elevated levels of CORT in losers retard metabolic recovery resulting in higher lactate levels in losers, whereas winners return to pre-fight levels within 60-min post-fight. Last, the CORT response has a net negative effect on metabolic recovery and may be implicated in the protracted suppression of aggressive behavior in losers.

Neonatal exposures to technical methoxychlor alters ovaries in adult mice.

Technical grade methoxychlor (MXC) is an estrogenic pesticide currently used for pest control in the US. To determine the long-term effects of technical MXC on ovaries and estrous cycles, neonatal mice received 14 daily intraperitoneal injections of sesame oil only, 10.0 micrograms estradiol-17 beta, or 0.05 mg, 0.1 mg, 0.5 mg, or 1.0 mg of technical MXC. At 3, 6, and 12 months, vaginal smears were examined for 12 d and ovaries collected. All technical MXC doses and estradiol increased the duration of vaginal cornification but only estradiol, 0.5, or 1.0 mg technical MXC induced ovarian atrophy, relative ovarian weight loss, and depletion of corpora lutea. Technical MXC doses of 0.05 or 0.1 mg produced the opposite effects; the ovaries remained heavy, large, and filled with corpora lutea. On the other hand, follicular cysts were recorded in all groups, except the 1.0 mg MXC group. These augmentary and inhibitory effects of MXC at low and high doses mimic the estrogen effects at low and high doses, and were probably due to the altered hypothalamic-hypophyseal function.

Group sex, sex change, and parasitic males: sexual strategies among the fishes and their neurobiological correlates.

Sexual selection, in the form of intrasexual competition and mate choice, has driven the evolution of a variety of sexual phenotypes amongst the vertebrates (Andersson, 1994). As a result, vertebrate species utilize many different approaches to acquire fertilizations. Humans and other primates show a wide range of sexual behaviors, but this range is dwarfed by the remarkable variation seen in advanced fishes. The goals of this review are (a) to acquaint the reader with the tremendous sexual diversity exhibited by fishes, (b) to demonstrate how this diversity provides unique opportunities to examine the neurobiological correlates of vertebrate sexual strategies, and (c) to highlight the parallels between the neuroendocrine correlates of the sexual strategies of fish with the mechanisms underlying sexual phenotypes in other vertebrates, showing the utility of fish studies for understanding sexual variation in general.

Aggressive encounters alter the activation of serotonergic neurons and the expression of 5-HT1A mRNA in the hamster dorsal raphe nucleus.

Serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN) have been implicated in stress-induced changes in behavior. Previous research indicates that stressful stimuli activate 5-HT neurons in select subregions of the DRN. Uncontrollable stress is thought to sensitize 5-HT neurons in the DRN and allow for an exaggerated 5-HT response to future stimuli. In the current study, we tested the hypothesis that following aggressive encounters, losing male Syrian hamsters would exhibit increased c-Fos immunoreactivity in 5-HT DRN neurons compared to winners or controls. In addition, we tested the hypothesis that losers would have decreased 5-HT1A mRNA levels in the DRN compared to winners or controls. We found that a single 15-min aggressive encounter increased c-Fos expression in 5-HT and non-5-HT neurons in losers compared to winners and controls. The increased c-Fos expression in losers was restricted to ventral regions of the rostral DRN. We also found that four 5-min aggressive encounters reduced total 5-HT1A mRNA levels in the DRN in losers compared to winners and controls, and that differences in mRNA levels were not restricted to specific DRN subregions. These results suggest that social defeat activates neurons in select subregions of the DRN and reduces message for DRN 5-HT1A autoreceptors. Our results support the hypothesis that social stress can activate 5-HT neurons in the DRN, reduce 5-HT1A autoreceptor-mediated inhibition, and lead to hyperactivity of 5-HT neurons.

Diurnal patterns and sex differences in cortisol, 11-ketotestosterone, testosterone, and 17beta-estradiol in the bluebanded goby (Lythrypnus dalli).

