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From Kabelik, D., Crews, D. Hormones, Brain, and Behavior in Reptiles. In: Pfaff, D.W andJoëls, M. (editors-in-chief), Hormones, Brain, and Behavior 3rd edition, Vol 2.

Oxford: Academic Press; 2017. pp. 171–213.Copyright © 2017 Elsevier Inc. All rights reserved.

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2.10 Hormones, Brain, and Behavior in ReptilesDavid Kabelik, Rhodes College, Memphis, TN, USADavid Crews, University of Texas at Austin, Austin, TX, USA

� 2017 Elsevier Inc. All rights reserved.This chapter is a revision of the previous edition chapter by D. Crews, N. Sanderson, B.G. Dias, volume 2, pp. 771–818, � 2009, Elsevier Inc.

2.10.1 Introduction 1722.10.2 Diversity in Sex Determination, Sexual Differentiation, and HormoneLBehavior Relationships 1732.10.2.1 Temperature-Dependent Sex Determination 1732.10.2.1.1 Mechanisms of Temperature-Dependent Sex Determination 1732.10.2.1.2 Organizing Influence of Incubation Temperature in the Leopard Gecko 1752.10.2.2 Functional Associations in Hormones, Gamete Production, and Mating Behavior 1792.10.2.3 Alternative Mating Strategies and Tactics 1812.10.2.3.1 The Red-Sided Garter Snake 1822.10.2.3.2 The Tree Lizard 1822.10.2.3.3 The Side-Blotched Lizard 1842.10.2.3.4 Fence Lizards 1852.10.2.3.5 Other 1862.10.2.4 Parthenogenetic Lizards 1862.10.2.5 Sex Steroids and Behavior: Other Reptiles 1872.10.2.5.1 Turtles 1872.10.2.5.2 Crocodilians, Tuatara, and Amphisbaenians 1882.10.3 Neuroanatomical Substrates of Sexual and Aggressive Behaviors in Reptiles 1892.10.3.1 Hormone Receptor Expression 1892.10.3.2 Intracranial Hormone Implants 1912.10.3.3 Morphological Parameters 1912.10.3.3.1 Sexual Dimorphisms 1912.10.3.3.2 Seasonal Variation 1932.10.3.4 Metabolic Indicators of Neural Activity 1942.10.3.5 Focal Lesions 1942.10.4 Neurochemical Bases of Sexual and Aggressive Behavior in Reptiles 1952.10.4.1 Nonapeptides 1952.10.4.2 Monoamines 1972.10.4.3 Nitric Oxide 2012.10.4.4 Steroidogenic Enzymes, Cofactors, and Receptors 2022.10.5 Regulation of Sex Steroid Hormone Receptors 2032.10.6 Conclusions and Future Directions 205References 206

Abbreviations2-DG 2-deoxyglucose5-HIAA 5-hydroxyindoleacetic acid5-HT 5-hydroxytryptamine (serotonin)5-HTP 5-hydroxytryptophanAH anterior hypothalamusAmbIX/VIImv motor nucleus of the facial nerveAmbX motor nucleus of the facial nerveAR androgen receptorAVPv anteroventral periventricular nucleusAVT arginine vasotocinBNST bed nucleus of the stria terminalisCO cytochrome oxidaseCREB cAMP response element-binding proteinDA dopamine

DHT dihydrotestosteroneDVR dorsoventricular ridgeE2 estradiolEB estradiol benzoateEPI epinephrineER estrogen receptorFTP female-producing temperatureGFP green fluorescent proteinGSD genotypic sex determinationHSP hormone-sensitive periodL-NAME L-NG nitro arginine methyl esterLS lateral septumMAN magnocellular nucleus of the anterior nidopalliumMPT male-producing temperature

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00027-4 171

Hormones, Brain and Behavior, Third Edition, 2017, 171–213


http://dx.doi.org/10.1016/B978-0-12-803592-4.00027-4

NE norepinephrineNOS nitric oxide synthaseNPY neuropeptide YNS nucleus sphericusP progesteronePOA preoptic areaPOAH preoptic area-anterior hypothalamusPostOv postovulatoryPR progesterone receptor

PreOv preovulatoryPvPOA periventricular preoptic areaqPCR quantitative real-time polymerase chain reactionsiRNA short interfering RNAT testosteroneTH-ir tyrosine hydroxylase immunoreactiveTSD temperature-dependent sex determinationTSP temperature-sensitive periodVMH ventromedial hypothalamus

2.10.1 Introduction

The living group of animals we refer to as reptiles includes essen-tially all the amniote vertebrates except the mammals and thebirds. It is represented by four living orders: the Squamata(lizards, snakes, and amphisbaenids), the Testudines (turtlesand tortoises), the Crocodilia (crocodiles, alligators, etc.), andthe Sphenodontia (tuataras) (Figure 1). In order to make thistaxon monophyletic, it would be necessary to include theapproximately 10 000 species of birds (Padian and Chiappe,1998), but for the purposes of this chapter, discussion will belimited to the familiar ‘reptile’ forms. These animals offer twolarge advantages from an experimental standpoint. The firstadvantage is the enormous diversity of patterns of sex determina-tion and sexual differentiation observed across reptilian species,enabling some particularly illuminating ‘natural experiments,’some of which are discussed below. Among the diverse patternsrepresented are temperature-dependent sex determination(TSD), mixed genotypic- and temperature-dependent sex-deter-mining mechanisms, reproduction by obligate parthenogenesis(all-female species), and distinct alternate male phenotypeswithin a species. The second advantage to the study of reptilesis the presence of many phylogenetically primitive characters.Additionally, because mammals and birds arose from turtle-like and crocodilian-like ancestors, respectively, the study of

reptiles allows for phylogenetic comparisons to these highlyinvestigated taxa.

This chapter will discuss how these advantages have beenexploited to yield new insights into the nature of the neuroendo-crine control of behavior in vertebrates in general. We begin witha short review of diversity in sex determination, sexual differen-tiation, and hormone�behavior relationships observed inreptiles. This is followed by discussion of some of the neuralmechanisms involved and the methods that have been success-fully applied to elucidating them. In each of these sections wewill focus on both within-sex and between-sex differences inthe neurochemistry of brain areas subserving sexual and aggres-sive behaviors in reptiles. Finally, we summarize some researchdirections that are likely to prove especially promising.

Modern reptiles exhibit phenotypes that are in many wayssimilar to what the ancestral amniote vertebrate must havebeen like. These characteristics include ectothermy, oviparity,and the lack of a well-developed cerebral cortex. The presenceof neural structures homologous to those found in mammalsand birds, coupled with the lack of complex cortical develop-ment, makes modern reptiles useful for examining basicbehavioral control mechanisms in vertebrates. Such compara-tive research has revealed that the areas in the limbic forebraininvolved in the regulation of sexual and aggressive behaviorsare ancient and highly conserved among vertebrates. Thisresearch has also demonstrated that differences in the distri-bution of sex steroid-concentrating neurons are rare, butdifferences in the regulation of steroid hormone receptorsare common. Further, species differences in plasma levels ofsex hormones are paralleled by differences in behavioralsensitivity to these hormones as well as by differences in theregulation of genes coding for steroid hormone receptors.Other features modern reptiles likely share with the firstamniote vertebrates are mechanisms of sex determinationthat are variable in terms of the important cues (genotypevs environment) and in the type of genotypic sex determina-tion (GSD) that is displayed within groups (male vs femaleheterogamety).

This combination of diversity and conserved charactersprovides a variety of ‘natural experiments’ with which to askquestions about basic principles in sex determination andsexual differentiation (Crews and Gans, 1992). Table 1 pres-ents examples of some of the questions that reptiles are partic-ularly useful in addressing. For instance, the diversity inreptilian characteristics allows for a variety of comparisonsbetween species exhibiting differing patterns of development

Figure 1 Phylogeny of amniote vertebrates. Mammals are believed tohave arisen from turtle-like therapsid reptiles approximately 350 myaand modern birds from crocodilian-like archosaurs approximately250 mya.

172 Hormones, Brain, and Behavior in Reptiles



and, for many processes there are sufficient numbers of species,including outgroups, to generate adequate sample sizes forcomparisons. For example, viviparity has evolved from ovipar-ity perhaps 100 or more times independently in reptiles (Black-burn, 1999; Guillette, 1991). Meanwhile, the conserved natureof many reptilian characteristics can lead to renewed study inmammals to further our understanding of the neuroendocrinecontrol of mammalian sexual behavior. For example, thediscovery that progesterone (P) is important in the control ofmale-like pseudocopulatory behavior in parthenogenetic whip-tail lizards led to studies with rats and transgenic mice that,together, revealed the importance of P and its receptor in thecontrol of sexual behavior in males (discussed below in detail).The lack of a well-developed cortex in reptiles is experimentallyadvantageous in many ways. Greenberg et al. (1979) refer toreptiles as ‘walking limbic systems’ and note the value of inter-pretations not being subject to the complications presented bya well-developed cortex. Modern reptiles are likely also primi-tive in the neural circuitry that mediates mounting and intro-mission behavior and sexual receptivity. The study of sexualbehaviors has been important for our understanding of behav-ioral neuroendocrinology generally and an understanding ofthe ancestral state of neuroendocrine mechanisms controllingthese behaviors should help us better understand how theyhave evolved and function in birds and mammals.

2.10.2 Diversity in Sex Determination, SexualDifferentiation, and Hormone�BehaviorRelationships

Reptiles show an extraordinary diversity in the patterns of sexdetermination and sexual differentiation. In addition to GSD,many reptiles possess primitive (e.g., TSD) or specialized(e.g., obligate parthenogenesis; alternative mating tactics) traitsthat have added new dimensions to our understanding ofreproductive neuroendocrine mechanisms underlying repro-duction in vertebrates in general. One important benefit ofthe diversity seen in reptiles relates to studying variation insexual behavior. Sexual behaviors and aggressive behaviorsoften show discontinuous variation between the sexes in birdsand mammals, although there is typically considerable

individual variation within the sexes (Crews, 1998). Incontrast, many reptiles show more continuous variation inthese behaviors. Examples include species with TSD thatshow substantial behavioral variation within sexes across incu-bation temperatures, GSD species that are responsive to incu-bation temperature to the extent that individuals with a malegenotype may become functional females, all-female speciesin which individuals alternate between the display of female-and male-like pseudosexual behavior during the course of theovarian cycle, and species that exhibit distinct alternate malephenotypes. Viewing sexuality as a continuous variable shouldfacilitate thinking about how modern states of sexuality inbirds and mammals arose. Our goal in this section is to high-light diversity in reptilian sex determination and differentiationpatterns and the research opportunities this diversity presents.

2.10.2.1 Temperature-Dependent Sex Determination

Many reptiles exhibit GSD, with some species exhibiting maleheterogamety (XX:XY) likemammals,while others exhibit femaleheterogamety (ZZ:ZW) like birds. However, in all crocodilians(alligators, crocodile, caiman, etc.),most turtles (allmarine turtlesand tortoises and many freshwater turtles), and some lizards(various Geckkonid and Agamid species), gonadal sex is estab-lished by the temperature experienced by the incubating egg(Viets et al., 1994; Janzen and Krenz, 2004; Figure 2). Oncethought to be restricted to oviparous species lacking sex chromo-somes, recent reports have extended TSD to oviparous and vivip-arous species with both XX:XY and ZW:ZZ sex-determiningsystems (Robert and Thompson, 2001; Shine et al., 2002;Warnerand Shine, 2008; Quinn et al., 2007). For example, in the CentralBeardedDragon (Pogona vitticeps) females are ZWwhilemales areZZ (female heterogamety) (Quinn et al., 2007). Incubation athigh temperatures can override genotype to create genetic malesthatare functional females. Fecundity in these ‘sex-reversed’malesis higher than in ZW females incubated at a female-producingtemperature (Holleley et al., 2015).

2.10.2.1.1 Mechanisms of Temperature-Dependent SexDeterminationThree basic patterns of TSD have been documented (Figure 2;Crews, 1994; Valenzuela and Lance, 2004). In the first pattern,

Table 1 Questions and possible comparisons using reptiles as model systems

Questions Comparison Example

How do behaviors and their underlying brainmechanisms change in speciation?

Ancestor vs descendant species Cnemidophorus inornatus vs C. uniparens

What is the effect of ploidy on neuroendocrinemechanisms?

Diploid vs triploid species Cnemidophorus inornatus vs C. uniparens

Are there functional differences in genotypicsex-determining systems?

Male heterogamety vs femaleheterogamety

C. inornatus vs Thamnophis sirtalisparietalis

Are there functional differences in genotypicsex-determining systems (GSD) vs temperature-dependent sex-determining (TSD) systems?

GSD lizard vs TSD lizardGSD turtle vs TSD turtle

C. inornatus vs Eublepharis maculariusTrionyx spniferus vs Trachemys scripta

Causal mechanisms and functional outcomesof different phenotypes of the same sex?

Alternative reproductive patterns Urosaurus ornatus

Uta stansburiana

What has the environment altered the functionalrelationship between gamete growth, sex steroidhormone secretion, and mating behavior?

Associated vs dissociated reproduction Anolis carolinensis vs Thamnophis spp.

Hormones, Brain, and Behavior in Reptiles 173



relatively high incubation temperatures produce males, whereasrelatively low temperatures produce females (Figure 2(a)); thesecond pattern is simply the reverse (Figure 2(b)). The thirdpattern is more complex in which intermediate temperaturesproduce males and high and low temperatures produce females(Figure 2(c)). Sensitivity to temperature is restricted to the mid-trimester of development, or the temperature-sensitive period(TSP) (Crews, 1996, 2003). TSD is believed to be ancestral tothe GSD characteristic of birds and mammals (Crews, 1994;Janzen and Krenz, 2004).

Sex steroid hormones are implicated in the process of TSD,and estrogens in particular appear essential in ovary determina-tion (Crews et al., 1994, 1996a; Lance, 1997; Pieau and Dorizzi,2004; Sarre et al., 2004; Wibbels et al., 1998). For example, estro-gens applied exogenously to red-eared slider turtle (Trachemysscripta) eggs incubating at a male-producing temperature over-ride the temperature effect and female hatchlings result (Crewset al., 1991; Wibbels and Crews, 1992). Exogenously appliedinhibitors of aromatase override a female-producing incubationtemperature, and male hatchlings result (Crews and Bergeron,1994; Wibbels and Crews, 1994). Similarly, testis determinationcan bemanipulated by exogenously applied dihydrotestosterone(DHT), a nonaromatizable androgen, and its derivatives as wellas by reductase inhibitors (Crews et al., 1996a). Importantly,temperature and hormones act synergistically, further indicatingthat steroid hormones are undoubtedly a part of TSD in bothmales and females (Crews, 1996; Crews et al., 1994, 2006;Wibbels et al., 1991).

The process of TSD is believed to have evolved by the retro-grade addition of regulatory elements upstream of establisheddevelopmental programs (Wilkins, 1995; Graves, 1995). Ifthis model is accurate, the various sex-determining systemscan be thought of as one evolutionarily conserved corenetwork for both testis and ovary determination regulated

by various taxon-specific upstream factors (McLaren, 1998).Support for this hypothesis is found in the conservation ofsex-determining genes among taxa (Crews and Bull, 2009);the same genes important in mammalian gonad determina-tion are found in other species and show strong similaritiesin their temporal expression patterns during gonadogenesis.

The developmental decision of ovary versus testis does notflow through a single gene but is instead determined bya ‘parliamentary’ system involving networks or cassettes ofgenes that have simultaneous inputs to several componentsof the downstream cascade. Systems with different balancesof the inherited and environmental influences could all operatethis way (as they could also operate through a single master),merely by varying the inputs to the networks. These cassettesfunction much akin to a ratchet (Crews and Bull, 2009;Figure 3). In this instance the cassette consists of a functionallyinterrelated suite of genes that, during the process of develop-ment, are progressive in nature, leading to more advancedtissue types that become increasingly distinct one from theother in form and function (i.e., testis vs ovary). It is significantthat this process is reversible initially; that is, the process can beredirected during the early, but not later, developmental stages.In the slider turtle this moment of lability corresponds to theoverlapping TSP and hormone-sensitive period.

