Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism
Introduction
Genomic rearrangements such as chromosomal inversions frequently provide the genetic variation necessary for the evolution of behavioral diversity associated with mating and reproduction (Chouteau et al., 2017; Gilburn and Day, 1999; Horton et al., 2014b; Küpper et al., 2016; Pearse et al., 2014). At the proximate level, these so-called “supergenes” may contribute to hormonal plasticity by capturing genes required for hormone production and sensitivity [e.g. (Horton et al., 2014a, Horton et al., 2020; Merritt et al., 2020)]. Over time, sequence evolution combined with selection on certain inversion haplotypes may further canalize hormonal profiles into a restricted range that becomes associated with specific behaviors and morphological traits.
Male mating success typically relies on secondary sexual characters that are paired with behaviors, such as territorial aggression and courtship displays. The hypothalamic–pituitary–gonadal (HPG) axis plays a pivotal role in the control of reproduction in vertebrates. At the apex of the HPG axis, gonadotropin-releasing hormone (GnRH) neurons fire synchronously and secrete GnRH to the pituitary in a pulsatile fashion. These pulses stimulate the rhythmic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary into the blood portal system (Fig. 1) [for details on birds see also (Li et al., 1994; Urbanski, 1984)]. The intermittent stimulation of gonadal tissue by gonadotropins then induces sex steroid synthesis and spermatogenesis in the male gonad.
Sex steroid hormones initiate and maintain social behaviors through genomic and non-genomic routes (Boonyaratanakornkit and Edwards, 2007). Among sex hormones, testosterone has been traditionally invoked to be largely responsible for differences in inter-male aggression (Goodson, 2005; Wingfield et al., 1987) although alternative more integrative mechanisms have been described recently (Goymann et al., 2019; Lipshutz et al., 2019; Wingfield et al., 2019). Testosterone can reach the brain via the bloodstream or by de-novo synthesis in neural tissue (Baulieu, 1997; do Rego and Vaudry, 2016; Tsutsui et al., 1999). Yet, the regulation of social behavior does not only include testosterone but also its androgenic and estrogenic metabolites. The conversion of testosterone into estradiol is a requirement for mediating aggressive and sexual behavior in birds (Ball and Balthazart, 2004; Fusani et al., 2001; Steimer and Hutchison, 1980; Watson and Adkins-Regan, 1989) and estradiol is a known potent activator for sexual behavior in birds (Cornil et al., 2006). Estradiol can also be synthesized from estrone that is made from aromatized androstenedione, a testosterone precursor. Of note, androstenedione is only a weak activator of the androgen receptor, compared to testosterone. Another sex steroid, progesterone, has the ability to mediate aggression in rodent models [e.g. (Erpino, 1975; Erpino and Chappelle, 1971; Schneider et al., 2003; Yang et al., 2013)], male tree lizards [Urosaurus ornatus (Weiss and Moore, 2004)], as well as in females of the sex-role reversed black coucals (Centropus grillii) (Goymann et al., 2008). In the rufuos hornero (Furnarius rufus) a South American ovenbird, the ratio between testosterone and progesterone has been shown to be a more useful predictor of outcomes of short-term agonistic encounters than testosterone alone (Adreani et al., 2018).