The primary goals of this study were to evaluate diurnal patterns of and sex differences in the levels of cortisol, 11-ketotestosterone, testosterone, and 17beta-estradiol in the sex-changing bluebanded goby Lythrypnus dalli. Steroid hormones were collected from water samples and analyzed by enzyme immunoassay. During the breeding season, hormones were sampled from both males and females at seven time points between 0600 and 2000 h. When comparing each time point separately, there were significant overall time effects for cortisol and 17beta-estradiol. Cortisol concentrations were lowest at the 0800-1000 h sampling point and showed a qualitative peak in late morning (1000-1200 h). Concentrations of 17beta-estradiol were elevated at the last sampling point (1800-2000 h). Broader temporal trends were revealed for testosterone and 11-ketotestosterone concentrations, both of which were elevated in the morning. There were no sex differences in overall hormone concentrations or temporal profiles for cortisol, 11-ketotestosterone, or testosterone. Males and females showed similar diurnal patterns of 17beta-estradiol but females had significantly higher water-borne 17beta-estradiol levels than males. The results show the presence of diurnal changes in steroid hormone levels in male and female bluebanded gobies. The lack of sex differences in androgens suggests that males of this species, and perhaps other bi-directional sex-changing species in which males do not exhibit prominent secondary sexual characteristics, do not require persistent elevations in 11-ketotestosterone or testosterone to maintain the male phenotype. Although the role of 17beta-estradiol in maintaining sex differences in sexually plastic species is unclear, our results suggest that, of the hormones measured, 17beta-estradiol has the greatest potential for future studies interested in this question.

Elevated 11-ketotestosterone during paternal behavior in the Bluebanded goby (Lythrypnus dalli).

The relationship between androgens and paternal behavior is not straightforward, potentially because of the diversity of tasks a male must undertake to maximize reproductive success, notably alternating between courtship, aggression, and offspring care. In some species, these events are separated in time, but in others they are coincident. The endocrine profiles of species that simultaneously court, parent, and defend a nest, such as male bluebanded gobies (Lythrypnus dalli), are not well understood. We sampled a potent fish androgen, 11-ketotestosterone (KT), at different life history stages (experienced parenting males, experienced males not actively parenting, inexperienced males with their first clutch, and females), to examine this relationship. We found that experienced parenting L. dalli males have the highest KT levels of any group, while none of the other groups differed significantly. Males showed elevated KT levels when they had eggs compared to when they did not. Our data suggest that KT facilitates at least some aspects of parental care in L. dalli.

Neuronal correlates of sex/role change in labrid fishes: LHRH-like immunoreactivity.

Gonadotropins have been implicated in the proximate control of socially induced sex and role change in labrid fishes. Since the release of gonadotropins is controlled by Gonadotropin Releasing Hormones, immunocytochemical techniques were used here to determine the qualitative and quantitative distribution of Luteinizing Hormone Releasing Hormone (LHRH, the primary Gonadotropin Releasing Hormone in mammals) in the brain of a sex-reversing labrid fish, Thalassoma bifasciatum. Fibers showing LHRH-like immunoreactivity were distributed throughout the brain, with densest concentrations in the olfactory bulb, the telencephalic area dorsalis and preoptic area of the forebrain, the medial posterior tuber and nucleus of the saccus vasculosus of the hypothalamus, and the tectum and tegmentum of the midbrain. Somata positive for LHRH were found in the anterior and posterior divisions of the ganglion of the nervus terminalis, the preoptic area, and the periventricular rostral midbrain. There were no qualitative differences in the distribution of LHRH-like immunoreactive cells and fibers among the different sexual phases. However, there were quantitative differences in the number of LHRH-like immunoreactive cells in the preoptic area, a brain region known to be involved both in sexual development and in the control of sexual physiology and behavior in adults. Based on this, and results in other teleosts, it seems likely that central control of sex reversal and expression of alternative reproductive behaviors in labrid fishes is more directly related to quantitative changes in the number and/or biosynthetic activity of LHRH-positive somata and their fiber projections.

Social and neural modulation of sexual plasticity in teleost fish.

Teleost fishes are the 'champions' of sexual plasticity among vertebrates. Several species have two male reproductive morphs with distinct suites of behavioral, somatic, neuronal, endocrinological, and life history traits. Here, we consider recent studies of the social and neural modulation of sexual plasticity for such species with a focus on two neuropeptides, gonadotropin releasing hormone (GnRH) and arginine vasotocin (AVT, teleostean analogue of mammalian arginine vasopressin). The major premise of this review is that phenotypic changes in GnRH and AVT expression in the brain can orchestrate events leading to changes in either sexual status or the expression of morph specific display behaviors important in reproduction.