The genes involved in both the core sex-determining cassetteand the hormone-sensitive cassette have had their expressionpatterns analyzed throughout gonadogenesis by whole-mountand sectioned in situ hybridization as well as quantitative real-time polymerase chain reaction (qPCR) in several species. Thered-eared slider turtle is the best-studied model (Ramsey andCrews, 2009; Shoemaker and Crews, 2009; Matsumoto andCrews, 2012), but excellent information is available for the snap-ping turtle (Rhen and Schroeder, 2010).Under female-producingtemperature (FPT) or ovary determination, Rspo1 and FoxL2 aremodulated differently than under male-producing temperature(MPT), while under MPT or testis determination, Dmrt1, Sox9,Mis, and Sf1 are modulated differently than under FPT (Ramseyand Crews, 2007a,b, 2009; Ramsey et al., 2007; Shoemakeret al., 2007a,b, 2010; Fleming and Crews, 2001). Wnt4 appearsto be regulated at both FPT and MPT. As described below,embryos shifted from FPT to MPT exhibit a rapid activation ofDmrt1, Mis, and Sf1 gene expression, while Sox9 upregulation isdelayed. Conversely, shifting embryos fromMPT to FPT upregu-latesRspo1 soon after the shift, followed by FoxL2 andWnt4upre-gulation during the end of the TSP. Cassette #2 genes (aromatase,AR, ERa, and ERb are present throughout gonadal development,but the intensity of expression differs according to incubationtemperature; e.g., aromatase is expressed at higher levels at FPTthan at MPT. As gonadal development continues, ERa and ARlevels are equivalent between MPT and FPT midway throughthe TSP, but both exhibit a spike in expression at FPT toward tothe end of TSP (Ramsey and Crews, 2007b).

As mentioned, administration of exogenous estrogen over-rides male-producing temperature effect, leading to ovariandevelopment in slider turtles. However, the gonads ofestrogen-treated embryos exhibit a slightly different pattern ofgene expressions in aromatase, ERa and ERb from the gonadsincubated at FPT even though the outcomes of bipotentialgonads are uniform. At FPT, both aromatase and ERa are upregu-lated while ERb is downregulated; in contrast, administration of

Figure 2 Response of hatchling sex ratio to incubation temperature invarious egg-laying reptiles. These graphs represent only the approxi-mate pattern of the response and are not drawn according to any singlespecies. The three patterns recognized presently are (a) only femalesproduced from low incubation temperatures, males at high tempera-tures, (b) only males produced from low incubation temperatures,females at high temperatures, and (c) only females produced at thetemperature extremes, with male production at the intermediate incuba-tion temperatures. Genotypic sex determination also occurs in reptileswith the result that the hatchling sex ratio is fixed at 1:1 despite incuba-tion conditions.




E2 causes both ERa and ERb to be downregulated in themedullary compartment, possibly indicating a negative feed-back effect of estrogen on ERa expression while aromataseexpression patterns remain the same (Ramsey and Crews,2007a,b, 2009).

The ability of estrogens and their mimics to override theMPT signal to direct ovarian development in slider embryossuggest the possible interaction between estrogen and coresex-determining gene expressions. Several studies indicatethat estrogen has a suppressive effect on the expressions oftestis-determining genes (Dmrt1 and Sox9) and an inducibleeffect on the expression of ovary-determining genes (Rspo1).Estradiol treatment in developing embryos significantlysuppresses Dmrt1 mRNA expression in the slider (Ramseyand Crews, 2007b; Murdock and Wibbels, 2006).

Dmrt1 is a key activational node of the sex-determiningcassette and, along with Sox9, is a master regulator of the testic-ular pathway much as FoxL2 as a regulator of the ovarianpathway (Ramsey and Crews, 2009; Shoemaker and Crews,2009; Matsumoto and Crews, 2012). These functions havebeen demonstrated in studies in which their expression is over-expressed or knocked down in in vitro gonadal explants. Forexample, studies with the slider turtle have utilized Dmrt1-green fluorescent protein (GFP) overexpression vector at FPTat which Dmrt1 expression was usually suppressed and shortinterfering RNA (siRNA) at MPT at which Dmrt1 expressionusually is activated. In such studies it is essential that the effectsof gene manipulation are validated by qPCR to confirm mRNAlevels, and GFP fluorescence to confirm protein levels.

2.10.2.1.2 Organizing Influence of Incubation Temperature inthe Leopard Gecko2.10.2.1.2.1 Effects on the General PhenotypeAnimals with environmental sex determination, such as lizardswith TSD, are particularly suitable for developmental studiesdesigned to distinguish between genetic and hormonal influ-ences on adult sexual behavior. The leopard gecko (Eublepharismacularius), in particular, has proven to be an excellent model

because the investigator has precise control of the critical envi-ronmental variable (in this case, incubation temperature) thatdetermines the sexual phenotype of the gonad, and hence itsproducts. Since these animals exhibit the third pattern of sexdetermination (discussed above), the sex ratio varies withtemperature, but individuals of both sexes are produced atmost incubation temperatures. By incubating eggs at thesevarious temperatures and then following individuals as theyage, we have found that incubation temperature accounts formuch of the phenotypic variation seen among adults bothbetween (sexual dimorphisms) and within (individual differ-ences) the sexes (Crews et al., 1998; Sakata and Crews, 2004a).

In the leopard gecko, incubation of eggs at 26 �C producesonly female hatchlings, whereas incubation at 30 �C producesa female-biased sex ratio, and incubation at 32.5 �Cproduces a male-biased sex ratio; incubation of 34–35 �Cagain produces virtually all females (Sakata and Crews,2004a; Figure 4). Hence, females from eggs incubated at26 �C are referred to as low-temperature females, whereasthose females from eggs incubated at 34 �C are referred to ashigh-temperature females; the two intermediate incubationtemperatures are referred to as female-biased (30 �C) andmale-biased (32.5 �C) temperatures. Adult leopard geckos aresexually dimorphic, with males having open secretory poresanterior to the cloaca. In low-temperature females these poresare closed, whereas in females from a male-biased temperaturethey are open (Gutzke and Crews, 1988). Head size is alsosexually dimorphic, with males having wider heads thanfemales, yet within females, those from a male-biased temper-ature have wider heads than do those from a low temperature(Gutzke and Crews, 1988). Similarly, although males are thelarger sex, incubation temperature has a marked effect ongrowth within a sex. Females from a male-biased temperaturegrow faster and larger than do females from a female-biasedtemperature and become as large as males from a female-biased temperature (Tousignant and Crews, 1995).

In leopard geckos, as in other TSD reptiles, administrationof exogenous estrogen to the incubation egg early in

Switch/trigger

Cassettes offunctionallyrelated genes

Sexualdifferentiationgenes

Puregenetic

Mixed environmentaland genetic

Pureenvironmental

Chromosomal

(e.g., temperature)

(ZZ:ZW or XX:XY)

(X:autosome ratio)

Figure 3 The switches/triggers once regarded as mutually exclusive (e.g., species with temperature-dependent sex determination lacking sex chro-mosomes; sex-determining genes), are now known to be present simultaneously in some species (e.g., sex-determining mechanisms in somespecies as having a genetically predisposed response to temperature) (left portion of figure). This in turn engages gene ‘cassettes’ of functionallyrelated genes that interact (middle portion of figure, with each corner representing a gene in a network). The nature of these interactions changesthrough development and a cassette can engage other cassettes of integrated gene assemblies. This parliament of interacting gene networks in turnhave simultaneous inputs to several components of the downstream cascade represented by sex-typical morphological, physiological, and ultimately,behavioral, traits (right side of figure). Modified from Crews, D., Bull, J.J., 2009. Mode and tempo in environmental sex determination in vertebrates.Semin. Cell Dev. Biol. 20, 251–255.




development will overcome a male-determining temperatureeffect and results in females; the opposite will occur if an aro-matase inhibitor is administered to an egg incubating ata female-producing temperature, which results in maleoffspring. Using this method it is possible to separate the actionof temperature on brain organization from that arising fromthe type of gonad that is formed. Such studies reveal thatsome traits are influenced by temperature, others by gonadalsecretions, and still others by amixture of the two. For example,female leopard geckos from estrogen-treated eggs incubated atthe male-biased temperature do not differ in growth rates fromunmanipulated females from the same temperature, indicatingthat it is incubation temperature, not gonadal hormones, thatregulates body growth.

At hatching, circulating concentrations of sex hormones arealready different between males and females, and this sexdifference increases throughout life until, as adults, concentra-tions of T in males are approximately 100 times higher than inadult females (Gutzke and Crews, 1988; Tousignant and Crews,1995; Rhen et al., 2005). However, the endocrine physiology ofthe adult varies in part due to the temperature experiencedduring incubation (Coomber et al., 1997; Tousignant et al.,1995; Figure 5). For example, plasma estrogen levels are signif-icantly higher in males from a female-biased temperaturecompared to males from a male-biased temperature. Amongfemales, circulating estrogen levels are significantly higher,and androgen levels significantly lower, in low-temperaturefemales compared to females from a male-biased temperature.

Incubation temperature also has a major influence on thenature and frequency of the behavior displayed by the adultleopard gecko. Females usually respond aggressively only ifattacked, whereas males will posture and then attack othermales but rarely females (Gutzke and Crews, 1988; Floreset al., 1994). However, males from a female-biased temperature

are less aggressive than males from the higher, male-biasedtemperature and, although not as aggressive as males fromthat same incubation temperature, females from a male-biased temperature are significantly more aggressive towardmales than are females from a low or female-biased tempera-ture. These same females show themale-typical pattern of offen-sive aggression and, as is the case for body growth, females fromestrogen-treated eggs incubated at the male-biased temperatureare as aggressive as their unmanipulated counterparts.

Incubation temperature also influences the ability of exoge-nous testosterone (T) to induce aggression. Following ovariec-tomy and T treatment, low-temperature females do not exhibitincreased levels of aggression toward male stimulus animals,whereas females from male-biased temperatures return to thehigh levels exhibited while gonadally intact (Flores and Crews,1995). Similarly, males from the male-biased embryonictemperature scent mark more than do males from the female-biased embryonic temperature when treated with DHT or T;treatment with E2 decreases submissive behavior in malesfrom amale-biased embryonic temperature compared to malesfrom a female-biased embryonic temperature (Rhen andCrews, 1999; Huang and Crews, 2012; Figure 6). Lastly, geckosfrom different incubation temperatures exhibit significantdifferences in dopaminergic activity (Dias et al., 2007). Suchdata suggest that incubation temperature influences how theindividual responds to steroid hormones in adulthood.

Courtship is a male-typical behavior. In a sexual encounter,the male will slowly approach the female, touching thesubstrate or licking the air with his tongue. Males also havea characteristic tail vibration, creating a buzzing sound, whenthey detect a female. Intact females have never been observedto exhibit this tail-vibration behavior, regardless of their incu-bation temperature. However, if ovariectomized females fromlow and male-biased temperatures are treated with T, they

Figure 4 Pattern of temperature-dependent sex determination in the leopard gecko, Eublepharis macularius. The right panel portrays the effect ofincubation temperature on sex ratio: extreme temperatures produce females, whereas intermediate temperatures produce different sex ratios. Sincethe effects of incubation temperature and gonadal sex co-vary, any difference between individuals could be due to the incubation temperature of theegg, the gonadal sex of the individual, or both factors combined. To assess the contribution of each, they must be dissociated. By studying same-sexanimals that differ only in the incubation temperature experienced reveals the effects of temperature (left panel), whereas comparing males andfemales from the same incubation temperature reveals the effects of gonadal sex.




will begin to tail-vibrate toward female, but not male, stimulusanimals; males appear to regard such females as male becausethey are attacked (Flores and Crews, 1995).

Attractiveness is a female-typical trait and is measured bythe intensity of a sexually active male’s courtship behaviortoward the female. Females from a male-biased temperatureare less attractive than are females from lower incubationtemperatures (Flores et al., 1994). Interestingly, attractivenessin high-temperature females is greater than that of femalesfrom male-biased temperatures and not different from that oflow-temperature females. Long-term castrated males are attrac-tive and initially courted by intact males, but on olfactory inspec-tion they are attacked. This suggests that both sexes can produceboth a female-typical attractiveness pheromone and a male-typical recognition pheromone, as does the red-sided gartersnake (Mason et al., 1989). As is the case with females, incuba-tion temperature influences sensitivity to exogenous hormonesin males. Estrogen treatment will induce receptive behavior incastratedmales if they were incubated at a female-biased temper-ature, but not if they were incubated at a male-biased tempera-ture. Mate preference is also influenced by incubationtemperature, with males from a male-biased temperature prefer-ring to associate with females from a lower incubation tempera-ture while males from a female-biased incubation temperatureprefer females from a higher incubation temperature.

2.10.2.1.2.2 Effects on the BrainAs might be predicted, these behavioral differences among andbetween male and female leopard geckos from different incu-bation temperatures are reflected by differences in the neural

substrates regulating these behaviors, including the size andmetabolic activity of different limbic areas. One exciting devel-opment in our ability to assess sex differences in the neuralsubstrates of sexual behavior has been the introduction ofmetabolic mapping techniques using cytochrome oxidase(CO) histochemistry (Sakata et al., 2005). CO catalyzes therate-limiting step in oxidative respiration in brain tissue andlevels of this enzyme provide a useful indicator of the totalmetabolic capacity (Wong-Riley, 1989).

As with brain nucleus volume, a variety of factors influencemetabolic capacity in the brains of leopard geckos. These includeincubation temperature, age, and sexual experience. Incubationtemperature affects CO activity in both females and males,although the effect varies depending on the brain nucleus beingconsidered. Females from amale-biased incubation temperaturehave increased metabolic capacity of the anterior hypothalamus(AH), external amygdala, dorsolateral hypothalamus, dorsoven-tricular ridge (DVR), nucleus sphericus (NS), lateral septum (LS),and striatum but do not increase capacity in the posterior hypo-thalamus or periventricular preoptic area (PvPOA) (Figure 7).Of particular interest is the finding that young females froma male-biased incubation show greater CO levels in the medialpreoptic area (POA) than young females from a female-biasedincubation temperature. Females from amale-biased incubationtemperature are more aggressive and less sexually attractive thanfemales from a female-biased incubation temperature. CO levelsin male leopard geckos are also influenced by incubationtemperature, with males from a female-biased incubationtemperature having greater levels of CO in the POA and ventro-medial hypothalamus (VMH) than males from a male-biased

Figure 5 Circulating levels of steroid hormones vary between males and female leopard geckos (Eublepharis macularius) as well as between individualsfrom different incubation temperatures. (a). Ratio of the plasma levels of total androgens (A) and estrogens (E) in adult female (dashed line) and male (solidline) leopard geckos) from different incubation temperatures. (b). Circulating concentrations of corticosterone (individuals indicated by circles). Data fromCoomber, P., Crews, D., Gonzalez-Lima, F., 1997. Independent effects of incubation temperature and gonadal sex on the volume and metabolic capacity ofbrain nuclei in the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination. J. Comp. Neurol. 380, 409–421; Gutzke,W.H., Crews, D., 1988. Embryonic temperature determines adult sexuality in a reptile. Nature 332, 832–834; Crews, D., unpublished.




incubation temperature (Sakata and Crews, 2004b). Such find-ings are reminiscent of the finding that inmice, males positionedbetween two females in utero are more sexually active than theirmale siblings positioned between twomales (cf, Clark andGalef,1995).

Age and sexual experience are also important factors deter-mining metabolic activity in limbic nuclei in both male andfemale geckos. For example, age is associated with a decreasein the size of the POA and VMH in males, but not in females.CO activity increases in the POA, NS, and external amygdala(reptilian counterparts to the mammalian amygdala) withage in males, but the precise effects of age on CO levels indifferent brain nuclei in female leopard geckos vary with incu-bation temperature, indicating a complex interaction of thesefactors (Coomber et al., 1997).

