Activational vs. organizational effects of sex steroids and their role in the evolution of reproductive behavior: Looking to foot-flagging frogs and beyond

Highlights

• Sex steroids organize vertebrate reproductive behavior during development.

• This process evolves within and among species, creating new behavioral traits.

• We know little about the diversification of organizational mechanisms.

• Foot-flagging frogs are ideal to study such mechanisms, given their development.

• Organizational effects likely underlie waving displays in these animals.

Abstract

Sex steroids play an important role in regulation of the vertebrate reproductive phenotype. This is because sex steroids not only activate sexual behaviors that mediate copulation, courtship, and aggression, but they also help guide the development of neural and muscular systems that underlie these traits. Many biologists have therefore described the effects of sex steroid action on reproductive behavior as both “activational” and “organizational,” respectively. Here, we focus on these phenomena from an evolutionary standpoint, highlighting that we know relatively little about the way that organizational effects evolve in the natural world to support the adaptation and diversification of reproductive behavior. We first review the evidence that such effects do in fact evolve to mediate the evolution of sexual behavior. We then introduce an emerging animal model – the foot-flagging frog, Staurois parvus – that will be useful to study how sex hormones shape neuromotor development necessary for sexual displays. The foot flag is nothing more than a waving display that males use to compete for access to female mates, and thus the neural circuits that control its production are likely laid down when limb control systems arise during the developmental transition from tadpole to frog. We provide data that highlights how sex steroids might organize foot-flagging behavior through its putative underlying mechanisms. Overall, we anticipate that future studies of foot-flagging frogs will open a powerful window from which to see how sex steroids influence the neuromotor systems to help germinate circuits that drive signaling behavior. In this way, our aim is to bring attention to the important frontier of endocrinological regulation of evolutionary developmental biology (endo-evo-devo) and its relationship to behavior.

Introduction

Reproductive behavior in animals is as diverse as it is spectacular. This is especially true with respect to behavioral traits that help animals compete with rivals and court potential mates. But, how do new behavioral traits arise in the first place, and how do they evolve to such extreme ends? Answers to these questions are multifaceted, and they have been the focus of study of several fields of biology for decades (Ryan, 2021; Westneat et al., 2010). Most often, those who study the evolution of reproductive behavior do so in ecological and/or functional contexts. Iconic examples include studies that explore why male bowerbirds construct large bowers to attract mates (Borgia, 1995; Kusmierski et al., 1997; Uy and Borgia, 2000), or how complex head-bobbing displays evolve in Anolis lizards (Ord et al., 2001, Ord et al., 2002). Other fascinating work in this area centers on the evolution of alternative reproductive tactics (Bailey et al., 2010; Wolff and Cicirello, 1990) and the factors that mediate divergence in mating systems (Anholt et al., 2020; Bowyer et al., 2020). However, we should also move outside of the ecological/functional context when considering the evolution of such traits, and instead focus on them from a mechanistic or physiological point of view (Bass and Chagnaud, 2012; Fischer and O'Connell, 2017; Fuxjager and Schlinger, 2015; Hoke et al., 2019; Jourjine and Hoekstra, 2021; Schwark et al., 2022). This approach is equally essential to understanding the evolution of reproductive behavior because the neural and neuromuscular systems that underlie reproduction set the stage on which selection acts to influence how the reproductive phenotype (including its behavioral underpinnings) change through time.

Consider sex steroids, which are integral to the evolution of vertebrate reproductive behavior (as well as other traits outside the realm of reproduction). This class of hormone, which includes androgens, estrogens, and progestins, mediates much of the behavior associated with copulation, courtship, and sexual aggression (Adkins-Regan, 2005; Crews and Moore, 1986). Thus, if selection alters any of these traits, then it must also modify underlying sex steroid systems. One factor complicating this process is that steroid hormones themselves are highly conserved with respect to their molecular structure (Schuppe et al., 2020). In other words, a steroid hormone like the androgen testosterone (T) is identical in most species (however, in the case of T, fish are a notable exception because they have a slightly different bioactive androgen called 11-ketotestosterone, or 11-KT) (Adkins-Regan, 2005). This means that selection does not likely alter reproductive behavior by influencing steroid hormone structure, but instead influences behavior by altering other facets of the machinery that underlie steroid hormone signaling (Hau, 2007).

In recent years, biologists have begun to take a deep dive into the mechanisms by which sex steroid systems evolve to facilitate behavioral adaptation and diversification (Adkins-Regan, 2008; Cox, 2020; Fuxjager et al., 2018; Fuxjager and Schuppe, 2018; Hau, 2007; Ketterson et al., 2009; Lipshutz et al., 2019). This work largely focuses on how selection might change the way sex steroids are detected in a target tissue, and how the effects of sex steroid action are transduced. Researchers have predominantly used adult animals for this work, and thus have considered the so-called “activational” effects of steroid hormone action, which refer to the ability of steroids to “dial up” or “dial down” behavioral output. Although this approach can reveal core insights about how steroid systems evolve, it does not shed light on all the other ways that sex steroids can regulate behavior. Behavioral traits are also shaped by processes that occur early in life and that have long-lasting consequences. Here, we mean the “organizational” effects of sex steroids on reproductive traits, which in the case of behavior refers to the role that steroid action plays during development to help shape neural circuits that are then activated during adulthood to produce reproductive behavior. The relationship between these two processes has long driven research in the field of behavioral endocrinology, and continues to this day to do so (Adkins-Regan, 1983; Adkins-Regan, 2012; Arnold, 2009b; Arnold and Breedlove, 1985; Beatty, 1979; Goy and Phoenix, 1972; Phoenix et al., 1959). If modifications to sex steroid signaling machinery underlie the evolution of reproductive behavior, then we might expect these effects to occur at two levels—during and after development (See Fig. 1).

In the current paper, we explore the idea raised above more thoroughly. We begin by providing a basic primer of activational vs. organizational effects of sex steroids on animal sexual behavior. We explore the literature that suggests that sex steroid signaling machinery underlies behavioral adaptation, while highlighting why we know that many of these effects arise through developmental processes. We end with a deeper exploration of an emerging animal model—the Bornean rock frog (Staurois parvus)—that might shed some light on this issue. This species has evolved a novel gestural waving signal called the “foot flag,” which it uses to compete with rivals for access to mates (Hödl and Amézquita, 2001; Preininger et al., 2013b). When males are given exogenous T, they begin to foot flag more frequently and produce displays with a rounder shape (Anderson et al., 2021a; Mangiamele et al., 2016). Moreover, studies show that the foot flag's evolution is marked by a significant increase in androgen receptor (AR) expression in the thigh muscles that actuate waving movements (Anderson et al., 2021c; Eigerman and Mangiamele, 2022; Mangiamele et al., 2016; Smith et al., 2021). Frogs are widely used to study developmental processes, largely because they undergo a metamorphic change from tadpole to frog. In doing so, researchers can probe the mechanisms that make this transition happen. Here, we compare select behavioral, endocrine, and neural traits between juvenile and adult S. parvus in an attempt to disentangle the effects of muscular AR on display production during adulthood from behavioral mechanisms that are likely innate or that arose during a critical developmental moment in the frog's life. Overall, our aim is to begin to understand how selection can drive the evolution of the reproductive phenotype by altering not only how hormones mediate processes in the nervous system, but also how hormones help set up the nervous system in the first place.