Gonadal steroids affect LHRH preoptic cell number in a sex/role changing fish.

In diandric sex-reversing fishes, sexually active males and females (primary phase) regularly transform into an alternative reproductive morph, terminal-phase males, that are morphologically and behaviorally distinct. The transformation from primary to terminal phase is associated with a twofold increase in the number of luteinizing hormone-releasing hormone (LHRH) immunopositive cells in the forebrain preoptic area, a region involved in both the initial development and daily control of reproductive physiology and behavior. We now show that implants of 11-ketotestosterone induce increases in LHRH cell number in both primary phase sexes to the level observed in field-collected terminal phase males. Conversely, gonadal steroids had no effect on the number of LHRH preoptic cells in terminal phase males, suggesting that this is indeed a terminal stage in the development of this species. These results demonstrate that transition to the terminal phase by both sexes involves a parallel and convergent change in LHRH cell number, which utilizes an evolutionarily conserved mechanism of sexual differentiation: the inductive effects of gonadal steroid hormones.

Male sexual polymorphism, alternative reproductive tactics, and androgens in combtooth blennies (pisces: blenniidae).

In species in which intense intermale competition for the access to females is present males of lower competitive ability may adopt alternative reproductive tactics (ART) to get access to mates. These ART translate in many cases into male sexual polymorphism, with individuals following distinctly different tactics. Usually two alternative male morphs can be recognized in species with ART: (1) bourgeois males that compete for access to mates invest in typically male behaviors, such as building elaborated nests or displaying ornaments; and (2) parasitic males that take advantage of the success of the bourgeois males in attracting females and attempt "sneaker" fertilizations (e.g., sneaker and satellite males). In combtooth blennies (Blenniidae) the co-occurrence of ART and male sexual polymorphism has been described for two temperate species: the peacock blenny, Salaria pavo, and the Azorean rock-pool blenny, Parablennius sanguinolentus parvicornis. Interestingly, while in the peacock blenny the alternative male morph adopts a sneaker tactic, in the rock-pool blenny parasitic males act as satellites to nest-holder males. Thus, this variation in the ART expressed in these two closely related species allows for a comparative study of the proximate and ultimate factors affecting the expression of the two ART. In this article we summarize the available information on androgen levels in bourgeois and parasitic males of natural populations of the two species and of recent studies on the effect of exogenous administration of androgens on tactic switching in parasitic males of the two species. The information is discussed within the frame of the relative plasticity hypothesis, which predicts that plastic alternative morphs should show differences in hormone levels and that the administration of sex steroids should be effective in promoting the switch from the parasitic to bourgeois tactic. The evidence is only partly consistent with this hypothesis. Alternatively, a social transduction hypothesis that better fits the available data on androgens and ART in teleost is proposed. It states that the observed differences in androgen levels between alternative morphs should not be interpreted as an organization vs activation effect of steroids, but rather as the limited vs lifelong responsiveness of the neuroendocrine axis to social regulation.

GnRH cell size and number in a teleost fish with two male reproductive morphs: sexual maturation, final sexual status and body size allometry.

Gonadotropin releasing hormone-like immunoreactive (GnRH-ir) cells in both the ganglion of the terminal nerve (TN) and the preoptic area (POA) have been implicated in the development and maintenance of reproductive behavior and physiology in teleost fishes. One marine species, the plainfin midshipman, Porichthys notatus, exhibits two sexually mature male morphs (types I and II) which differ with respect to size at sexual maturation, gonad/body weight index, reproductive tactic and vocal motor traits. Type II males become reproductively active at a smaller body size than either females or type I males. Immunocytochemical techniques and quantitative analyses were used here to determine the size and number of GnRH-ir cells in the TN and POA amongst field collected juveniles, sexually mature females, and type I and II males. Mean GnRH-ir cell size and number in the TN did not vary across the entire range of specimens. However, mean GnRH-ir cell size and number in the POA were 50-100% greater in sexually mature adults compared to juveniles. Analyses of covariance indicated that increases in cell number, but not cell size, could be explained solely on the basis of changes in body size. However, regression analyses showed that body size had a significant influence on increasing cell number only in the juvenile-type I male transition and the juvenile-female transition, not in the juvenile-type II male transition. The latter suggests that type II males, unlike the other adult morphs, have 'escaped' from a body size constraint imposed on increasing GnRH-ir cell number in the POA. There were also significant differences among the adult morphs in the size of GnRH-ir POA cells that could not be explained on the basis of differences in body size but, rather, appear to reflect differences in the temporal onset of sexual maturation. Together, the data suggest that the timing of changes in POA phenotype may provide a proximate mechanism permitting the development of alternative male reproductive morphs.