Sexual experience also influences the metabolic capacity oflimbicnuclei in leopardgeckos, again incomplexways (Coomberet al., 1997; Sakata and Crews, 2003, 2004a,b; Sakata et al., 2000,2002). Several nuclei, including the VMH and AH, have higher

metabolic capacities in sexually experienced males compared tosexually inexperienced males. In contrast, there is no differencewithexperience in thePOAor severalother limbicnuclei inmales.On the other hand, sexually experienced female leopard geckosshow a higher CO abundance in the POA compared to sexuallyinexperienced females regardless of incubation temperaturehistory, while the results were mixed for other nuclei.

Sakata et al. (2000) assessed functional connectivity amonglimbic nuclei in the leopard gecko by analyzing covariancepatterns in metabolic capacity, as revealed by quantitative COhistochemistry (Figure 8). As indicated above, incubationtemperature during embryonic development influences anindividual’s aggressive and sexual behaviors in adulthood.For example, an increase in incubation temperature results inan increase in adult aggressivity in both males and females.Correlated with this are increased amounts of CO in the AHand both the septum and POA. Similarly, female-typical sexualbehaviors decline with increasing incubation temperature, andthe correlations between the VMH and both the DVR andseptum were significant only in females. Correlations amongpreoptic, hypothalamic, and amygdalar areas tend to be distrib-uted across both sexes, suggesting that there may exist sharedpathways underlying the expression of male-typical andfemale-typical behaviors (Crews et al., 2006; Figure 9).

Figure 6 Incubation temperature influences sensitivity to exogenoushormones in adult male leopard geckos (Eublepharis macularius). Illus-trated are the effects of embryonic incubation temperature and adulthormone treatment on mounting (upper panel) behavior and scentmarking (lower panel) of castrated male leopard geckos. Individualsreceived a Silastic implant containing cholesterol, 17b-estradiol (E2),dihydrotestosterone (DHT) or testosterone (T). Data from Rhen, T.,Crews, D., 1999. Embryonic temperature and gonadal sex organizemale-typical sexual and aggressive behavior in a lizard withtemperature-dependent sex determination. Endocrinology 140,4501–4508.

Figure 7 Relationship between sex ratio, aggressive behavior, andcytochrome oxidase activity in the amygdala in female leopard geckos.Sex in the leopard gecko is determined by the incubation temperatureof the egg; the sex ratio produced at the temperatures indicated is re-flected in the bar graph. At the low (26�C) and high (34�C) incubationtemperature females are 100 or 95% of the offspring; a female-biased(30�C) and male-biased (32.5�C) sex ratio is produced at the interme-diate temperatures. The proportion of females responding aggressivelytoward a courting male is indicated by squares. Cytochrome oxidaseactivity in the nucleus sphericus and external amygdala of females fromthese same incubation temperatures are shown by inverted trianglesand circles, respectively. In reptiles, the nucleus sphericus and externalamygdala are homologous to the medial and basolateral amygdala ofmammals, respectively; as in mammals, both areas are involved in thecontrol of aggression. Thus, embryonic experience with temperatureaffects the level of aggressive behavior and brain metabolism in theamygdala of adult females.




Thus, a variety of factors including gonadal sex, age, sexualexperience, and incubation temperature influence the volumeand metabolic capacity of brain nuclei and the connectivityamong these nuclei in leopard geckos. The dominant influence,however, is incubation temperature. This work provided thefirst unequivocal demonstration that factors other thangonadal sex and gonadal hormones can influence the sexualdifferentiation of the brain in vertebrates.

2.10.2.2 Functional Associations in Hormones, GameteProduction, and Mating Behavior

Species that evolved under different constraints presumablyexhibit different patterns of reproduction and therefore are likely

to have fundamentally different neuroendocrine mechanismscontrolling their reproduction and associated behaviors (Crews,1984, 1987). The three elemental components of the reproduc-tive process in vertebrates are gametes, steroid hormones, andbehavior. In species in which the pattern of gonadal activity isassociated temporally with mating, as occurs in many mammalsand birds, these elements are functionally associated. This hasled to the long-standing assumption that increasing levels ofgonadal steroid hormones in the circulation activates matingbehavior. That is, that there is a fundamental functional associ-ation among these three basic components. However, studiesindicate that the dependence of mating behavior on sexhormones depends upon the reproductive pattern exhibitedthat, in turn, depends upon various ecological, phylogenetic,

Figure 8 Incubation temperature and adult experience shapes the functional connections between limbic areas in the leopard gecko. Presented arediagrams of the significant correlations among the anterior hypothalamus (AH), nucleus sphericus (NS), preoptic area (POA), septum (SEP), andventromedial nucleus of the hypothalamus (VMH). All correlations are positive. Top six figures: note that the correlation between the AH and SEP issignificant only in relative aggressive individuals of both sexes. Bottom two figures: note how sexual experience as an adult can modify the connec-tions imposed by incubation temperature.




developmental, and physiological constraints on the organism.Beginning with studies of reptiles (Crews, 1984; Crews et al.,1984) and more recently with other vertebrates (Crews, 1987),it has become clear that there is no intrinsic linkage betweenthe production of gametes, the secretion of gonadal steroidhormones, and the expression of sexual behavior. A number ofstudies now have shown that sexual behavior need not dependupon increased levels of sex steroid hormones and, further,that males and females of a particular species may regulatesimilar reproductive (behavioral) events by using different prox-imate cues and mechanisms. Indeed, of the six relationshipspossible among these three elements, only one can be regardedas fundamental, namely that gametes cannot be produced inde-pendent of steroid hormone secretion (Crews, 1984, 1987). Aninteresting question is whether the other associations arederived, and evolved independently in the different genera,and therefore are analogous, or were present in the commonancestor of each Class (e.g., mammals) and therefore are

homologous. This will only be determined through comparativeanalysis.

The display of reproductive behavior in reptiles and othervertebrate groups shows one of three basic temporal relation-ships to gamete production (Figure 10). The most commonrelationship is termed an associated reproductive pattern.Animals displaying this pattern exhibit sexual behavior whentheir gonads are actively producing gametes and steroidhormone levels are elevated. Reptilian examples of this patternare the green anole lizard (Anolis carolinensis), the sea turtles dis-cussed in the next part of this section, and many of the otherspecies discussed in this chapter.

The display of mating behavior may also be temporallyuncoupled from gamete production. This is termed a dissociatedreproductive pattern. The most thoroughly reptilian example ofthis pattern remains the red-sided garter snake, Thamnophis sir-talis parietalis (Crews, 1983). This species is discussed at lengthbelow.

Figure 9 Between sex and within sex differences in metabolic capacity in interconnected limbic nuclei of adult leopard geckos. Left Panel depictsthe limbic landscapes of males and females both from a 32.5 �C incubation temperature; the bottom graph indicates the difference between theneural landscapes of the sexes. Positive peaks (those above plane) indicate nuclei that change in relation to baseline brain metabolic rate in males,whereas negative peaks indicate nuclei in which change was more positive in females. Right Panel depicts the limbic landscapes of males, the topgraph of males from a 32.5 �C incubation temperature and the middle graph of males from 30 �C incubation temperature (middle panel); the bottomgraph indicates the difference between the landscapes, revealing the effect of incubation temperature within a sex on metabolic activity in the adultbrain. Illustrated is the least squared means of cytochrome oxidase (CO) activity. Positive peaks (those above plane) indicate nuclei that change morein males from a 32.5 �C incubation temperature, whereas negative peaks (those below plane) indicate nuclei that change more in males from a 30 �Cincubation temperature. AH (anterior hypothalamus), VMH (ventromedial hypothalamus), AME (external amygdala), SEP (septum), PP (periventricularnucleus of the preoptic area), POA (preoptic area), NS (nucleus sphericus). Orientation of nuclei in each landscape in the two panels is different toshow best the patterns of change.




The third possible temporal relationship between gameto-genesis and reproductive behavior is a constant reproductivepattern. This pattern is characterized by maintenance of repro-ductive readiness (mature sperm and ovarian follicles), butwith actual reproduction and the display of associated behav-iors limited to typically short and unpredictable periodswhen environmental conditions are such that reproductioncan be successful. While this pattern has not been documentedin any reptile, it does characterize wild populations of zebrafinches in unpredictable desert environments (Sossinka,1980; Allen and Hume, 1997) and remains a possibility forreptiles facing similar challenges.

2.10.2.3 Alternative Mating Strategies and Tactics

Many species of reptiles show alternate male phenotypes thatexhibit discontinuous variation in male morphology, physi-ology, and behavior (Moore, 1991; Rhen and Crews, 2002).Alternative male phenotypes are also found in other vertebrategroups, including fishes, amphibians, birds, and in mammals,as well as in many invertebrates. Often, one male phenotype inthese systems is similar to females and the other shows exagger-ated male characters. As with the leopard gecko with TSD, suchsystems have the advantage of allowing comparisons ofdiffering behavioral phenotypes without the confound ofa difference in gonadal sex (Moore, 1991; Rhen and Crews,2002). For this reason, it has been suggested that alternatereproductive phenotypes present a valuable opportunity toexplore the physiological and neural mechanisms underlyingindividual variation in behavior and morphology. Historically,research on alternative reproductive phenotypes in reptiles hasfocused primarily on behavioral and ecological correlates,

though new research is helping us understand the neuroendo-crine bases of these phenotypes.

Males and females also represent alternate reproductivephenotypes, but sex comparisons face an important confound-ing factor: the groups being compared differ in both behaviorand the type of gonad they possess. Alternate reproductivephenotypes within a sex avoid this complication since thegroups being compared do not differ in type of gonad andare therefore valuable models for exploring the bases of ubiq-uitous individual variation within the sexes. In order for thisapproach to be a useful one conceptually and operationally,the physiological mechanisms operating to generate differencesin behavior and morphology between alternate within-sexphenotypes should be similar to those documented to producebetween-sex differences.

The Relative Plasticity Hypothesis of Moore (1991) proposesthat fixed differences between alternate phenotypes are due toorganizational actions of steroid hormones while more plasticdifferences are due to activational influences of these hormones(Figure 11). Moore et al. (1998) further refined this hypothesisto account for cases where permanent phenotypic effects mightrequire actions of the relevant hormones only during criticaldevelopmental windows. An expansion of this model by Rhenand Crews (2002) incorporates other dimensions that includeall known alternative reproductive tactics.

Next, we review three well-characterized models of alternatereproductive phenotype variation from a physiological perspec-tive: the red-sided garter snake, the tree lizard (Urosaurus ornatus),and the side-blotched lizard (Uta stansburiana). We also considera genus of iguanid lizards, Sceloporus, in which variation in malephenotypes is common both within and across closely relatedspecies, as well as some examples from other species.

Figure 10 Vertebrates display a variety of reproductive patterns. Here gonadal activity is defined as the development of eggs and sperm and/orincreased sex steroid hormone secretion. Individuals exhibiting the associated reproductive pattern (solid line) live in temperate regions whereseasonal cycles are regular and prolonged; in such species, the gonads are fully developed at the time of mating and circulating levels of sexhormones are maximal. Individuals exhibiting the dissociated reproductive pattern (dashed line) live in extreme environments in which seasonalchanges are regular, but the length of time available for breeding is limited; in such species, the gonads are small and sex steroid hormone levels arelow at the time of mating. Individuals exhibiting a constant reproductive pattern (hatched line) live in harsh environments where breeding conditionsare completely unpredictable; in such species, the gonads are maintained at nearly maximal development so that when breeding conditions do arise,breeding can occur immediately. Just as the reproductive cycles have adapted to the environment, so too have the neuroendocrine mechanisms sub-serving breeding behavior. The temporal uncoupling of sexual behavior and gonadal recrudescence in vertebrates exhibiting these different reproduc-tive patterns is reflected in the dynamics of their hormone-brain-behavior relationship. NB. The dimension of reproductive pattern is depicted asmutually exclusive extremes only for the sake of argument; intermediate forms are known to exist.




2.10.2.3.1 The Red-Sided Garter SnakeThe first physiological studies exploring alternate male pheno-types focused on the red-sided garter snake. As described above,this species exhibits a reproductive pattern in which peak levelsof gonadal hormones are temporally dissociated from thedisplay of reproductive behavior (Crews, 1991; Figure 12).Males emerge from winter hibernacula before females andvigorously court and attempt copulations in multimale ‘matingballs’ as females emerge (Crews, 1983). Two pheromonesunderlie male courtship behavior (Mason et al., 1989). One isan estrogen-dependent and T-inhibited attractivity pheromoneproduced by females that elicits vigorous courtship from males(Parker and Mason, 2012, 2014). A second pheromone,produced by males, identifies them as male and hence not theobject of courtship.

A small proportion of males (termed ‘she-males’) producethe attractivity pheromone, which characterizes females,appearing to confuse other males in mating aggregations intoexpending effort attempting tomate with the she-males (Masonand Crews, 1985). It is thought that, in addition, the she-malesderive benefits from being surrounded by the mating ball dueto attainment of heat generated from the friction of movementby males, and by the reduction of predation risks by crows due

to being at the center of a mating ball. Subsequent studiesrevealed that as males emerge from winter conditions, theyinitially may produce the female pheromone (Shine et al.,2001; LeMaster et al., 2008). This, however, proved to be a tran-sient phase. In collecting and holding all males for several days,it has become clear that the initially attractive males revert backto their normal unattractive state, while the she-males remainattractive for days, weeks, and even 1 year later (RT Mason,22 January 2016, unpublished communication).

The comparison of males with she-males presents theopportunity for much more examination of the neuroendo-crine regulation of sexual behaviors. She-males have highercirculating concentrations of T (Mason and Crews, 1985) andgreater abundance of aromatase in the skin (Krohmer, 1989),which presumably converts the endogenous T to estrogen,thereby stimulating production of the female attractivenesspheromone. The difference in circulating T between malesand she-males may also translate to differences in hormonalregulation of neural function, an idea supported by the findingthat preoptic area-anterior hypothalamus (POAH) morphom-etry differs between males, females, and she-males, with she-males possessing larger, and males smaller, POAH volumesthan females (Krohmer et al., 2011). Neural aromatase activityalso shifts from predominance in the olfactory region duringspring courtship to the septal-POAH in the fall, suggestingthat the aromatization of androgens into estrogens is alsoinvolved in the regulation of pheromone detection and repro-duction in this species (Krohmer et al., 2010).

Glucocorticoids have recently also been shown to exerteffects on red-sided garter snake behavior, causing a shift inpreference from courtship to feeding, and thus likely playinga role in the control of seasonal differences in behavior (Lut-terschmidt and Maine, 2014). The underlying mechanismlikely involves neuropeptide Y (NPY) (Morris and Crews,1990).

2.10.2.3.2 The Tree LizardThe tree lizard is the most thoroughly studied lizard speciesthat exhibits alternative mating strategies. In this species, themales possess colored dewlaps that are extended during bothaggressive and sexual interactions (Thompson and Moore,1991a; reviewed in Moore et al., 1998). The color of thisdewlap varies among males and shows at least nine geographicvariants with one to five variants occurring in any given loca-tion (Thompson andMoore, 1991b). Experiments where malesare raised in a common laboratory environment indicate thatthe basis of this dewlap color variation is either genetic ormaternal in origin since the orange and orange-blue pheno-types develop in approximately the same proportions as areobserved in the wild source populations (Thompson et al.,1993; Hews et al., 1997). Most attention has focused on oneArizona location in which two male morphs exist, one withorange-blue dewlaps (‘orange-blue males’) who are site-attached and hold territories encompassing the territories ofthree to four females, versus males with orange dewlaps(‘orange males’) who are more nomadic under poor habitatconditions (i.e., in drought years) and sedentary with smallhome ranges in good habitat conditions (Figure 13). Orange-blue males are often more aggressive both in the laboratoryand in nature (Thompson and Moore, 1991a, 1992), though

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Figure 11 Two hypotheses accounting for alternative reproductivetactics within a sex in vertebrates. Panel A: Schematic representation ofthe Relative Plasticity Hypothesis (RHP) of Moore (1991) for thehormonal basis of intrasexual variation in reproductive behavior. PanelB: Schematic representation of the Orthogonal Hypothesis of Rhen andCrews (1999) for the hormonal basis of intrasexual variation in repro-ductive behavior. Here the RHP is a subset of the possible hormonalmechanisms underlying variation in reproductive behavior, but theOrthogonal Hypothesis also includes species that do not conform to theRHP.




