Localization and divergent profiles of estrogen receptors and aromatase in the vocal and auditory networks of a fish with alternative mating tactics

Abstract

Estrogens play a salient role in the development and maintenance of both male and female nervous systems and behaviors. The plainfin midshipman (Porichthys notatus), a teleost fish, has two male reproductive morphs that follow alternative mating tactics and diverge in multiple somatic, hormonal and neural traits, including the central control of morph-specific vocal behaviors. After we identified duplicate estrogen receptors (ERβ1 and ERβ2) in midshipman, we developed antibodies to localize protein expression in the central vocal-acoustic networks and saccule, the auditory division of the inner ear. As in other teleost species, ERβ1 and ERβ2 were robustly expressed in the telencephalon and hypothalamus in vocal-acoustic and other brain regions shown previously to exhibit strong expression of ERα and aromatase (estrogen synthetase, CYP19) in midshipman. Like aromatase, ERβ1 label co-localized with glial fibrillary acidic protein (GFAP) in telencephalic radial glial cells. Quantitative PCR revealed similar patterns of transcript abundance across reproductive morphs for ERβ1, ERβ2, ERα and aromatase in the forebrain and saccule. In contrast, transcript abundance for ERs and aromatase varied significantly between morphs in and around the sexually polymorphic vocal motor nucleus (VMN). Together, the results suggest that VMN is the major estrogen target within the estrogen-sensitive hindbrain vocal network that directly determines the duration, frequency and amplitude of morph-specific vocalizations. Comparable regional differences in steroid receptor abundances likely regulate morph-specific behaviors in males and females of other species exhibiting alternative reproductive tactics.

Comparative Neurobiology of Sound Production in Fishes

“…These sex and morph differences were later confirmed by quantitative PCR (qPCR) of midshipman aromatase in isolated hindbrain tissues with the VMN removed ( Fig. 2.11) (Fergus and Bass 2013). These results suggest that differences in constitutive hindbrain aromatase activity could prevent testosterone from masculinizing the vocal pattern generator in females and type II males.…”

“…Sex-and male morphspecific mRNA expression profiles of aromatase (Aro) and estrogen receptor (ER) subtypes, quantified by real-time PCR, for vocal motor nucleus (VMN) dissected in toto and for surrounding hindbrain region without VMN. Taken from Fergus and Bass (2013) Aromatase-ir and mRNA expression were abundant throughout the vocal network, including the forebrain POA, midbrain PAG and hindbrain VMN (Forlano et al 2001). Dense aromatase-ir containing glial cells around the dorsolateral margin of VMN extended their processes throughout the nucleus in a radial-like pattern.…”

Melatonin action in a midbrain vocal-acoustic network

“…Each spike reflects the synchronous firing of vocal motor neurons whose activity is patterned by hindbrain premotor nuclei (Bass and Baker, 1990;Chagnaud et al, 2011;Chagnaud et al, 2012). Hence, fictive calls are a reliable proxy for assessing hormonal influences mediated by specific receptors on a discrete vocal network that directly determines natural call properties (Forlano et al, 2005;Forlano et al, 2010;Goodson and Bass, 2000a;Goodson and Bass, 2000b;Goodson et al, 2003;Fergus and Bass, 2013;Remage-Healey and Bass, 2004;Remage-Healey and Bass, 2007). Given the importance of day length on regulating reproductive physiology, melatonin's role as the main time-keeping hormone among vertebrates, and evidence of its interaction with the hypothalamo-pituitary-gonadal axis (Bhattacharya et al, 2007;Falcón et al, 2007;Falcón et al, 2010), the midshipman presents a tractable model for investigating potential melatonin action on the excitability of neural networks regulating courtship behaviors.…”

“…Subsequent studies show androgen and estrogen receptor subtypes in the PAG (Forlano et al, 2010;Fergus and Bass, 2013), and high concentrations of the estrogen-synthesizing enzyme aromatase in the medial PAG (see Forlano et al, 2005). Presumably, endogenous steroids could act upon midbrain-specific receptors to increase vocal network excitability.…”

FoxP2 Expression in a Highly Vocal Teleost Fish with Comparisons to Tetrapods

“…This could include the observed overlap between fish and mammals in FoxP2 expression in the extended amygdala/BNST and preoptic area, sites that are especially rich in steroid-and other hormone-signaling pathways [e.g., see Goodson and Bass, 2001]. Double label, in situ immunohistochemical experiments in midshipman of aromatase or steroid receptors [e.g., Forlano et al, 2001;Fergus and Bass, 2013] with FoxP2 could help to establish the identity of the FoxP2-expressing cells in these overlapping regions.…”