GnRH-like immunoreactivity in the peripheral olfactory system and forebrain of Atlantic salmon (Salmo salar): a reassessment at multiple life history stages.

Neurons with gonadotropin releasing hormone-like immunoreactivity (GnRH-ir) were identified within the peripheral olfactory system of Atlantic salmon (Salmo salar) at multiple life history stages. Within the forebrain, GnRH-ir somata were found in the preoptic area and in the caudomedial olfactory bulb in a position comparable to the ganglion of the nervus terminalis of other teleosts. Somata positive for GnRH were also found throughout the rostro-caudal extent of the olfactory nerve, and clustered within the medial component of the olfactory nerve as it arises from the olfactory epithelium. Results from tract tracing experiments with horseradish peroxidase indicate that at least some cells in this cluster project to the retina, suggesting that they too are part of a terminal nerve ganglion as anatomically defined in other vertebrates. We suggest that the presence of a distinct cluster of terminal nerve ganglion cells in the immediate vicinity of the olfactory epithelium may point to a peripheral site of neuromodulatory control in the olfactory system in salmon and perhaps in other teleosts as well.

Hypothalamic arginine vasotocin mRNA abundance variation across sexes and with sex change in a coral reef fish.

Gonadal hormones are important mediators of sexual and aggressive behavior in vertebrates. Recent evidence suggests that the peptide hormones arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin (AVP) often critically mediate these gonadal hormone effects on behavior and have direct influences on behavioral variation. Behavioral differences between sexes, across reproductive states, and even among closely related species are correlated with differences in central AVT/AVP systems in many species. We report differences in hypothalamic AVT mRNA levels between distinct alternate male phenotypes and with female-to-male sex change in the bluehead wrasse (Thalassoma bifasciatum), a teleost fish. The aggressively dominant and strongly courting male phenotype has greater numbers of AVT mRNA producing cells in the magnocellular preoptic area of the hypothalamus than females. Levels of AVT mRNA within these cells in dominant males are also approximately three times female levels whereas the non-aggressive male phenotype has AVT mRNA levels approximately twice female levels. Behavioral sex change is very rapid in this species and is not dependent on the presence of gonads. Conversely, rapid increases in sexual and aggressive behavior during sex change are closely paralleled by approximate fourfold increases in hypothalamic AVT-mRNA levels. The behavioral plasticity shown by bluehead wrasses in response to social environment might be mediated in part by a neuropeptide, AVT, with changes in the gonads and gonadal hormones as the result rather than the cause of behavioral dominance.

The effects of estradiol on gonadotropin-releasing hormone neurons in the developing mouse brain.

The hypothalamic-pituitary-gonadal (HPG) axis plays a critical role in the control of reproduction. Two key hormonal components of the HPG axis are gonadal steroids and gonadotropin-releasing hormone (GnRH). Gonadal steroids are known to organize the development of neural substrates which control adult reproductive behavior; GnRH is required for normal reproductive structure and function. The possibility that gonadal steroids may produce organizational changes in the pattern of GnRH staining observed in the brain is investigated through the use of injections of estradiol to neonatal mice and subsequent GnRH immunocytochemistry at 2 months of age. Our results indicate that the number of GnRH-immunoreactive (GnRH-ir) cells is normally lower in females than males. Estradiol did not affect the number of GnRH-ir cells in females, but significantly increased the number of GnRH-ir cells in males, suggesting that early exposure to estradiol results in masculinization of the GnRH axis of males.

Muscle fiber type correlates with innervation topography in the rat serratus anterior muscle.