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Figure 12 The major physiological and behavioral events in the annual reproductive cycle of the red-sided garter snake in Canada. Animals spendmost of the year underground. In the spring, they emerge and mate before dispersing to summer feeding grounds. In the female, mating initiatesgonadal growth as well as changes in the hormone profile of the female. Young are born in late summer. Since all metabolic processes slow downduring the cold months, androgen levels in the male will be elevated in the spring if he entered hibernation with elevated levels (dotted lines);however, androgen levels usually are basal on emergence (solid line). Sperm are produced during the summer after mating and are stored in the vasdeferens (heavy squiggle line next to testis) over winter.

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Figure 13 Moore and coworkers model for organizational and activational influences on male phenotype development in tree lizards. Both testos-terone and progesterone acting during early development affect adult dewlap coloration. Plastic switches between satellite and nomad tactics in adultmales of the less aggressive orange morph are hypothesized to be due to an interplay of T and corticosterone. Redrawn from Moore, M.C., Hews,D.K., Knapp, R., 1998. Hormonal control and evolution of alternative male phenotypes: generalizations of models for sexual differentiation. Am. Zool.38, 133–151.




not in all studies. Weiss and Moore (2004) and Kabelik et al.(2006) found no aggression differences between morphs; theseparticular studies were conducted during a prolonged drought,when population densities of all morphs were well below theirusual levels, suggesting complex and not fully understood regu-lation of these behaviors across environments.

The effects of gonadal steroid and glucocorticoid hormonesin male tree lizards support the predictions of Moore’s relativeplasticity hypothesis. Adult T and corticosterone levels do notdiffer between orange-blue and orange males and neithercastration nor androgen manipulations alter dewlap colorexpression in adult males (Kabelik et al., 2006; Moore, 1988;Moore and Marler, 1987; Moore et al., 1998). In contrast,castration of neonatal male tree lizards increases the propor-tion developing as orange males while T implants given onhatching or 30 days thereafter increase the proportion devel-oping as orange-blue males (Hews et al., 1994). By 60 daysposthatch, T implants are ineffective in altering dewlap color,indicating a defined early critical period for the developmentof this trait as has been shown for behavioral organization bysteroid hormones in mammals. Patterns of plasma androgensin developing male tree lizards do suggest a possible bimo-dality in male androgen levels during the period in whichdewlap color develops (Moore et al., 1998), but much morestriking is a clear bimodality in P levels on the day of hatch.This possible role for P in determining morph type has experi-mental support: single injections of P on the day of hatchsignificantly increased the proportion of males developing asorange-blue males (Moore et al., 1998). This organizationalrole for P may point to an unrecognized developmental rolefor this steroid in other vertebrate systems. It is also reminiscentof the important role played by P in stimulating male-typicalsexual behavior andmale-like pseudosexual behaviors in whip-tail lizards discussed below. More recently, P has been shownto promote an aggressive phenotype in both forms of castratedand implanted morphs (Weiss and Moore, 2004), and thiseffect is likely independent of androgenic pathways as the Pgroup failed to show the same effects of increased limbicmorphology as did an experimental group treated with T(Kabelik et al., 2008c).

Corticosteroids play important roles in short-term behavioralresponses in male tree lizards, supporting the relative plasticityhypothesis and adding to a growing body of information indi-cating these steroids are important mediators of behavioral plas-ticity in this and other species. Corticosterone levels vary in bothorange and orange-blue males depending on habitat conditions,being higher in dry years (Moore et al., 1998). In the aggressiveorange-blue males, corticosterone levels are temporarily higherin losers of long-term laboratory dominance interactions butshow the opposite pattern in winners of short-duration encoun-ters (Knapp and Moore, 1995). In the field, the less aggressiveorange males show both less intense aggressive behavior andgreater corticosterone elevations in response to an aggressiveencounter than do orange-blue males (Knapp and Moore,1996). Males of the two morphs also differ in their responseto exogenous corticosterone. Both morphs show decreases incirculating T, but this decrease is greater in the subordinateorange males. Knapp and Moore hypothesize that the greatersensitivity of T levels to elevations in corticosterone in subordi-nate orange males accounts for the fact that these males switch

between roving and sedentary satellite patterns of space usedepending on habitat conditions, while changes in space useare not seen in the more aggressive orange-blue males (Knappand Moore, 1997).

The corticosterone response of male tree lizards to predators,however, does not differ between morphs. Thaker et al. (2009a)demonstrated that while both morph types show similarincreases in corticosterone to apredator (Sonoran collared lizard,Crotaphytus nebrius), themales of the orangemorphdisplaymoreprolonged subsequent antipredator behaviors (i.e., increasedhiding duration and flight initiation distance). In another study,Thaker et al. (2009b) demonstrated the causality of corticoste-rone in increasing antipredator behaviors in both morph types,with corticosterone treatment increasing hiding durations anddisplay frequency while decreasing latency to first display(Figure 14). These studies further suggest that even when circu-lating corticosterone concentrations across morphs are equiva-lent, the orange morph is more sensitive to this hormone,suggesting a possible difference in neural glucocorticoid receptordensities in the relevant brain regions. Alternately, this greatersensitivity to glucocorticoids in orange males may be mediatedwithin the periphery, as the orangemale morph possesses lowerconcentrations of corticosterone-binding globulins in theplasma than does the orange-bluemorph (Jennings et al., 2000).

Finally, female tree lizards are also polymorphic andevidence is now emerging for differences in mate choice prefer-ences between female morphs (Lattanzio et al., 2014).Although female tree lizards show very low aggression levels,these findings pave the road for further research into the neuro-endocrine regulation of sexual behaviors across female treelizard morphs.

2.10.2.3.3 The Side-Blotched LizardThe tree lizard work finds support from studies in the side-blotched lizard. This species has three male morphs that aredistinct in both morphological characteristics and behavior,and these differences are genetically determined (Sinervo andLively, 1996; Sinervo et al., 2001). Unlike tree lizards, there issome plasticity in male morph type in side-blotched lizards inthat the female-mimic yellow-throated males can becomemate-guarding blue-throated males, but not the ultradominantorange-throated morph. These morph differences seem at leastpartly to be the result of differential regulation of T by gonado-tropins in a morph-specific manner (Mills et al., 2008). Theorange-throated morph has higher plasma levels of T, loweryear-to-year survivorship, greater endurance, higher activitygenerally, and occupies larger home ranges that overlap the areasused by more females than either the yellow- or blue-throatedmales (Sinervo et al., 2000). A series of studies have shownthat corticosterone decreases aggression by adult males interraria (Denardo and Licht, 1993) and both home range sizeand activity levels in the field (Denardo and Sinervo, 1994a),even when corticosterone is given in combination with T. Thiseffect of corticosterone on male home range appears to dependon interactions with neighboring males (Denardo and Sinervo,1994b). Corticosterone-implanted males decreased home rangesize if some neighboring males were saline-implanted, but not ifall neighboring males received corticosterone implants. Testos-terone implants can increase home range size in male side-blotched lizards if not given with corticosterone (Denardo and




Sinervo, 1994b). Experimentally elevating T levels in yellow- andblue-throated males to those found in orange-throated malesalso increases both endurance and access to females in nature(Sinervo et al., 2000).

More recent research on side-blotched lizards has expandedinvestigation into the realm of the brain, demonstrating thatthe male morphs differ in relative hippocampal (homolog)volume in relation to territory/home-range size and that ratesof neurogenesis within the hippocampus is related to habitatcomplexity in the territorial morphs (LaDage et al., 2009, 2013).

2.10.2.3.4 Fence LizardsAlthough not as well studied from the standpoint of endocrinephysiology, other groups of reptiles present opportunities forexploring the mechanisms underlying behavioral variationwithin and across species. One particularly promising groupis the speciose lizard genus Sceloporus, or the fence lizards. Thereis a large body of behavioral and ecological information forSceloporus species and both phylogenetic relationships in thegenus as a whole (Wiens, 1993) and the evolution of dimor-phic coloration and behavior within and between specieshave received attention (Wiens et al., 1999; Wiens, 2000).Finally, there is a growing body of information on the endo-crine bases of the development of sexual phenotypes andhormonal consequences of social interactions in this genus.

As with alternate male types in the tree lizard, various Scelo-porus species show geographic variation in male coloration(Rand, 1990; Wiens et al., 1999). There are also effects of steroidhormones on coloration in Sceloporus. Both orange facial colorand a blue ventral coloration are influenced by T implants inadult red-lipped western fence lizards (Sceloporus undulatus eryth-rocheilus), with effects on ventral coloration being greater inmales than females (Rand, 1992). In contrast, no effects wereseen on the final size or intensity of blue throat or ventral patchesin striped plateau lizards (Sceloporus virgatus) when T implantswere given to hatchling males and females (Abell, 1998). Arecent comparison of androgen receptor (AR) density inS. undulatus and S. virgatus also revealed that a male-biased sexdifference is present in the POA and VMH in the former specieswhile no sex difference exists in the latter (Hews et al., 2012).This latter finding suggests that receptor distribution differencesacross Sceloporus species and sexes not only underlie differencesin body coloration but also in social behavior.

Androgens affect aggressivebehavior inbothmale and femaleSceloporus. Moore (1986) found elevated T associated with terri-torial behaviorduring thenonbreeding season inmalemountainspiny lizards, Sceloporus jarrovi. This territorial aggression isaffected by both castration and T replacement (Moore,1987a,b). It is possible that aromatization is important foraggression in mountain spiny lizards, at least in females, sinceaggressiveness in females is correlated with seasonal elevationsin both T and estradiol (E2) (Woodley and Moore, 1999a), butovariectomized, T-implanted females lacked some elements ofaggressive behavior observed in sham-operated controls(Woodley and Moore, 1999b). Elevated T and the associatedterritorial behavior have substantial energetic and survival costsin male S. jarrovi that appear to result from increases in energyexpenditure without compensating increases in energy intake(Marler and Moore, 1989, 1991; Marler et al., 1995).Comparable energetic and growth costs of experimentallyelevated T levels are seen in male northern fence lizards(S. undulatus hyacinthus) (Klukowski et al., 1998).Corticosterone concentrations are also correlated with territorysize in S. undulatus, perhaps due to the need to mobilize larger

Figure 14 Male tree lizards treated with corticosterone dermalpatches exhibited (a) more rapid antipredator responses, (b) longerhiding durations, and (c) more antipredator displays than did animalstreated with control patches. Black bars denote males of the orange-blue morph, while white bars denote orange males. Different lettersabove bars denote significant hormone treatment effects. DifferentRoman numerals above bars denote significant differences betweenmorphs. Bars represent means � one standard error. Sample sizes aredisplayed within the bars. Modified from Thaker, M., Lima, S.L., Hews,D.K., 2009b. Acute corticosterone elevation enhances antipredatorbehaviors in male tree lizard morphs. Horm. Behav. 56 (1), 51–57.




energy stores to patrol a larger territory; the benefit of that extraenergy expenditure seems to be an increase in the opportunityto sire offspring, as offspring number correlates strongly withcorticosterone level and territory size (John-Alder et al., 2009).

Sceloporus males can also show the short-term steroidhormone responses to encounters described above for treelizard and side-blotched lizard males. Male S. undulatus showT elevations in response to a series of staged laboratory encoun-ters with other males during the breeding season, but not inresponse to similar staged encounters with females or othermales outside the breeding season (Smith and John-Alder,1999). Corticosterone levels in males are increased by bothmale and female encounters, while females do not showhormonal responses to either male or female encounters.

Species in the genus Sceloporus were also recently used to testthe hypothesis that species/populations with short breedingseasons show a diminished glucocorticoid stress response, sothat onlymassive stressorswouldderail their infrequent opportu-nity at propagation. This hypothesis was tested by exposing indi-viduals to varied durations of handling stress followed by bloodcollection and hormone analysis. However, no differences in thecorticosterone stress responses to the stressor were detectedbetween a short-breeding-season, single-clutching species, S. vir-gatus, and S. undulatus and S. occidentalis, species with longerbreeding seasons and multiple annual clutches, thereforeproviding evidence against this hypothesis (Hews and AbellBaniki, 2013).

2.10.2.3.5 OtherBaird and Hews (2007) examined the relationship between T,DHT, corticosterone, and aggressive behavior in territorial(older) and nonterritorial (younger but mature) male commoncollared lizards (Crotaphytus collaris). These authors found thatconcentrations of the aforementioned steroid hormones do notvary between the morphs, or with behavioral expression. Inter-estingly, when neighbors died, aggressive display ratesincreased in the nonterritorial males, but rather than experi-encing coinciding increases in hormone levels, these individ-uals actually experienced decreases in their circulatingcorticosterone and T concentrations (the latter was solelya trend, but in the opposite direction of the behavior). Thisfinding emphasizes the complexity of steroidal regulation ofbehavior and a lack of a direct correspondence between aggres-sive display and circulating steroid levels.

This complex relationship between circulating steroidhormone levels and aggression is echoed in a study by Husakand Lovern (2014) examining the relationship between T andaggression across 18 Anolis species inhabiting four Caribbeanislands. As in the genus Sceloporus, many cross-species compar-isons are possible in the diverse genus Anolis. These researcherspredicted that species with higher aggression levels would bethose that possessed higher baseline T concentrations. Instead,on three of the four islands, the circulating concentrations of Twere inversely correlated with aggression.

2.10.2.4 Parthenogenetic Lizards

In four families of lizard (agamid, gekkonid, teiid, and lacertidlizards) there are species that consist only of females that

reproduce by cloning. There are scattered observations ofparthenogenesis in snakes, but no further studies have beenreported.

Among the whiptail lizards (Cnemidophorus spp.; the genuswas recently renamed to Aspidoscellis) fully one-third of thespecies reproduce by obligate parthenogenesis. The beststudied to date is the triploid desert-grasslands whiptail, Cnemi-dophorus uniparens. This particular species arose from the hybridmating between two sexual species and restriction analysis ofmitochondrial DNA indicates that two-thirds of its genomecomes from the little striped whiptail, C. inornatus (Densmoreet al., 1989; Conant and Collins, 1998; Figure 15).

During the breeding season males of the little striped whip-tail lizards will mount receptive females; neither males norfemales exhibit the heterotypical sexual displays. However,the descendant parthenogen C. uniparens displays both male-like mounting behavior and female-like receptivity, termed‘pseudosexual’ behavior during discrete phases of the ovariancycle (Crews and Fitzgerald, 1980; Moore et al., 1985a,b;Figure 16). This behavior is identical, excepting intromission,with that observed during mating in its sexual ancestor,C. inornatus.

The hormonal regulation of both sexual (ancestral species)and pseudosexual (descendant parthenogenetic species)behaviors as well as the downstream gene expression in discretebrain nuclei has been extensively studied (Crews, 2005). Inboth the parthenogens and female C. inornatus, circulatinglevels of E2 increase during the preovulatory (PreOv or follic-ular) phase and reach peak levels at the time of ovulation; Plevels, which are low during most of the postovulatory(PostOv) phase, are at their highest following ovulation andthen decline rapidly; androgens are not detectable in the circu-lation at any time of the ovarian cycle (Moore and Crews,1986). The only apparent exception is that in the unisexualspecies the levels of circulating E2 are lower (Moore and Crews,1986; Moore et al., 1985b; Young and Crews, 1995).

Female C. inornatus will stand and be receptive to themounting and copulation of sexually active males only whenthose females have large, preovulatory follicles, but will beaggressive toward males following ovulation (Lindzey andCrews, 1988b); receptivity can also be induced by exogenousestrogen in ovariectomized females but P does not inducemounting behavior (Young and Crews, 1995). In the partheno-gens, PostOv individuals will approach and investigate anotherindividual and attempt to mount. If PreOv, the individual willstand and be receptive to the mounting and pseudocopulationensues. During pseudocopulation the male-like individual willswing its tail under that of the other individual, and assumea genus characteristic donut-like position that places theircloacal openings in apposition.