The evolution of diversity in mating repertoires is largely facilitated by testosterone regulation (Ball and Balthazart, 2004; Balthazart et al., 2003; Day et al., 2007; Fusani et al., 2014; Oliveira et al., 2008). Therefore, knowledge of the physiological limits of androgen responsiveness is key to understanding how behavioral diversity can evolve, especially in association with reproductive behaviors. Species that exhibit alternative reproductive tactics (ARTs) are ideal study systems for the proximate regulation of mating behavior because courtship, aggression and secondary sexual traits are in many cases dissociated from male gonadal function (Oliveira et al., 2008). GnRH challenges have proven particularly effective at unmasking when hormonal differences are largely regulated by the social context and interactions therein, and not by an actual physiological limitation to testosterone production (Apfelbeck and Goymann, 2011; Barron et al., 2015; Cain and Pryke, 2017; Goymann and Flores Dávila, 2017; Goymann and Wingfield, 2004; Jawor et al., 2006; Spinney et al., 2006). During a GnRH challenge, a standardized amount of GnRH is administered to animals to examine the ability of the pituitary to respond to GnRH and the ability of the gonads to produce and secrete testosterone. In some species with reversible ranks or morphs, the entire HPG axis of subordinate males is suppressed by the social context. For example in the African cichlid Astatotilapia burtoni, the presence and agonistic behavior of dominant males actively keeps other males in a subordinate status (Maruska and Fernald, 2011). Subordinate males experience substantial shrinkage of GnRH neurons, decreased circulating 11-ketotestosterone and reduced spermatogenesis, which leads to smaller testes when the HPG axis is under social suppression for weeks (Davis and Fernald, 1990; Francis et al., 1993). However, this can be rapidly reversed if the social environment changes (Burmeister et al., 2005; Maruska et al., 2013). In contrast, in species with genetically determined ARTs, sneaker phenotypes are usually fixed for a lifetime (Oliveira et al., 2008). Such sneaker males tend to have low circulating levels of testosterone but their gonads are of similar size or even larger than those of territorial males, probably because this maximizes their fertilization success with fewer mating opportunities (Fu et al., 2001; Gross, 1996; Taborsky, 1998). This is possible because spermatogenesis actually requires low levels of testosterone to be initiated and maintained (Kustan et al., 2012; Oliveira et al., 2001, Oliveira et al., 2008). Understanding how genetic changes are able to produce fixed alternative morphs with distinct hormonal profiles is key to building the bigger picture of how morph-specific behaviors with a genetic basis can evolve.
A prominent inversion polymorphism that captures several genes involved in steroid synthesis and metabolism is responsible for three genetically determined male mating morphs - Independents, Satellites, Faeders - in the ruff, Philomachus pugnax (Küpper et al., 2016; Lamichhaney et al., 2016). During the breeding season, each morph has a distinct appearance and behavior (Hogan-Warburg, 1966; van Rhijn, 1973). Independent males have dark colored plumage and are overtly aggressive, whereas Satellites and Faeders, the two other ARTs, seldom exhibit aggressive behavior. Satellite males have light colored plumage and form temporary coalitions with Independent males. Faeders, the smallest of the three morphs, lack ornamental plumage and do not perform male-typical courtship behaviors but rather adhere to a pure sneaking strategy and have testes that are up to 2.5 times larger than those of Independent males (Jukema and Piersma, 2006). Faeder males have been documented to mount females, as well as other males in the wild, and mate successfully in captivity (Jukema and Piersma, 2006; Küpper et al., 2016; Lank et al., 2013). These morphological and behavioral differences are linked to striking hormonal differences in testosterone among the morphs: Independent males have high levels of testosterone compared to Satellites and Faeders (Küpper et al., 2016). Conversely, Satellites and Faeders have high levels of androstenedione, a testosterone precursor, compared to Independent males. Androstenedione, on its own, may act as a weak activator of androgen receptors and additionally, may be converted to testosterone or estrogen in target tissues including the brain (Soma et al., 2003). Indeed, experimentally elevating androstenedione in Independents results in increased aggression but is ineffective at eliciting any aggressive behavior in Satellites (albeit it increases courtship behavior in Satellites, Morgan, 2009).
Remarkably, the determination of morph type is controlled by a 4.5 Mb autosomal inversion. Independents have two copies of the ancestral non-inverted haplotype, whereas Satellites and Faeders are both heterozygous for the inversion. Satellites and Faeders each have different inversion haplotypes: the Faeder inversion haplotype arose first and the Satellite haplotype is the result of subsequent recombination between the Faeder inversion and one or several ancestral Independent alleles (Küpper et al., 2016; Lamichhaney et al., 2016). The presence of several genes in the inversion region (i.e., HSD17B2, SDR42E1, CY5B5) with known involvement in sex steroid synthesis and metabolism suggests an inversion-based direct effect on hormone production and regulation. In view of the relatively high levels of circulating androstenedione in inversion morph males compared to Independent males, the HSD17B2 gene, which is located within the inversion region and encodes the enzyme that converts testosterone back to its precursor androstenedione, has been suggested as a candidate that could provide a direct explanation for this hormonal difference among morphs (Küpper et al., 2016; Lamichhaney et al., 2016). Yet, pilot data did not provide clear evidence of a key role for testicular expression differences in HSD17B2 among morphs (Loveland et al., in preparation). This suggests that perhaps a more fruitful avenue to understanding how the inversion disrupts sex steroid synthesis requires examining the entire HPG axis, and in particular its ability to mount maximal androgenic hormonal responses, rather than just gene expression differences between morphs in testicular tissue. Because the ability to produce testosterone may be a crucial trait directly controlled by genes within the inversion, identifying where within the HPG axis this disruption occurs is a necessary first step towards deciphering the underlying molecular mechanisms that generate morphological, neurobiological and behavioral differences across morphs.
