Research reportReproductive status regulates expression of sex steroid and GnRH receptors in the olfactory bulb
Introduction
Animals must integrate signals from their external and internal environments to make appropriate and context-dependent behavioral decisions. One key player in this process, the olfactory system, is important for vertebrate behavior, since many taxa use chemical communication in their courtship and dominance displays [1], [2], [3], [4]. In many fish species, for example, males determine female reproductive state through compounds released by gravid or pre-ovulatory individuals [5], [6], [7], male urine can be used as a form of sexual signaling to denote social rank [3], [8], [9], [10], and chemical cues are used in mother–young social bonds [11], [12]. How might the internal physiological state of an individual influence its perception of these chemical cues? Steroid hormones and neuropeptides are known to fine-tune sensory responsiveness to behaviorally relevant cues [4], [13], [14], [15], providing a potential link between internal state and external signals, but the mechanisms and site of action for such modulators in the olfactory system are not well understood.
Previous studies in fishes and other vertebrates show that steroids and the modulatory neuropeptide gonadotropin-releasing hormone (GnRH) can influence olfactory processing, and that these effects often vary between sexes or across reproductive seasons [13], [14], [16], [17], [18], [19], [20], [21]. For example, more olfactory receptor neurons responded to GnRH application during the breeding season compared to the non-breeding season in the mudpuppy Necturus maculosus [17], [22], olfactory responsiveness differed between sex hormone-treated male and female goldfish Carassius auratus [18], and steroid-related response differences were reported in several mammal species [19]. For any modulator to influence the olfactory system, however, cognate receptors must be present in the tissues implicated, so quantification of receptor levels can be used as a proxy for physiological action. One potential mechanism to explain changes in olfactory responsiveness is variations in steroid and GnRH receptor abundance within processing centers such as the olfactory bulb. However, little is known about how receptor levels change with reproductive state or social status in the olfactory system of any vertebrate. Here, we tested for changes in receptor mRNA levels of key modulators in the first-order olfactory processing center of the brain, the olfactory bulb, using the highly social African cichlid fish Astatotilapia burtoni to test the hypothesis that receptor levels vary with reproductive and social state.
Cichlid fishes use multiple sensory cues (e.g., visual, olfactory, auditory, mechanosensory) to coordinate their complex social behaviors, and show great diversity in reproductive and parental care strategies, which makes them excellent models for discovering how hormones influence sensory function. The African cichlid A. burtoni is endemic to shallow shore pools of Lake Tanganyika and lives in a lek-like social system where males exist in one of two phenotypes: (1) dominant territorial males (∼10–30% of population) that are brightly colored, aggressively defend a spawning territory, and actively court and spawn with females; and (2) subordinate non-territorial males that are drab-colored, frequently chased by dominant males, and do not court females [23]. Males can rapidly and reversibly switch between dominant and subordinate phenotypes depending on the composition of the social environment. Importantly, this social transformation in males causes a suite of behavioral and physiological changes along the reproductive axis [24]. Females do not have a similarly organized social system, but typically school with subordinate males and enter the territories of dominant males only to eat and spawn. After spawning, females rear the developing young in their mouths (mouth brooding) for ∼2 weeks before releasing them, and then physiologically recover (∼25–30 days) before spawning again. While visual cues are important for social behaviors in this species [23], [25], [26], olfactory cues may also provide crucial information on sex, reproductive condition, and dominance status. Previous studies in A. burtoni show that olfactory signals are used for perception of social information [25], [27], and that the olfactory system is responsive to putative pheromone compounds [28], [29], [30]. In the closely related tilapia Oreochromis mossambicus, chemical communication is also used by both males and females for reproductive and dominance behaviors [6], [31]. These studies provide support for the importance of olfactory-mediated behaviors in A. burtoni, but whether the olfactory system might be influenced by the animals’ internal physiological state remains unknown.
We do know from previous studies that GnRH receptors and sex steroid receptors are found within the olfactory bulb of several teleosts including A. burtoni [Munchrath and Hofmann, pers. comm. [32], [33], [34], [35], [36]], and thus the olfactory bulb could be an important substrate for neuropeptide and steroid-mediated olfaction. Here we asked whether social status and/or reproductive state influenced mRNA levels of GnRH receptors, sex steroid receptors, and aromatase in the central olfactory system of male and female A. burtoni, and whether these changes were correlated with circulating steroid levels. Our results show that mRNA levels of multiple sex steroid receptor subtypes, GnRH receptor subtypes, and aromatase in the olfactory bulb of a cichlid fish vary with sex, social status in males, and reproductive condition in females. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid action, and that changing receptor expression may provide a mechanism to influence context-dependent olfactory responsiveness.
Section snippets
Animals
Laboratory-bred adult male and female A. burtoni, derived from wild-caught stock in Lake Tanganyika, Africa, were maintained in aquaria under environmental conditions that mimic their natural equatorial habitat (28 °C; pH 8.0; 12 h light:12 h dark with full spectrum illumination; constant aeration), and fed cichlid pellets and flakes (AquaDine, Healdsburg, CA, USA) each morning. Aquaria contained gravel-covered bottoms with terra cotta pots cut in half to serve as spawning territories. All
GSI and circulating steroid levels
GSI and circulating steroid levels for these animals were previously reported as part of a separate study [38] and those values are summarized here in Table 1. GSI differed among all three reproductive phases in females (KW, H = 28.10, p < 0.001, Dunn's test, p < 0.05), and was 2-fold greater in dominant males compared to subordinate males (Student's t-test, t = −6.50, p < 0.001). Serum E2, T, and 11-KT levels differed among all three female reproductive stages (ANOVA or KW, all p < 0.001). In males,
Discussion
Our results show that mRNA levels of multiple sex steroid and GnRH receptor subtypes in the olfactory bulb of a cichlid fish vary with sex, social status in males, and reproductive state in females. To our knowledge, this is the first quantification of mRNA levels of GnRH receptors, sex steroid receptors, and aromatase specifically in the olfactory bulb in any non-mammalian vertebrate. While previous studies in many vertebrates showed that both GnRH and steroids can influence olfactory
Acknowledgements
We thank Brian Grone, Julie Desjardins, Russ Carpenter, and two anonymous reviewers for their insightful comments that improved the manuscript. This research was funded by National Institutes of Health (NIH) NRSA F32NS061431 to KPM and NIH NS 034950 to RDF.
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