In the parthenogens the P surge following ovulation medi-ates the male-like pseudosexual behavior. Although androgensare not detectable in the general circulation (Moore et al.,1985b), the whiptail brain is capable of de novo neurosteroido-genesis (Dias et al., 2009) and may explain why T is more effec-tive than DHT in stimulating both mounting behavior and PRabundance in the PvPOA of unisexual whiptails (Godwin et al.,2000; Wade et al., 1993). Implantation of P directly into thePOA, but not in the VMH, will elicit mounting in ovariecto-mized parthenogens (Crews, 1988).




Castrated male C. inornatus do not mount and copulatewith females but will do so after androgen treatment (Lindzeyand Crews, 1986). However, about 25–50% of castratedmales will respond to exogenous P with both mounting andcopulation (P-sensitive males), the remainder do notrespond to exogenous P (P-insensitive males) (Lindzey andCrews, 1986, 1988a). This effect of P is mimicked by R5020,a nonmetabolizable progesterone receptor (PR) agonist, and

abolished by the anti-progestin RU486, suggesting thatprogestins mediate this behavior at the level of PR ratherthan via progestin metabolites such as androgens (Lindzeyand Crews, 1988a). Within a reproductive season, thisphenotype is not plastic in that P-insensitive males do notbecome P-sensitive or vice versa. Progesterone-sensitive andprogesterone-insensitive C. inornatus males differ in theirneural substrates controlling sexual behavior (see Section2.10.4.3).

2.10.2.5 Sex Steroids and Behavior: Other Reptiles

Reptiles display diverse relationships between circulatingsteroid hormones and reproductive behavior; see reviews byWhittier and Tokarz (1992) for female reptiles and by Mooreand Lindzey (1992) for male reptiles. Findings related to thistopic also appear elsewhere in this chapter as they relate toTSD, parthenogenesis, and alternate reproductive phenotypes.We briefly review information presented in these earlier contri-butions by taxon and cover information that has been pre-sented since these reviews were published.

As with other aspects of brain�behavior relationships inreptiles, steroid hormone effects on behavior are best under-stood in lizards and snakes. This is primarily due to the easeof husbandry and adaptability of many of these species to labo-ratory conditions. Many lizards are also very amenable tostudies in the natural habitat. Recent exceptions to this focuson lizards and snakes are studies on sea turtles and tuataras.Both are of interest in part because of their endangered status.The hope is that better information on their reproductivebiology may be applied to aiding in their conservation.

2.10.2.5.1 TurtlesThis group has been the subject of a great deal of research relatedto TSD, but relatively little work has addressed hormone�behav-ior relationships in turtles. It is known that both luteinizinghormone and follicle-stimulating hormone rise during thebreeding period in female green sea turtles (Chelonia mydas; Lichtet al., 1979, 1980). Progesterone and T also rise during thisperiod, although E2 does not significantly. Patterns in the logger-head sea turtle (Caretta caretta) show similarities where P, T, andcorticosterone all decline over the course of the mating seasonthrough repeated nesting episodes (Wibbels et al., 1990;Whittieret al., 1997). Sea turtles are most easily sampled during the nest-ing period when they emerge onto beaches. Less information isavailable on steroid levels during other seasons. Rostal et al.(1998) approached this problem by sampling from captiveKemp’s Ridley sea turtles (Lepidochelys kempi) under semi-natural conditions. Male Kemp’s Ridley turtles show T peaksseveral months prior to mating and these levels decline slightlyby the mating season in March; they decline sharply followingthe cessation of breeding. Females exhibit peak levels of T, E2,and P at the time of mating. Both T and E2 decline sharply aftermating, while P declines more slowly.

Social interactions also influence circulating steroid levels ingreen sea turtles during the mating period. Jessop et al. (1999a)found that green sea turtle females have higher levels of plasmacorticosterone at nesting beaches (‘rookeries’) with a highdensity of other nesting females than at comparable low densitynesting beaches. A combined measure of plasma androgens

Evolution of the triploid Cnemidophorus uniparens

C. inornatus

C. inornatus F1 hybrid

C. gularis

F1 diploid parthenoform

Triploid C. uniparens

X

X

Figure 15 Most whiptail lizard species (genus Cnemidophorus,recently renamed Aspidoscellis) are gonochoristic, having both male andfemale individuals that reproduce sexually. However, one-third of the 45species of whiptail lizards are unisexual, consisting only of individualsthat reproduce by true parthenogenesis. The parthenogenetic speciesarose fully formed from the hybrid mating of two sexual whiptailspecies. Indeed, in many instances, we know which species wereinvolved. The best-studied parthenogen is C. uniparens. The maternalancestor of C. uniparens is the Little Striped Whiptail, C. inornatus. Thepaternal ancestor is still under dispute, with some favoring C. gularis

and others favoring C. burti. Whatever the paternal species, it is knownthat C. uniparens arose from the F1 hybrid mating in a backcross withC. inornatus.




showed no difference with nesting female density in this study.In contrast, male green sea turtles do show effects on plasmaandrogens related to social interactions (Jessop et al., 1999b).Males near, or actually mounting, females have elevatedandrogen levels, whereas males that are the recipient of aggres-sion from rival males or males that exhibit injuries from othermales while courting have lower circulating levels of androgen.

Although there are data on seasonal cycles in gonadalsteroids hormones for other turtles and tortoises (e.g., Callardet al., 1978; Lewis et al., 1979; Sarkar et al., 1996; Mahmoudand Licht, 1997; Schramm et al., 1999; Shelby et al., 2000), nostudies have addressed the behavioral correlates of this variation.The single exception is work in musk turtles (Sternotherus odora-tus) where there is some evidence of both photoperiod andandrogen control of sexual behavior (Mendonça, 1987a,b).

2.10.2.5.2 Crocodilians, Tuatara, and AmphisbaeniansThe 21 species of crocodilians represent the most primitiveextant members of the archosauromorph lineage that includesmodern birds and the extinct dinosaurs. As with turtles,

considerable information is available regarding steroidhormones and the process of TSD for crocodilians (representedby the American alligator, Alligator mississippiensis). Some infor-mation is also available regarding gametogenic cycles andcirculating steroid hormones in the group (Guillette et al.,1997). However, no experimental work has yet addressed therelationship of steroid hormones in crocodilians to behaviorand there are relatively few studies on the mating behavior ofthe group generally (e.g., Compton, 1981; Webb et al., 1983;Thorbjarnarson and Hernandez, 1993; Tucker et al., 1998).

Data on tuataras, limited to a single extant species represent-ing the order Sphenodontida, are similarly limited. Femaletuatara exhibit a prolonged reproductive cycle, carrying eggs inthe oviduct for 6–8 months and nesting only once every 4 yearson average (Cree et al., 1992). Tuataras appear to exhibit an asso-ciated reproductive pattern. Gametogenesis and T levels inmalesfollow an annual cycle, being low during the winter, rising in thespring, and peaking in mid-summer to early autumn during themating period. Female tuatara show elevated levels of E2 and Tduring vitellogenesis, which decrease after ovulation when P

Figure 16 Relationships among male-like and female-like pseudosexual behavior, ovarian state and circulating levels of estradiol and progesteroneduring different stages of the reproductive cycle of the parthenogenetic whiptail lizard. The transition from receptive to mounting behavior occurs atthe time of ovulation (arrow). Also shown are the changes in abundance of the gene transcripts coding for estrogen receptor (ER) and progesteronereceptor (PR) in the preoptic area (POA) and the ventromedial hypothalamus (VMH), brain areas that are involved in the regulation of male- andfemale-like pseudosexual behaviors. Redrawn from Crews, D., Coomber, P., Gonzalez-Lima, F., 1997. Effects of age and sociosexual experience onthe morphology and metabolic capacity of brain nuclei in the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex deter-mination. Brain Res. 758, 169–179.




levels rise. Females also show elevations in plasma arginine vaso-tocin (AVT) during oviposition relative to during the nestdigging and guarding stages that are likely associated with ovidu-cal contractions (Guillette et al., 1991).

As when this topic was last comprehensively reviewed(Whittier and Tokarz, 1992; Moore and Lindzey, 1992), noinformation is available regarding the relationship of steroidhormones to either reproduction or behavior in amphisbae-nians. However, it has recently been suggested that an interplaybetween steroids and social communication in amphisbae-nians may exist, given that males respond with chemosensoryand aggressive responses to squalene, a biochemical precursorto cholesterol and thus all steroid hormones, as they do to pre-cloacal secretions (López and Martín, 2009).

2.10.3 Neuroanatomical Substrates of Sexual andAggressive Behaviors in Reptiles

The neural pathways subserving sexual and aggressive behav-iors in reptiles appear to involve a network of limbic nucleithat, in many cases have clear homologs in the more complexnervous systems of birds and mammals. For instance, the finalcommon pathway for male-typical mounting and intromissionbehavior appears to be the POAH while that of female-typicalreceptive behavior is the ventromedial portion of the hypothal-amus (VMH; comparable to the ventromedial nucleus of thehypothalamus of rodents), as is the case in rodents (DeVriesand Simerly, 2002). Both of these areas are rich in steroidhormone receptors and their activity and neurochemistryrespond to both steroid hormones and environmental signals.Several kinds of studies have been performed to establish theroles of various brain areas in controlling sexual behaviors,including the critical importance of the POAH in regulatingmale-typical, and VMH in regulating female-typical, suites ofreproductive behaviors. Examples of five such types of experi-ment are described below.

2.10.3.1 Hormone Receptor Expression

An obvious property of a brain area involved in the control ofa hormone-influenced behavior is that it ought to expressreceptors for that hormone. Receptor expression has beenstudied in reptiles by several methods, including autoradiog-raphy with radiolabeled ligands, immunohistochemistry, andin situ hybridization. Accumulation of tritium-labeled E2, T,and DHT is found at a variety of sites in the brain of green anolelizards (Morrell et al., 1979). Halpern et al. (1982) performeda similar study with labeled E2 and T in the brain of red-sidedgarter snakes. These studies, similar to those in mammals andother vertebrates (see Morrell and Pfaff, 1978), reveal substan-tial E2 binding in the POA, AH, amygdaloid nuclei, ventrome-dial and posterior hypothalamic nuclei with lighter labeling inseveral areas including the septum, torus semicircularis, centralgray, and some brainstem areas. In the green anole binding oftritiated T and DHT are very similar to patterns for tritiated E2,but there are some differences in pallial and the mesencephalictegmental area.

Later, a series of studies explored the location and hormonaland social regulation of the mRNAs encoding the estrogen

receptor-a (ERa), AR, and PR in whiptail lizards (reviewed inCrews, 2005; Young and Crews, 1995). These studies eluci-dated the brain regions where each of these receptor mRNAsis expressed, variation in receptor mRNA abundances over thecourse of the ovarian cycle, species differences in expressionlevels and behavioral correlates of this expression, sex andspecies differences in the hormonal regulation of this expres-sion, developmental influences on the sexual differentiationof hormonal responsiveness in receptor mRNA expression, aswell as documenting social influences on ER- and PR-mRNAabundance in hypothalamic nuclei. Regulation of receptorexpression is discussed in a later section, and the discussionthat follows will be limited to the neuroanatomical distribu-tion of hormone receptors.

As in other groups of vertebrates, reptiles show what Pfaffand coauthors termed a ‘lawfulness’ of steroid receptor distri-bution in the brain (Pfaff et al., 1994). Indeed, this conserva-tion in distribution provides evidence of neural homologiesas with the expression of AR in the lizard DVR and the avianmagnocellular nucleus of the anterior nidopallium (MAN)(see below). In situ hybridization studies have documentedexpression of ER and AR in the same regions as previouswork using tritiated label to identify neurons concentratingsex steroid hormones, although some additional regions notfound using steroid autoradiography have been described(see below).

Strong ER mRNA expression is found in the whiptail peri-ventricular and medial POA, AH, periventricular nuclei of thehypothalamus, septal nuclei, the optic tectum, and the dorsal,posterior, and VMH (Figure 17). Weaker labeling is found inthe dorsal cortex, near the nucleus accumbens, the lateral andmedial septal areas, and the supraoptic nucleus. Previouswork with green anole lizards suggested little difference inandrogen- and estrogen-concentrating neurons in the brain(Morrell et al., 1979). However, this previous study used triti-ated E2 and T, respectively, and it is possible that some ofthe T label was aromatized to E2 (see Wade, 1997, 2005)and then bound the ER. This is discussed at greater lengthbelow.

Progesterone receptor mRNA expression is also widelydistributed through the brain of whiptail lizards (Figure 17).Especially strong expression is seen in the VMH and both theperiventricular and medial POA. Strong labeling is also foundin the medial septum, lateral POA, central amygdala, AH, theposterior, dorsal and VMH, the lentiformis thalamis pars plicta,and the torus semicircularis. Lower levels of PR mRNA areevident in the lateral hypothalamic area, near the premammi-lary nucleus, and in parts of the optic tectum.

In both the whiptail lizard and the leopard gecko, ARmRNAexpression occurs in the DVR, external nucleus of the amygdala,medial POA, AH, LS, dorsolateral anterior nucleus, VMH, peri-ventricular nuclei of the hypothalamus, and especially the pre-mammilary nucleus (Young et al., 1994; Rhen and Crews,2000; Figure 17). This distribution reveals differences frompreviously documented distributions in the brains of greenanole lizards and garter snakes based on steroid autoradiog-raphy. Specifically, AR mRNA is more abundant than ERmRNA in the LS and ER mRNA does not occur in the externalamygdala of whiptail lizards. Of particular interest froma comparative perspective is sexual dimorphism in expression




of AR mRNA in the DVR of the lizard, which may have a func-tion similar to that of MAN in songbirds (Young et al., 1994).There are also some strong similarities between the patterns ofAR expression in whiptail lizards and those in mammals.

Mapping ARgene expression through in situhybridization hasalso been employed more recently in green anole lizards toexamine the distribution of AR mRNA expression (Rosen et al.,2002). Theseworkers foundARmRNA inmany of the same areasexpressing thismessage in the brain ofwhiptail lizardswith somedifferences. The regions of similarity include the POA, septum,amygdala, striatum, premammilary nucleus, VMH, and torussemicircularis and brainstem motor nuclei. In contrast to thepatterns observed in whiptail lizards, AR mRNA expression is

not localized to the DVR of green anole lizards in this study.Beck and Wade (2009a,b) also examined the expression of ERamRNA in the embryonic, perinatal, and adult green anole fore-brain, finding more mRNA in the adult female POA and amyg-dala than in adult males, and mRNA expression several timesgreater in the developing VMH than in the POA and amygdalaof both sexes during development and in adulthood. Cohenet al. (2012) further examined the expression of ERb mRNA ingreen anoles, finding widespread forebrain expression, includingthe POA, VMH, and amygdala, with greater densities of ERb-expressing cells in the VMHand amygdala of females thanmales.