In this study, we aimed to investigate the physiology and molecular mechanisms that maintain hormonal differences in adult male ruffs during the breeding season. First, we performed a GnRH challenge to characterize the physiological range of testosterone that each morph can produce. We tested whether inversion morphs were capable of mounting a typical testosterone surge following a GnRH injection. We measured testosterone, androstenedione and corticosterone to examine the response range of each hormone during the GnRH treatment. Second, in a separate set of untreated birds, we measured the expression of key genes in the pituitary and gonads to assess hormonal sensitivity and production within the HPG axis. We measured the expression of genes required for pituitary sensitivity to sex steroids, gonadotropin-releasing hormone sensitivity (GnRH receptors), gonadotropin production (FSH) and gonadal sensitivity to gonadotropins (LH and FSH receptors), as well as the expression of the gene encoding the steroidogenic acute regulatory protein (StAR) which is essential for making cholesterol available for the synthesis of steroid hormones. We hypothesized that morph differences in the expression of any of these genes would indicate which tissue types and processes (i.e. hormone production or sensitivity) along the HPG axis would be responsible for the low circulating testosterone levels observed in inversion morphs.
Section snippets
Birds and housing
We used a total of 69 adult ruff males from a captive breeding flock at Simon Fraser University, with a mean age ± standard error (SE) of 5.44 ± 0.62 yrs. This captive population was originally established from eggs collected near Oulu, Finland, in 1985, 1989 and 1990 (Lank et al., 2013). We conducted all experiments and sample collection during three breeding seasons (see specific dates in relevant sections below). Males were housed in an outdoor aviary in a same-sex pen with visual access to
GnRH challenge in Independent males
The GnRH challenge resulted in different circulating testosterone concentrations in saline and GnRH-injected Independent males (Table S2, Fig. 2A). We found a strong interaction between time and treatment (F(2,32) = 14.06, p < 0.001). Saline-injected birds had testosterone levels lower in post-injection samples relative to baseline, whereas, as predicted, GnRH-injected birds had higher levels of testosterone after 30-min post-injection relative to baseline (Dunnett's p = 0.006) (Fig. 2A). We
Discussion
Breeding ruffs show striking differences in behavior, appearance and androgen physiology across three morphs that only differ at the genomic level by a small autosomal inversion. Exactly how the inversion causes these hormonal and behavioral differences is unknown. In one set of birds, we tested the physiological range of androgenic production across the three morphs and in another set of untreated birds, we analyzed gene expression along the HPG axis to discern which tissue types and processes
Conclusions
We investigated how inversion morphs respond hormonally to exogenous GnRH in male ruffs. We found that the ability to synthesize testosterone is severely disrupted in the two inversion morphs, Faeders and Satellites. There was no evidence for widespread reduced functioning or throughput in early stages of the testosterone synthesis pathway, as inversion morphs show over-expression of STAR, which provides substrate for synthesis of sex hormones. Given the absence of clear differences across
Acknowledgements
We would like to thank Stephanie Roilo for bird care and technical assistance in sample collection, Aaron Walchuk, Soma Marton, Gabi Trainor for aviary maintenance, Soma Marton for technical assistance in the GnRH challenge experiment, Hubert Schwabl for discussions in the early planning stages of this project, Monika Trappschuh for hormone sample processing and Antje Bakker for assistance with the Bioanalyzer and two anonymous reviewers for constructive feedback on a previous version of the
Funding
This study was funded by the Max Planck Society and the National Research Council of Canada.
CRediT authorship contribution statement
JLL conceived the GnRH-related and gene expression elements of the project; JLL, CK and DL designed experiment conditions and sample collection; JLL, LG and DL performed the GnRH challenge experiment; WG supervised hormone measurements; MG provided lab space and reagents, JLL collected and processed all samples and analyzed the data. JLL wrote the initial draft with input from CK, all authors edited and approved the final manuscript.
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