Matthew S. Grober

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.

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.

Social interactions tune aggression and stress responsiveness in a territorial cichlid fish (Archocentrus nigrofasciatus)

We examined the relative influences of pre-fight housing condition, contest intensity, and contest outcome in modulating post-fight stress hormone concentrations in territorial male convict cichlids (Archocentrus nigrofasciatus). Individuals were housed either in isolation or in semi-natural communal tanks. Pairs of male cichlids that differed considerably in body mass were selected from the same housing regime. Pre-fight water-borne cortisol levels were obtained before allowing the dyad to interact until contest resolution, after which time post-fight cortisol levels were obtained from the winner and loser. There were no outcome-related differences in post-fight cortisol concentrations following escalated or non-escalated contests, a result that held true for both housing regimes. Pre-fight cortisol levels were significantly higher than post-fight cortisol levels, suggesting that initial confinement in a beaker for the water-borne hormone samples was a stressor, but that the animals acclimated quickly to confinement. Fights involving previously isolated participants were significantly more intense than those involving group-housed animals, which we explain as being a function of established relationships between social isolation, heightened acute cortisol responsiveness, and the expression of excessive aggressive behavior. Only group-housed losers demonstrated the ability to modulate aggression or hypothalamic-pituitary-interrenal (HPI) activity in a graded fashion to acute increases in cortisol or changes in contest intensity, respectively. We discuss a variety of factors that could disrupt the ability of isolates to appropriately modulate interactions between social behavior and the HPI axis, and we examine a number of functional hypotheses underlying the sensitivity of group-housed losers to changes in contest dynamics.

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.

A model for social control of sex change: interactions of behavior, neuropeptides, glucocorticoids, and sex steroids.

The optimal regulation of vertebrate sexual development and reproductive function involves integration of internal physiological signals, indicative of an individuals sexual status and capability for reproduction, with signals from the external environment. While these environmental cues are diverse, and oftentimes species-specific, the induction of sexual readiness is typically carried out through the same basic components of the hypothalamic-pituitary-gonadal axis conserved among vertebrates. Therefore, species exhibiting diverse patterns of reproduction can contribute to the understanding of the general mechanisms underlying the expression of adult sexual phenotypes. The bluehead wrasse, Thalassoma bifasciatum, is a tropical coral reef fish that displays social control of sex change, whereby dominant males inhibit sex change in other members of the social group using aggressive interactions. In many fish species and vertebrates in general, individuals that lose these interactions often experience increased serum glucocorticoids, which can have a subsequent impact on their physiology and behavior. We discuss glucocorticoid regulation of both neuropeptide gene transcription and the major steroid biosynthetic pathways as potential mechanisms involved in the regulation of sex change in the bluehead wrasse. We present a model describing behavioral regulation of sex change in the bluehead wrasse and then describe the potential mechanistic roles of glucocorticoids, gonadal steroids, and neuropeptides in generating the changes predicted by the model. Through the use of alternative model systems it is possible to observe novel interactions among the neuroendocrine axes that regulate major life history events, like reproduction. These insights may then shed light on similar functional mechanisms underlying behavioral regulation of reproduction in all vertebrates.

Forebrain AVT and courtship in a fish with male alternative reproductive tactics

In this paper, we present the results of cellular and molecular studies on the neuroendocrine correlates of male sexual polymorphism in a population of the blenniid fish Salaria pavo (Risso). Bigger and older males defend nests, whereas smaller and younger males mimic female nuptial coloration and behavior to gain access to nests and sneak fertilizations. In this population, sex-role reversal in courtship also occurs (i.e., females are the courting sex). Immunocytochemistry and in situ hybridization were used to examine the production of arginine vasotocin (AVT) peptide and messenger RNA, respectively. The expression of AVT mRNA on a per-cell basis was correlated with mating behavior, rather than with sex morphotype, which suggests that the greater level of AVT mRNA expression in females and sneakers is correlated with the production of courtship behavior. On the other hand, the number and size of AVT peptide-producing cells in both male types is higher or larger, respectively, than in females, which suggests that it is correlated with sex morphotype, rather than with mating behavior.

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.

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.

An integrative approach to sex change: social, behavioural and neurochemical changes in Lythrypnus dalli (Pisces)

Abstract. This study examined three aspects of protogynous sex change in Lythrypnus dalli (Gobiidae): (1) social influences on the rate of sex change, (2) the sequence of behavioural changes, and (3) neuroendocrine changes. Social groups consisted of either four females, or four females with a male who was subsequently removed. Sex change occurred most rapidly in male- removed groups when the sex changer was larger than other females. Sex changers in female only groups and sex changers not larger than other females in male-removed groups changed sex at similar rates. These differences may be explained by two factors that affect dominance: prior knowledge of the social group and greater size. Sex changers were dominant to other females prior to male removal, and larger sex changers increased displacement rates three-fold immediately after male removal. Sex changers in the other groups did not show this increase in displacements. This early establishment of dominance accounts for the overall difference in the rate of sex change. Prior to spawning, however, all sex changers increased displacements and performed male-typical displays. Arginine vasotocin-immunoreactive forebrain cells of sex changers were similar in size to field-collected males, and larger than field-collected females. Previously nesting males also changed sex in male-only groups, but at slow rates. These data are combined with those of existing studies to generate an integrative model of sex change in this goby.

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.