Immunocytochemical studies such as those of Moga et al.(2000) have used an antibody directed at a conserved sequence

Figure 17 Distribution of cells expressing steroid receptor mRNA in selected sections of the brain of whiptail lizards. Shown are the positions ofcells expressing mRNA for estrogen receptor (ER, column 1), progesterone receptor (PR, column 2), and androgen receptor (AR, column three) inthe right half of brain sections. Solid circles indicate heavily labeled cells and hollow circles indicate lightly labeled cells. Abbreviations: AC, anteriorcommissure; AME, nucleus externus amygdale; DH, nucleus dorsalis hypothalami; LFB, lateral forebrain bundle; LHA, lateral hypothalamic area; LPA,lateral preoptic area; MPA, medial preoptic area; NS, nucleus sphericus; NSL, nucleus septalis lateralis; NSM, nucleus septalis medialis; PH, nucleusperiventricularis hypothalami; PP, nucleus periventricularis preopticus; SC, nucleus suprachiasmaticus; SO, nucleus supraopticus; VMH, nucleus ven-tromedialis hypothalami. Redrawn from Young, L.J., Lopreato, G.F., Horan, K., Crews, D., 1994. Cloning and in situ hybridization analysis ofestrogen- receptor, progesterone-receptor, and androgen receptor expression in the brain of whiptail lizards (Cnemidophorus uniparens andC. inornatus). J. Comp. Neurol. 347, 288–300.




in the N-terminal domain of the AR protein (residues 1–21 ofthe rat AR) to map AR-ir in S. undulatus. This method allowsdistinguishing between staining in the nucleus and cytoplasmand also reveals AR-ir in axons and dendrites with a highanatomical specificity. Androgen receptor immunoreactivity inthe brain of S. undulatus showed good, but not complete,agreement with the distribution of AR determined in otherspecies and by other methods described above. Androgenreceptor staining was found in males in the medial and dorsalcortices, medial septum, and several cell groups in theamygdala as well as the adjacent bed nucleus of the striaterminalis (BNST). These investigators also found nuclearstaining in the medial POA, periventricular hypothalamus, andventromedial, premammillary, and arcuate nuclei. AR-ir fiberstaining occurred throughout the AH and POA and in a varietyof other diencephalic areas. Females showed nuclear AR-ir infewer areas than males, with nuclear staining only in theventral posterior amygdala and VMH (the ventral posterioramygdala appears homologous to the external amygdala ofwhiptail lizards based on anatomy and AR expression; Mogaet al., 2000). Fiber staining in male and female fence lizardswas broadly similar. The sex difference in distribution foundhere for S. undulatus could represent a species difference ora difference in the sensitivity of the immunocytochemicalmethod employed relative to the in situ hybridizationapproach used in other studies. Male whiptail lizards do showhigher levels of AR mRNA in the medial POA than females(Godwin et al., 2000), just as females show higher levels ofestrogen-induced PR mRNA expression in the VMH (Crewset al., 2004; Wennstrom et al., 2003). The results reported forfence lizards could be very similar if females do express ARmRNA in the medial POA, but AR protein at levels too low tobe detected by immunocytochemistry. A difference insensitivity of the technique could also account for the lack ofAR-ir observed in the dorsal lateral thalamic nucleus andanterior dorsal ventricular ridge, a result that also contrastswith the described distribution of AR mRNA in whiptaillizards (Young et al., 1994).

Thus, in situ hybridization has proven very useful for deter-mining sites of steroid receptor synthesis, but immunocyto-chemical work further exploring regions of steroidresponsiveness has been limited by the availability of anti-bodies that bind specifically to target proteins in reptile tissue.Advances in this area enabling immunocytochemical workexploring colocalization of different receptor types within cellswould be particularly useful.

2.10.3.2 Intracranial Hormone Implants

Sex steroid receptors are widely distributed in areas of the brainthat play critical integrative roles in sociosexual behavior,including the POAH and VMH. The role of these brain areasimplicated in hormone-dependent behaviors can be tested byintracranial implantation of minute amounts of steroidhormones directly into the candidate region of animalsdeprived of systemic sex steroids by gonadectomy. Thisapproach has been shown to effectively restore male-typicalsexual behaviors in rats and other mammals (reviewed inDeVries and Simerly, 2002). Implantation of androgensdirectly into the POAH reinstates courtship and copulatory

behaviors in castrated male green anoles (Morgentaler andCrews, 1978). Likewise, intracranial implantation of androgeninto the POAH will induce copulatory behaviors in both cas-trated male whiptails C. inornatus (Rozendaal and Crews,1989), and the parthenogenetic C. uniparens (Mayo and Crews,1987). Intracranial implantation of P rather than androgen isalso effective in restoring courtship and copulatory behaviorin a subset of C. inornatus males that are sensitive to intraperi-toneal P implants (Crews et al., 1996b).

As with male-typical sexual behavior, implantation ofestrogen directly into the VMH of ovariectomized female andunisexual whiptail lizards reinstates receptive behavior (Wadeand Crews, 1991). This finding agrees well with the morerecently characterized distribution and regulation of both ERand PR in this brain region.

2.10.3.3 Morphological Parameters

Another simple prediction is that a brain area involved in thecontrol of a behavior will be influenced structurally by param-eters known to affect that behavior. An area involved in medi-ating a seasonal behavior would be expected to changeseasonally, an area involved in a sexually dimorphic behaviorought to exhibit sexual dimorphism in structure, and so on.Several examples of such differences in brain nuclei or somasizes are present in reptiles.

2.10.3.3.1 Sexual DimorphismsIn the sexual species of whiptail lizard, C. inornatus, males havelarger POAH than do females while females have a larger VMH(Crews et al., 1990). These sexual dimorphisms in size areunder the control of gonadal androgens in the male(Figure 18). That is, castration of breeding animals results inboth a reduction in the area of the POA, and an enlargementof the VMH, whereas androgen replacement therapy reversesthese effects of castration (Wade et al., 1993). It is significantthat only the male shows these responses to hormonal manip-ulation. These overall differences in nucleus size are correlated

Figure 18 Schematic representations of the volumes of the sexuallydimorphic areas in the brain relative to body size in sexual and parthenoge-netic whiptails. To aid in comparison, the volume of the anteriorhypothalamus-preoptic area (AH-POA) and the ventromedial nucleus of thehypothalamus (VMH) of female Cnemidophorus inornatus is represented asa dashed outline in other drawings to indicate significant differences.




with differences in soma size within these areas. MaleC. inornatus have larger soma sizes in the POA while femaleshave larger soma sizes in the VMH (Wade and Crews, 1992).Interestingly, the brains of the all-female C. uniparens showpatterns similar to those of females of the sexual species despitethe fact that these females regularly show male-like pseudosex-ual behavior. This finding is also true when the parthenogeneticfemales are sex reversed using fadrozole, an inhibitor of aroma-tase that effectively induces male development in C. uniparens(Wibbels and Crews, 1994; Wennstrom and Crews, 1995;Wennstrom et al., 1999). This dissociation between brain andbehavior indicates that, while sometimes useful, measurementsof brain nucleus volume and soma size often do not reflectspecific functions. This topic will be further addressed belowwhen we discuss metabolic capacity, neurotransmitter func-tion, and regulation of steroid hormone receptor expression.

Information is also available on sexual dimorphisms inboth the brainstem and limbic system of green anoles. Sexualand aggressive behaviors in green anoles are well characterized(Crews, 1975b; Crews et al., 1978; Andrews and Summers,1996; Propper et al., 1991). The control of these behaviors bygonadal steroid hormones in green anoles has also receiveda good deal of attention (Valenstein and Crews, 1977; Crewset al., 1978; Crews and Morgentaler, 1979; Tokarz and Crews,1979, 1980, 1981; Jones et al., 1983). Males perform ‘pushups’ as in many male lizards and are also able to greatly extenda red-pigmented portion of skin on the ventral side of the neck,termed the dewlap, through flexion of the hyoid apparatus(Crews, 1975a); although female green anoles have similarpigmentation in the gular region and use this as an aggressivesignal, neither the skin or the hyoid is as well-developed as inmales (Crews, 1975b). Both of these signals are used in socialcontexts with dewlap extension as a courtship signal beingshown only in males while female headbobs, with or withoutdewlap extension, are regarded as a signal of submission duringmale–female interactions (Crews, 1975b). The muscleprimarily responsible for dewlap extension, the ceratohyoideusmuscle, is innervated from the nucleus ambiguus (AmbX) aswell as the glossopharyngeal portion of the AmbX and theventral portion of the motor nucleus of the facial nerve(AmbIX/VIImv). Neurons in both brain regions are larger inmales than in females (Wade, 1998). Motor neuron numberdoes not vary by sex or across the breeding and nonbreedingseasons, but nerve cross-sectional area and both muscle fibersize and number are greater in males than females (O’Bryantand Wade, 1999). However, there is no consistent relationshipbetween either the breeding/nonbreeding season or androgentreatment (T propionate) and these characteristics in males.Testosterone implantation into juvenile females 30 days post-hatching increases cartilage length and size of the dewlapmuscle fibers compared to sham-implanted controls (Lovernet al., 2004), suggesting that T exposure during developmentserves to masculinize components of the neuromuscularsystem. Another study found that, unlike some other vertebratesystems where the sexes differ in their display behavior, greenanoles show no sex difference in the dendritic arborization ofmotoneurons in AmbX or AmbIX/VIImv (O’Bryant andWade, 2000). These findings suggest that changes in this systemduring adulthood do not underlie sex or seasonal differences inthe dewlap extension behavior. O’Bryant and Wade propose

that this may be due to the fact that, while extension of thedewlap is associated with male courtship, females will alsolower the hyoid, thereby exposing the patch of red. Takingadvantage of the natural variation in copulatory behavior(studs vs duds), it has been reported that dewlap extension ispositively correlated with size of the associated muscle aswell as soma size in the amygdale (Neal and Wade, 2007b).The same study also suggests that androgen-sensitive tissuesin studs might be more responsive to T than those in duds,potentially explaining some of the behavioral variation.

In the tree lizard, males on average possess a larger POA andamygdala than females (Kabelik et al., 2006). A lateral portionof the ventromedial nucleus of the hypothalamus, which hasbeen linked to aggression (Kabelik et al., 2008a), was alsolarger in males, although the overall nucleus did not differbetween males and females. Testosterone level was shown tounderlie some of this neural plasticity, with male tree lizardstreated with T-filled Silastic implants possessing largeramygdaloid nuclei than controls or males receiving P-filledimplants (Kabelik et al., 2008c; Figure 19).

Figure 19 (a) A depiction of the location, in unilateral coronal brainsections, of the lateral septum (LS), preoptic area (POA), nucleus spher-icus (NS), amygdala (AMY), ventromedial hypothalamus (VMH), and habe-nula (HAB). (b) Compared to blank implant-treated subjects, testosteronebut not progesterone treatment increased the average cross-sectional areaof the NS and AMY in male tree lizards. Both testosterone and proges-terone had been found to increase aggressive display in the same study. *denotes a significant difference between homogenous groups, with thelatter denoted by lines above bars. Adapted from Kabelik, D., Weiss, S.L.,Moore, M.C., 2008c. Steroid hormones alter neuroanatomy and aggres-sion independently in the tree lizard. Physiol. Behav. 93 (3), 492–501.




The research described above focuses on species with thefamiliar pattern of GSD. The other end of the genotype-�environment spectrum is represented by species that exhibitTSD. In the leopard gecko, both low and high incubationtemperatures produce females while intermediate temperaturesresult in male determination and differentiation (Figure 4).Interestingly, sexuality covaries with incubation temperaturesomewhat independently of gonadal sex such that femalesproduced at higher temperatures are masculinized relative tofemales incubated at low temperatures. This variation is remi-niscent of the intrauterine position effect in mammals andmay provide a powerful experimental system for addressingthe causes and consequences of prenatal hormonal effects aswell as maternal effects on offspring phenotypes. Coomberet al. (1997) found that females from male-biased incubationtemperatures had larger POA volumes than those fromfemale-biased incubation temperatures. Indeed the variationwithin each sex across temperature morphs is greater than thevariation between the sexes from each incubation temperature.There are parallel differences in the VMH, but this varies withage; for example, old females have a larger POA and VMHthan do young females. Differences are also seen in maleswith age, but these results contrast with those found in females

(Crews et al., 1997). Young males show larger volumes for thePOA and VMH than do older males.

2.10.3.3.2 Seasonal VariationSeasonal variation in the size of brain areas has been docu-mented in a variety of vertebrate species, particularly in thesong system of many birds (see Chapter 2.11, NeuroendocrineRegulation of Reproductive Behavior in Birds by Ball and Balth-azart; and Chapter 2.12, Neural and Hormonal Control of Bird-song by Schlinger and Brenowitz, this volume). Many reptilesalso show seasonal reproduction.

Along with the sexual dimorphisms described above,Kabelik et al. (2006) also reported changes in tree lizard limbicnuclei volumes across the breeding season (Figure 20). It isnoteworthy that the authors conclude that the observed alter-ations in brain nuclei volume might not be involved in theregulation of aggressive interactions because the nucleuschanges do not correlate well with behavioral changes, furtheremphasizing the lack of a direct correspondence between struc-ture size and individual function.

Seasonal changes in limbic brain nucleus volumes have alsobeen reported in the green anole, where male and femalesubjects from the breeding season possess larger POA and

Figure 20 (a) Nissl-stained coronal brain sections depicting the lateral septum (LS), preoptic area (POA), nucleus sphericus (NS), amygdala (AMY), andventromedial hypothalamus (VMH). (b) Seasonal changes in brain volume were found in (top) male and (bottom) female tree lizards. Larger volumes wereseen during the breeding (May–June) or postbreeding periods (August–September) periods than during the pre-breeding period (Feb–March). The figuresare based on estimated marginal means following the inclusion of whole brain volume as a covariate. * denotes a significant difference between the threeperiods at p < 0.05, y denotes a strong trend at p ¼ 0.056. Bars represent means� one standard error. Adapted from Kabelik, D., Weiss, S.L., Moore,M.C., 2006. Steroid hormone mediation of limbic brain plasticity and aggression in free-living tree lizards, Urosaurus ornatus. Horm. Behav. 49, 587–597.




VMH volumes than do those from the nonbreeding season(Beck et al., 2008). Interestingly, while no volume differenceswere found across seasons in the amygdala, the number ofneurons in this region was found to be greater in nonbreedingseason relative to breeding season animals.

In the Canadian red-sided garter snake the volume of thePOA varies seasonally in females, but not in males (Crewset al., 1993). The POA of female snakes is smaller than thatof males during the hibernation period. The lack of variationin males may be related to the fact that, unlike the songbirdsthat are the focus of most studies, these garter snakes exhibita dissociated reproductive pattern in which seasonal matingbehavior and gonadal steroid hormone peaks are temporallyoffset (Crews, 1991).

2.10.3.4 Metabolic Indicators of Neural Activity

The involvement of a particular brain area in a particularbehavior is associated with an increase in metabolic activityin the area during expression of the behavior. The 2-deoxyglucose (2-DG) technique enables postmortem assess-ment of recent metabolic activity in the brain and can beused to compare groups of animals engaged in different behav-iors. The technique utilizes the dependence of neurons onglucose as a source of energy, and 14C radiolabeled 2-DG,analog of glucose that can be taken up by cells but not metab-olized. The labeled 2-DG accumulates in cells and this accumu-lation can be assessed by the relative amounts of radioactivitypresent in tissue sections (Cada et al., 1995). Rand and Crews(1994) found that the acute metabolic activity of the partheno-genetic C. uniparens depended on whether they were displayingmale-like or female-like pseudosexual behavior (Figure 21).Specifically, animals displaying male-like pseudocopulatorybehavior showed a sixfold greater accumulation of 2-fluoro-2-DG in the medial POA than did animals showing female-like behavior. Conversely, individuals showing female-likereceptive pseudosexual behavior exhibited a greater accumula-tion of 2-DG in the VMH.

The results from C. uniparens are paralleled across seasons inred-sided garter snakes. Male red-sided garter snakes thatactively court females show significantly higher 2-DG accumu-lation in the POAH than males who either are exposed tofemales but fail to court or males that are not exposed tofemales (Allen and Crews, 1992). Interestingly, even simplybeing exposed to females increases overall 2-DG accumulationin garter snake males. These results suggest both a generalizedarousal effect as well as a more specific effect of active courtingwhich is restricted to the brain region very directly involved inmediating this behavior; this finding is paralleled by resultsusing immediate early gene products as markers of neuralactivity, as discussed below.

Radiolabeled 2-DG has also been used to study patterns ofneural activity associated with the change from a receptive toan unreceptive state in female red-sided garter snakes(Mendonça and Crews, 2001). Upon emergence from hiberna-tion, females initially are receptive to male courtship behaviorbut become unreceptive immediately following mating. Femalesthat are courted and thenmated have significantly higher activityin the POA and significantly lower activity in the VMH comparedto females who are courted but not mated (Figure 22). Since

intromission during mating is responsible for the loss of sexualreceptivity in the female (Mendonça and Crews, 1990;Mendonça et al., 2003; Ross and Crews, 1977; Whittier andCrews, 1989; Whittier et al., 1985, 1987), injection of a localanesthetic (tetracaine or lidocaine) into the cloacal region desen-sitizes the female to mating stimuli (Mendonça and Crews,1990, 2001). Not only will this treatment prevent the mating-induced surge in estrogen levels in the plasma and subsequentovarian recrudescence but also the pattern of 2-DG accumula-tion in tetracaine-treated females is similar to courted butunmated females and to females exposed only to other females.These results suggest that in the female red-sided garter snakesensory input from the cloaca during mating alters patterns ofmetabolism in those brain areas most often associated withsexual receptivity. The increased activity in the POA accompa-nied by a decrease in activity in the VMH after mating supportsthe hypothesis that mating initiates a neuroendocrine reflex thatresults in a loss of receptivity in female red-sided garter snakes.

