A mechanism for rapid neurosteroidal regulation of parenting behaviour

Abstract

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11β-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11β-HSD activity.

Keywords: androgen; cortisol; fish; fitness; glucocorticoid; reproduction.

Figure 1.

Simplified pathway of steroidogenesis in fish. Testosterone is converted to KT via the sequential action of 11β-hydroxylase, which converts KT to 11β-hydroxytestosterone (11β-OHT), and 11β-HSD, which converts 11β-OHT to KT and cortisol to cortisone. Inhibition of 11β-HSD with CBX (solid grey ‘X’) can elevate cortisol and reduce KT (solid grey arrows) levels.

Figure 2.

Effect of CBX and 11β-OHT (an endogenous substrate) on 11β-HSD activity in adult male L. dalli (a) brain and (b) testes. Tissue supernatants were incubated in vitro at 25°C with 1 mM NAD⁺ as a co-substrate for 60 min. Controls were incubated with buffer + vehicle only. KT peak area ratio was calculated by dividing area of the KT peak by that of corticosterone (B), the internal LCMS standard (N = 3 males per group); **p < 0.01, ***p < 0.001.

Figure 3.

Effect of IP implants of CBX, an 11β-HSD inhibitor, on KT exuded in water (systemic levels) 1 h, 1 day and 4 days post-treatment of adult male L. dalli. Vehicle (unfilled bars): n = 5 and CBX (filled bars): n = 4; *p < 0.05, **p < 0.01.

Figure 4.

Effect of ICV injection of parenting male L. dalli on (a) latency to enter nest, (b) time spent in nest between 10 and 20 min and (c) number of parenting bouts between 10 and 20 min. Vehicle: n = 7; CBX: n = 9; CBX + KT: n = 9; cortisol: n = 9; *p < 0.05, **p < 0.01.

Figure 5.

Effect of ICV injection of parenting male L. dalli on agonistic interactions and parenting behaviour; (a) male approach rate, (b) male displacement rate and (c) male parenting. Vehicle: n = 7; CBX: n = 9; CBX + KT: n = 9; cortisol: n = 9; *p < 0.05, **p < 0.01.