Previous studies have reported that motoneurons from the sixth spinal nerve (C6) innervate the majority of muscle fibers in the rat serratus anterior (SA) muscle. The seventh spinal nerve (C7) innervates a limited number of SA fibers, increasing caudally. This topographic map is partially reestablished following denervation. In the present study, muscle fibers of the SA were stained with monoclonal antibodies for the muscle-specific fast myosin heavy chain (F-MHC) and slow myosin heavy chain (S-MHC) proteins. We found that the majority of fibers in the SA muscle stained for F-MHC antibody, and the percentage of muscle fibers staining for S-MHC antibody increased caudally. When newborn SA muscles were denervated and then reinnervated by the entire long thoracic (LT) nerve or only the C6 branch to the LT nerve, the reinnervated muscle had the normal proportion of muscle fibers expressing S-MHC protein. However, if the LT nerve was crushed and only C7 motoneurons allowed to reinnervate the SA muscle, a greater percentage of muscle fibers stained for S-MHC antibody than normal. We conclude that there is a correlation between muscle fiber type and innervation topography in the SA muscle of the rat.

Endocrine correlates of male polymorphism and alternative reproductive tactics in the Azorean rock-pool blenny, Parablennius sanguinolentus parvicornis.

In the Azorean rock-pool blenny male sexual polymorphism occurs. Larger and older males (M+ males) fully express male secondary sex characters (SSC), particularly an anal gland that produces a sex pheromone, whereas smaller and younger sexually active males do not express SSC (M- males). Two mating tactic types can be identified among M+ males: nest-holders that establish nests and court females and floaters that move around in the breeding area and try to achieve parasitic fertilizations and/or to take over nests. Two behavioral tactic types can also be identified within M- males: satellites that are associated with particular nests and actively participate in territorial defense (when females go inside the nest to spawn they try to enter to fertilize some of the eggs) and sneakers that do not help nest holders (when spawning occurs they also try to enter the nest to fertilize eggs). It was found that M+ males have significantly higher levels of 11-ketotestosterone (KT), but not testosterone (T), than M- males [M+ male androgen levels (mean +/- SE): total T = 11.6 +/- 3.0 ng ml(-1), total KT = 4.5 +/- 1.1 ng ml(-1); M- male androgen levels (mean +/- SE): total T = 9.6 +/- 1.0 ng ml(-1), total KT = 2.5 +/- 1.1 ng ml(-1)]. There were no differences in plasma T or KT among individuals using different mating tactics within the same male morph; that is, among M+ males, nest-holders did not differ in androgen levels from floaters [nest-holder androgen levels (mean +/- SE): total T = 12.3 +/- 4.4 ng ml(-1), total KT = 4.3 +/- 1.4 ng ml(-1); floater androgen levels (mean +/- SE): total T = 5.9 +/- 0.8 ng ml(-1), total KT = 3.4 +/- 0.3 ng ml(-1)], and among M- males, satellites did not differ in androgen levels from sneakers [satellite androgen levels (mean +/- SE): total T = 7.7 +/- 1.5 ng ml(-1), total KT = 1.3 +/- 0.3 ng ml(-1); sneaker androgen levels (mean +/- SE): total T = 8.3 +/- 1.6 ng ml(-1), total KT = 1.4 +/- 0.3 ng ml(-1)]. Thus, the observed differences appear to be correlated with the expression of different male morphotypes and not with the expression of different behavioral tactics within the morphotype. Androgen levels were not correlated with the behavior activity of nest-holders, except for a negative correlation between KT levels and parental behavior. Furthermore, nest-holder males that succeeded in having females spawn in their nests during the observation period had significantly lower KT levels than unsuccessful males. Since behavioral observations preceded blood sampling in time, it is suggested that these results indicate a negative relationship between KT and parental care, since successful males were parenting when blood samples were collected. Male SSC were better correlated with KT than with T and the use of total blood levels (i.e., free + conjugates) yielded higher correlation coefficients than when only the free fraction of each steroid was considered. Since conjugates are nonactive metabolites of the free androgen they should reflect active free steroids in a previous time. Thus, their incorporation into the hormonal measurements increases the time frame captured, and because steroids are released in a pulsatile way, this time-integrated measure can be more meaningful than the free steroids, which represent a snapshot of the hormone levels at a given point in time.