2.10.3.5 Focal Lesions

Electrolytic lesion experiments involve creating localizeddamage in candidate regions followed by behavioral assays toassess disruptions of function. Lesions of the POAH impaircourtship and copulatory behavior in male green anole lizards(Wheeler and Crews, 1978) and little striped whiptail lizards(Kingston and Crews, 1994), whereas lesions of the VMH inthe parthenogenetic whiptail lizard C. uniparens abolish

Figure 21 Metabolic activity during pseudosexual behavior in theunisexual lizard. Depicted is average change in 2-deoxyglucose (2-DG)uptake relative to whole brain/optic tract (WB/OT) in the brain of twoindividual lizards engaged in a pseudocopulation. Left column are lightmicrographs of brain section at the level of the POA (top row, solidarrow) and the VMH (bottom row, open arrow). Other columns arepseudocolor images where red denotes maximum accumulation of 2-DG and green the lowest accumulation. Middle column is the brain ofthe individual exhibiting male-like pseudosexual behavior (¼ courtship)while the right column is the brain of the lizard exhibiting female-likepseudosexual behavior (¼ receptive). Bottom indicates differences inaccumulation at each brain area. After Rand, M.S., Crews, D., 1994.The bisexual brain – sex behavior differences and sex- differences inparthenogenetic and sexual lizards. Brain Res. 663, 163–167.




receptive behavior; it is significant that only those lesions thatencompassed the area containing ER were effective (Kendricket al., 1995). Interestingly, POAH lesions also impair male-like pseudocopulatory behavior in the unisexual C. uniparens.This suggests that pseudosexual behavior in the descendantspecies of this pair is mediated by the same neural circuitsresponsible for copulatory and receptive behaviors in malesand females of its ancestral species.

2.10.4 Neurochemical Bases of Sexual andAggressive Behavior in Reptiles

The relationship of neurotransmitters and neuropeptides tosexual and aggressive behaviors in reptiles has received rela-tively little attention, but some progress has been made inunderstanding the neurochemical mechanisms underlyingsuch behaviors in anoles, tree lizards, and whiptails. Further-more, other signaling molecules such as nitric oxide (NO), aswell as other regulatory molecules such as steroid convertingenzymes, need to be considered.

2.10.4.1 Nonapeptides

Arginine vasotocin populations have been examined ina number of reptile species and generally exhibit stronghomology to those found in other amniotes (Hattori andWilczynski, 2009; Kabelik et al., 2013; Propper et al., 1992a).Some differences between species have been noted, butdisagreement about reptilian neuroanatomy and differencesin experimental procedures make it difficult to be certain ofthis variability, and further studies are needed to solidifyconclusions. A possible link between sociosexual behaviorsand AVT is suggested by Propper et al. (1992a) who reportthat AVT immunoreactivity in green anoles is generally ofgreater intensity in males than females. Hattori and Wilczynski(2009) also found increases in diencephalic AVT immunoreac-tivity in male green anoles that were the dominant individualin a stable dominance hierarchy, while decreased (from controlgroups) AVT immunoreactivity was found in animals that heldthe subordinate role. Dunham and Wilczynski (2014) foundthat intraperitoneal injections of AVT in male green anolesdecreased aggression in mirror trials (with one’s own reflec-tion), but not aggressive interactions among size-matchedmales; however, administering AVT in this manner led toa concomitant increase in circulating glucocorticoids, makingit unclear what was the causal variable to the reduction inaggression.

Immediate early genes, themost commonly examined beingc-fos, are expressed in response to recent neural activation andcan be visualized via in situ hybridization and immunohisto-chemistry. Like the 2-DG technique described above, visualiza-tion of the resultant mRNA or protein can be used to assessrecent neural activity in specific brain regions. However, usingimmunofluorescence or other multilabel approaches, one canfurther colocalize this activity marker to specific populationsof neurons. Kabelik et al. (2013) used immunofluorescence tocolocalize AVT and c-Fos immunoreactivity in male brownanoles (Anolis sagrei) and determined that AVT-producing cellswere activated during both sexual and aggressive behaviors.However, all populations of AVT-producing neurons did notact in synchrony. In particular, AVT-immunoreactive neuronsin the POA were activated primarily in sexual contexts,whereas populations in the BNST, paraventricular nucleus ofthe hypothalamus, and supraoptic nucleus were activatedduring both sexual and aggressive behaviors (Figure 23). Thelatter is somewhat at odds with findings in other amniotes,especially birds, where the BNST population tends to beactivated in sexual contexts and inhibited in aggressivecontexts (Goodson and Kabelik, 2009).

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Figure 22 Effect of mating on metabolic activity in the brain of femalered-sided garter snakes (Thamnophis sirtalis parietalis). Depicted is therelative change in 2-deoxyglucose (2-DG) uptake in different treatmentgroups relative to whole brain/optic tract (WB/OT). Note that tetracaine,a local anesthetic applied to the cloaca, abolishes the response to mating.The dashed line indicates background brain levels (e.g., zero differencefrom background). A positive change represents higher accumulation,a negative change lower accumulation than background. Asterisks repre-sents significant differences with level of the difference is indicated. PanelA depicts uptake of 2-DG in the preoptic area/optic tract (POA/OT). PanelB depicts uptake of 2-DG in the ventromedial hypothalamus/optic tract(VMH/OT). Redrawn from Mendonça, M.T., Daniels, D., Faro, C., Crews,D., 2003. Receptivity and 2-deoxyglucose uptake in female red-sidedgarter snakes. Behav. Neurosci. 117, 144–149.




No sex differences were described in vasopressin-like oroxytocin-like (presumably AVT and mesotocin) immunoreac-tivity in the brain of the chameleon, although differenceswere found for females across the ovarian cycle (Bennis et al.,1995). This variation in AVT-like immunoreactivity alsooccurs in female green anoles, where females with largepreovulatory follicles have higher AVT concentrations in thesupraoptic area than females with small preovulatory follicles(Propper et al., 1992b). In whiptail lizards, T administrationalso increases the number of AVT-ir cells in the POA offemale C. inornatus (Hillsman et al., 2007; Figure 24). Thereare a few reports in other reptiles regarding distribution ofAVT immunoreactivity (e.g., the turtle (Pseudemys scripta) andsnake (Python regius) (Smeets et al., 1990)).

Variation in AVT level with sex, reproductive state, hormonelevel, and seasonal cycle has also been detected in variousneural populations of the tree lizard (Kabelik et al., 2008b).In general, males of this species also exhibited greater AVT

Figure 23 Arginine vasotocin (AVT) neurons and fibers in the malebrown anole brain: (a) preoptic area, POA; (b) suprachiasmatic nucleus,SON; (c) bed nucleus of the stria terminalis, BNST; and (d) paraventricularnucleus, PVN. (e) Neural activity, as assessed by colocalization with Fos90-min post behavioral encounter, reveals that all of these populations ofvasotocinergic neurons exhibit increased activation during social encoun-ters in individuals that exhibited maximum behaviors for that category(biting of opponent during aggression trials, copulation during courtshiptrials). Note that activity within the parvocellular POA neurons was elicitedspecifically by sexual encounters, while activity within the magnocellularneurons of the PVN was elicited by aggressive encounters. Different lettersabove bars designate groups differing at p < 0.05. Other abbreviations:lfb, lateral forebrain bundle; III, third ventricle; oc, optic chiasm. Adaptedfrom Kabelik, D., Alix, V.C., Burford, E.R., Singh, L.J., 2013. Aggression-and sex-induced neural activity across vasotocin populations in the brownanole. Horm. Behav. 63 (3), 437–446.

Figure 24 Arginine vasotocin immunoreactive cells (AVT-ir) and fibersin the periventricular preoptic area (PP) and the bed nucleus of the striaterminalis (BNST) in a Virago Cnemidophorus uniparens (aromataseinhibitor created males) receiving a blank implant (Top Panel). Otherabbreviations: AC, anterior commissure; LFB, lateral forebrain bundle;OC, optic chiasm; SO, supraoptic nucleus. Bottom Left Panel:Testosterone treatment increases abundance of AVT-ir in the PP ofCnemidophorus inornatus. Effect size is large in both females (top) andmales (bottom), but reaches statistical significance only in females.Bottom Right Panel: Testosterone treatment increases abundance ofAVT-ir in the PP of Cnemidophorus uniparens treated as embryos witharomatase inhibitor (Viragos) (bottom), although not in C. uniparens

treated as embryos with ethanol (Parthenoform) (top). Mean � SEMshown with number of individuals in parentheses. Redrawn fromHillsman, K.D., Sanderson, N.S.R., Crews, D., 2007. Testosteronestimulates mounting behavior and arginine vasotocin expression in thebrain of both sexual and unisexual whiptail lizards. Sexual Devel. 1, 77–84.




immunoreactivity than did females, and AVT levels were higherwhen circulating T concentrations were increased (Figure 25).However, direct correlations were not found between AVTexpression and aggression levels in these studies, suggestingthat activation of AVT neurons (see c-Fos studies above) ismore directly related to individual differences in socialbehavior expression than is AVT chemoarchitecture. Large-scale differences in chemoarchitecture may neverthelessunderlie species differences in social behavior propensities.

Much less work has been carried out in reptiles on the non-apeptide mesotocin (nonmammalian homolog of oxytocin).Like AVT, mesotocin has been linked to egglaying, includingthe timing of nesting behavior in three-toed box turtles (Carret al., 2008). However, recent work in brown anoles by Kabelik

and Magruder (2014) also found positive correlations betweensexual behavior and c-Fos colocalization in mesotocin neuronsof the paraventricular nucleus of the hypothalamus. Thisfinding suggests that mesotocin may play a similar role in socialbehavior regulation in reptiles as oxytocin does in mammals(Zingg and Young, Chapter 3.13, Oxytocin).

2.10.4.2 Monoamines

Like the changes in AVT expression associated with status ina dominance hierarchy (see above), dominance interactionshave also been shown to influence monoamine metabolismin male lizards. For example, aggressive interactions increaseplasma epinephrine (EPI) and norepinephrine (NE) in male

Figure 25 (a) Arginine vasopressin (AVT) immunoreactivity within unilateral coronal brain sections from the tree lizard. (b) No morph differenceswere observed but males exhibited greater AVT fiber densities, as measured by optical density of signal within that region, than did females withinthe amygdala (AMY), bed nucleus of the stria terminalis (BNST), lateral septum (LS), nucleus accumbens (NAcc), and preoptic area (POA). Thecross-sectional soma sizes of magnocellular and parvocellular neurons from the paraventricular nucleus (PVN) did not differ between morphs orsexes. (c) Hormone manipulations of male tree lizards resulted both in increases to cross-sectional areas of both parvocellular and magnocellularneurons of the PVN, and in increases in the optical density estimates of fiber density within the AMY, BNST, LS, and the mixed fiber and cell densityin the PVN. Other acronyms: magn, magnocellular; parv, parvocellular; supraoptic nucleus, SON. Adapted from Kabelik, D., Weiss, S.L., Moore, M.C.,2008b. Arginine vasotocin (AVT) immunoreactivity relates to testosterone but not territorial aggression in the tree lizard, Urosaurus ornatus.Brain Behav. Evol. 72 (4), 283–294.




green anoles and these levels are higher in males winningencounters than in those males that lose (Summers andGreenberg, 1994). This response and the speed of the correlatedeyespot darkening are reduced by castration, suggesting an influ-ence of T. Both dominant and subordinate male anoles showchanges in central monoaminemetabolism following aggressiveencounters, but these changes are more pronounced in subordi-nates. Subordinate male green anoles show elevated ratios of5-hydroxyindoleacetic acid to 5-hydroxytryptamine (5-HIAA/5-HT) and the substrate for 5-HT, 5-hydroxytryptophan (5-HTP) (indicating enhancement of both 5-HT turnover andproduction) 1 h after an encounter with a dominant individual(Summers andGreenberg, 1995). The difference between subor-dinates and both dominants and control males diminishesthereafter. Dominant males show broadly similar patterns, butreturn to baseline turnover levels more rapidly. Serotonergicsignaling via the 5-HT2C receptor is thought to play a role inthe dominance observed as part of such agonistic interactions(Baxter et al., 2001). Neither the dopaminergic nor adrenergicsystems showed similar patterns, indicating this response isspecific to the serotonergic system. This serotonergic responseis also regionally specific. The nucleus accumbens and hippo-campal cortex show the most dramatic changes 1 h followingan aggressive interaction, but the medial and lateral amygdalashow a more delayed response with serotonergic activity peak-ing at 1 week postinteraction in subordinate males (Summerset al., 1998). The amygdalar region is important in regulatingsexual and aggressive behaviors in green anoles (Greenberget al., 1984).

Both plasma and brain region-specific alterations in mono-amine metabolism can also be induced in male anoles bymanipulating a key aggressive signal and exposing males toa mirror (Korzan et al., 2000a,b). Masking a male’s eyespotwith green paint suggests a less aggressive or subordinate oppo-nent when the animal observes this ‘opponent’ in a mirror.Males whose eyespots were painted green showed the highestfrequency of biting behavior. These males also showed elevatedplasma levels of dopamine (DA), EPI, and NE relative to iso-lated controls and males whose eyespots were painted black.In the brain, males with green-painted eyespots showedincreased serotonergic and adrenergic activity (but lower dopa-minergic activity) in the subiculum (dorsal cortex), hippo-campus, nucleus accumbens, and medial amygdala relative tomales whose eyespots were darkened (suggesting an aggressiveor dominant opponent). Feeding green anoles with cricketsinjected with L-DOPA increases dopaminergic activity (withoutaffecting the serotonergic and noradrenergic systems) in themedial amygdala and hippocampus coupled with a decreasein aggressive behavior (Höglund et al., 2005). The authorssuggest that this result might be due to D2 receptor signalingin the indirect basal ganglia system of the green anole. Theinvolvement of dopaminergic activity in aggressive behavioris corroborated by the observation that lizards viewing anopponent with artificially darkened eyespots exhibit decreasedaggression, assume a subordinate status with elevated DA levelsmeasured in the medial amygdala, lateral amygdala, and raphe(Korzan et al., 2006). Conversely, green anoles that view anopponent with hidden eyespots express the dominant pheno-type with increased aggression, and increased DA levels in thestriatum, nucleus accumbens, and substantia nigra/ventral

tegmental area. Increases in DA levels in different brain nucleiin subordinate as well as dominant animals suggest differentialbrain systems involved in phenotypic variation.

In a recent study, Kabelik et al. (2014) examined c-Fosinduction (a marker of recent neural activity) across catechol-amine populations throughout the entire male brown anolebrain (except for the retina and olfactory bulb). Theseresearchers found evidence of widespread increases in Fosinduction within several putatively dopaminergic populationsfollowing a sexual encounter with a female conspecific. Theseregions included portions of the POAH, a midline population(subdivision of the catecholaminergic A11 population) on theborder of the diencephalon and mesencephalon, and the maintegmental midbrain populations (ventral tegmental area, sub-stantia nigra, A8 population). Limited activation associatedwith sexual behavior was also detected in more caudal, nondo-paminergic catecholamine populations. During male–maleaggression encounters, Fos induction was also present in cate-cholaminergic neurons within the ventral tegmental area andthe inferior raphe region in animals exhibiting intense aggres-sion (biting of opponent rather than simply stereotypeddisplay from a distance). The limited amount of Fos inductionin dopaminergic neurons of brown anole males engaged inaggressive encounters, coupled with the findings of DA releaseduring agonistic encounters or even a perceived social threat(Watt et al., 2007) in the closely related green anole, suggestcomplex regulation of DA release in social conditions; thesebrown anole males may have seen their opponents as notvery threatening (they were size-matched, with opponentsslightly smaller than focal males) and the involvement of DArelease in limbic regions during aggression seems to dependon one’s dominance status and recognition of opponent,with some situations eliciting DA release, while others do notdo so, or even inhibit DA release (Ling et al., 2010). Alternately,some catecholamine release may occur during aggressiveencounters without activation of the cell soma and accompa-nying Fos induction (e.g., local release of neurotransmittersfrom vesicles already docked at release sites of axons).

Patterns of monoamine metabolism in mountain spinylizards are similar to those in anoles, where higher serotonergicactivity is seen in nonterritorial satellite (subordinate) malesrelative to territorial males (Matter et al., 1998). As with anoles,aggressive defense of territory in males results in increases inboth 5-HTP and 5-HIAA/5-HT ratios. These interactions alsoincrease activity of the central DA and EPI systems. Plasmalevels of NE and EPI also rise rapidly during restraint stress orfollowing territorial interactions (Matt et al., 1997).

The rapid alterations in serotonergic metabolism duringaggressive interactions in male green anoles may be modulatedby alterations in circulating steroid hormones. In green anoles,5-HT turnover, estimated from the ratio of 5-HIAA, a metaboliteformed after 5-HT release, to 5HT was enhanced in the hippo-campus and medial amygdala 20 min after males received lowdose systemic injections of corticosterone (1.6–2.0 mg kg�1),but not following tenfold higher corticosterone doses(16–20 mg kg�1) (Summers et al., 2000). Testosterone injections(1.6–2.0 mg kg�1) enhanced 5-HT turnover in the hippocampus,but not the medial amygdala. There were no changes found inseveral other brain regions or in the activity of other monoamin-ergic systems. The authors note the possibility that the injected T




could have been converted before having this effect. The greenanole brain does show both aromatase and 5a-reductase activity(Wade, 1997) and at least aromatase activity is important insome behavioral contexts (Winkler and Wade, 1998; Lathamand Wade, 2010). Aggressive interactions influence plasmasteroid levels in green anoles and other male lizards (Greenbergand Crews, 1990; Knapp and Moore, 1995, 1996). Testosteronelevels are also typically elevated during the breeding season andinfluence aggression (Moore and Crews, 1986; Moore, 1986,1988), although plasma T levels do not necessarily changefollowing an aggressive encounter (e.g., Moore, 1987b). Thesteroid hormonemediation of serotonergic metabolism demon-strated in green anole males may be part of a mechanismenabling an individual to respond to changing social situations.Plasma corticosterone levels are elevated in animals exposed toa video of a dominant male compared to an animal showna nonsocial video (Yang and Wilczynski, 2003). Repeated expo-sure of these lizards to the video of a dominant male elicitsa decrease in aggressive behavior that is not observed in animalsadministered metyrapone (an inhibitor of corticosteronesynthesis). Consequently, it has been postulated that the increasein corticosterone levels after an aggressive encounter mightserve to habituate an animal’s subsequent response (decreasedaggression) by influencing memory of the social interaction.

Differences are also found with social status in female anoles(Summers et al., 1997). Females housed with males singly didnot differ from isolated females. However, there were differencesamong females housed in groups of five with a male. In contrastto results obtained with green anole males, dominant females inthis experiment showed higher 5-HT andDA activity in the telen-cephalon than did subordinate females, while subordinatefemales showed higher serotonergic activity in the brainstem.It was suggested that the heightened serotonergic activity indominant females is more directly related to interactions withmales than those with other females.

Woolley et al. (2001) also showed that a DA receptor agonistfacilitates the display of courtship and copulatory behaviors inboth castrated sexual (C. inornatus) and ovariectomized

parthenogenetic (C. uniparens) whiptail lizards. In both speciestheD1 agonist SKF 81297 increases the proportion of individualsmounting and decreases the latency tomount. Moreover, there isa difference in sensitivity to the agonist between the species:mounting is elicited at a lower dose inC. uniparens than inC. inor-natus. This suggests that, as is the case for sensitivity to exogenousestrogen (see above), the triploid parthenogen is more sensitive,indicating that the parthenogen may have elevated levels of D1receptor in those limbic brain areas modulating courtshipbehavior. Not only does this work extend to reptiles the centralrole ofDA in themodulation of copulatory behavior, it also indi-cates that DA can elicit male-typical mounting behavior fromboth a ‘male’ (C. inornatus) and a ‘female’ (C. uniparens) brain.Measurements of the size and number of tyrosine hydroxylaseimmunoreactive cells (TH-ir) across the reproductive cycle infemales of both species reveal that in the PvPOA the somal sizeof TH-ir cells is larger in C. uniparens (Woolley et al., 2001;Figure 26). Further, while there is no change in the number ofTH-ir cells across the reproductive cycle in the ancestral species,in the parthenogen there are fewer cells prior to ovulation,when the individual is exhibiting receptive behavior, comparedto the postovulatory phase, when individuals are displayingpseudocopulatory behavior (Woolley and Crews, 2004;Woolley et al., 2004a,b; Figure 26). In other unpublishedstudies, we have found that the origins of dopaminergicafferents to the behaviorally relevant parts of the mPOA aresimilar to that of mammals. Using a combination of tracttracing and tyrosine hydroxylase ICC, castrated C. inornatusreceived injections of testosterone and DiI into the mPOA,a procedure that reliably restores copulatory behavior.Catecholaminergic cells were labeled and the tracer visualized.A significant contribution to the dopaminergic innervation ofthe mPOA from cell bodies is situated close to the thirdventricle. Some of these cells project to the external amygdala asin rodents (Figure 27). Somal area is an important determinantof connectivity and related to the extent of the dendritic arbor(Szaro and Tompkins, 1987) and the size of the neuronalreceptive field (DeVries and Simerly, 2002). Increases in the size

Figure 26 Neuron size and abundance of tyrosine hydroxylase in the periventricular preoptic area of female whiptail lizards. Panel A: Tyrosinehydroxylase immunoreactive (Th-ir) cells in Cnemidophorus. uniparens (uni, black bars) are larger than those of female C. inornatus (ini, open bars),presumably reflecting ploidy. Panel B: However, there is a greater number of TH-ir cells in the PvPOA of the descendant parthenogenetic speciesduring the postovulatory ovarian stage (POSTOV), when male-like pseudocopulatory behavior is being exhibited, compared to the preovulatory stage(PREOV); females of the ancestral sexual species show no change in cell number. Mean � SEM shown. After Woolley, S.C., Lydon, J., O’Malley,B.W., Crews, D., 2006. Genotype differences in behavior and the number of catecholamine producing cells between wild-type and progesteronereceptor knockout mice. Behav. Brain Res. 167, 197–204.




or number of neurons can increase the amount of synaptic inputto a particular nucleus (Rakic, 1975; Szaro and Tompkins, 1987;Tompkins et al., 1984). Thus, species differences in both the sizeand number of cells in limbic and midbrain nuclei may havedramatic functional consequences for both neural organizationand behavior.

Serotonergic neurotransmission is involved in gating male-as well as female-like pseudosexual behavior expressed by theparthenogenetic whiptail C. uniparens. Increasing 5-HT levelsin T-implanted C. uniparens via systemic administration ofa combination of the monoamine oxidase inhibitor,clorgyline, and the precursor of 5-HT, 5-HTP suppresses male-like pseudocopulation (Dias and Crews, 2006; Figure 28). Incontrast, administration of pCPA (a tryptophan hydroxylaseinhibitor) decreases 5-HT levels in the POA as well asincreasing male-like mounting behavior.

Serotonin injected into the POA of ovariectomized, T-implanted lizards expressing male-like pseudocopulationsuppresses male-like pseudosexual behavior (Dias and Crews,2008). In a similar experiment, female-like receptivity exhibitedby ovariectomized, E2-injected C. uniparens was suppressed byinjection of 5HT into the VMH (Dias and Crews, 2008).Pharmacological targeting of specific 5-HT receptors indicatesthat the 5-HT1A and 5-HT2A receptors are involved in male-likepseudocopulation and female-like receptivity, respectively. Insitu hybridization used to measure 5-HT1A and 5-HT2A receptor

Third ventricle

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Figure 27 Tract tracing and tyrosine hydroxylase immunocytochemistry (TH-ir) in the medial preoptic area (mPOA) of male whiptail lizards. Left:Tracer combined with testosterone was implanted into the mPOA. TH-ir neuron in the MPOA labeled green, identifying the cells that may providedopamine involved in modulation of male-typical copulatory behavior. Right: Retrogradely labeled cell in external amygdala which projects to themPOA.

Figure 28 Relationship between steroid hormones, serotonin manipula-tion and male-like pseudosexual behavior in the unisexual whiptail lizard(Cnemidophorus uniparens). pCPA, a tryptophan hydroxylase inhibitor thatreduces serotonin biosynthesis, induces male-like mounting in ovariecto-mized lizards receiving a blank (Bl) or testosterone (T) treatment, but notovariectomized lizards receiving estrogen (E). Animals were tested afterreceiving four daily injections of saline, four daily injections of pCPA begin-ning 2 days after the Saline Behavioral tests, and 2 weeks (Recovery).These data suggest that E prevent the pCPA induction of mountingbehavior. Data from Dias, B.G., Crews, D., 2006. Serotonergic modulationof male-like pseudocopulatory behavior in the parthenogenetic whiptaillizard, Cnemidophorus uniparens. Horm. Behav. 50, 401–409.




mRNA levels indicates that T and E2 regulate mRNA levels ofthese receptor subtypes in the POA and VMH.

More recent studies have measured 5-HT and receptormRNA levels in naturally cycling animals (O’Connell andCrews, unpublished data). These results closely parallel thedata generated in the hormonally manipulated conditions. Itappears that the hormonal regulation of 5-HT and receptorsubtype mRNA levels at the POA and VMH, and subsequentsignaling via these receptors at these brain nuclei allows forthe reciprocal inhibition that characterizes behavior associatedwith either hormonal state (e.g., the facilitation of male-likepseudocopulation in androgen-implanted animals whilefemale-like receptivity is simultaneously suppressed).

2.10.4.3 Nitric Oxide

Nitric oxide is produced in neurons from arginine by theenzyme nitric oxide synthase (NOS) and plays a critical rolein both peripheral and central control of reproductivebehavior. As indicated above, the POA is important for medi-ating mounting behavior both inmale C. inornatus and pseudo-copulatory behavior in C. uniparens; lesions in this regionabolish mounting behavior in sexually active animals.Implants of DHT or P, but not E2, restore mounting behaviorin gonadectomized animals (demonstrating the involvementof AR and PR, but not estrogen receptor or ER) (Godwin andCrews, 1997, 2002). As in other vertebrates, this brain regionhas high densities of steroid hormone receptors (see Section2.10.3.1), receives inputs principally from chemical sensorysystems, and projects to structures involved in copulatorybehavior. The rostral PvPOA, the homolog to the mammaliananteroventral periventricular nucleus, or AVPv, contains abun-dant sex steroid-concentrating neurons and the greatest concen-tration of neuronal NOS mRNA (Crews et al., 2009; Sandersonet al., 2008). Inhibition of nNOS, the neuronal enzyme thatproduces nitric oxide, prevents androgen-induced mountingbehavior in both mammals and whiptail lizards (Sanderson

et al., 2005). Administration of T to both C. uniparens andfemale C. inornatus induces mounting behavior and enhancesNOS expression, as indicated by NADPH diaphorase histo-chemistry (a marker for nNOS) and citrulline immunoreac-tivity (a marker of recent nitric oxide production) (Sandersonet al., 2008; O’Connell et al., 2011a,b, 2012).

Progesterone activates mounting behavior in both P-sensitivemale C. inornatus and in the unisexual C. uniparens throughconvergence onto the NOS signaling system in the PvPOA(Figure 29). In this instance progesterone activates genes thatin males of the sexual species are normally dependent onandrogens. In mammalian systems AR and PR bind to andactivate a common canonical steroid response element (Catoet al., 1987; Nelson et al., 1999). In addition to AR and PR,Class I nuclear receptors include the glucocorticoid receptor(GR) and mineralocorticoid receptor (MR). Of these, only ARand PR, but not the GR and MR are able to induce luciferaseactivity via the selective sex-limited protein androgen responseelement (Denayer et al., 2010). In P-sensitive morphs thisspecificity of action is partly dependent on the cellularexpression of the receptors. Thus, small changes in theregulation of steroid hormone receptors are capable ofconferring on one hormone (e.g., PR) new functionspreviously restricted to the other (e.g., AR). The interaction ofAR and PR determines the regulation of nNOS within thePvPOA of animals that display mounting behavior amongboth P-sensitive and P-insensitive morphs of the ancestralbisexual species, and in the descendant parthenogenetic species.

This responsiveness to the two different steroids and involve-ment in copulatory behavior suggest a possible involvement inthe neural control of pseudocopulation in C. uniparens whichnormally display male-like pseudosexual behavior under theinfluence of P, but can be made to display the same behaviorwithT treatment. The role ofNO in this behaviorwas investigatedby treatment with the arginine analog L-NG nitro argininemethylester (L-NAME), which inhibits NOS. Using a repeated-measuresdesign, ovariectomized, hormone-treated lizards that were

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Figure 29 Model showing the possible interactions between steroid hormone receptors and neurotransmitter modulation of the preoptic area (POA)and ventromedial hypothalamus (VMH) in the unisexual whiptail lizard (Cnemidophorus uniparens). In both brain areas steroid hormones regulate therelease of neurotransmitters, which then change the level of excitation/inhibition in the respective areas in a reciprocal fashion to result in eithermale-typical or female-typical copulatory behavior.




displaying robust pseudocopulatory behavior were treated withan intraperitoneal injection of 600 mg L-NAME in physiologicalsaline 1 h before testing with a receptive stimulus animal, orwith the same dose of the inactive isomer D-NAME (Sandersonet al., 2005). Following L-NAME administration, latencies toapproach, mount, and pseudocopulate were increased, and halfof the individuals failed to pseudocopulate at all, while thebehavior in D-NAME-treated animals was similar to baseline.

Another candidate in addition to DA (see above) for thelocus at which P might engage the cellular pathway normallyactivated by androgens is NO. Since both neurotransmittersare involved in T-dependent male-typical copulatorybehavior, and independently in P-dependent female-typicalbehavior, ectopic expression of PR in the POA of the brainsof parthenogenetic whiptails might enable P to activate male-like pseudocopulatory behavior via one or both of thesemolecules. Pharmacological blockade of NOS suppressespseudocopulatory behavior in whiptails (Sanderson et al.,2005, 2006) as it does in rats. Preoptic NOS is upregulatedby T exposure sufficient to activate male-like copulatorybehavior in female whiptails (Sanderson et al., 2006) and inmales (Sanderson et al., 2008), and also at the time of theovarian cycle when male-like copulatory behavior isdisplayed. Furthermore, analysis of citrulline productionsuggests that nitric oxide is synthesized more in the POA ofwhiptails displaying T-dependent male-typical copulatory

behavior than in controls deprived of sex steroids (andtherefore not copulating). The molecular mechanisms ofNOS upregulation have been studied in some detail, usinglaser capture microdissection (Figure 30) and quantitativePCR (qPCR) to examine the time course of NOS inductionfollowing T administration to gonadectomized whiptails. Forexample, if castrated male whiptails implanted with T aretested at four time points following the display of sexualbehavior, nNOS transcript abundance and parallel NADPHdiaphorase histochemistry indicate that nNOS is upregulatedby T in a pattern consistent with a role in mediatinghormonal gating of copulatory behavior (Figure 31).

2.10.4.4 Steroidogenic Enzymes, Cofactors, and Receptors

Unlike in many mammals and birds, androgens seem to be themain regulators of male sexual behavior in lizards such as greenanoles (Crews et al., 1978; Winkler andWade, 1998). However,it has been documented that DHT is insufficient to fully restorecourtship behavior in castrated males (Crews et al., 1978;Rosen and Wade, 2000) and that aromatase activity is greaterin the brains of males than females during the breeding season,with males possessing more aromatase-expressing cells thanfemales in the POA; these findings suggest that some involve-ment of estrogens is still likely in the regulation of male sexualbehavior (Cohen and Wade, 2010, 2011). Latham and Wade

Figure 30 Localization and expression of neuronal nitric oxide synthase (nNOS) in response to testosterone. Top Panel: mRNA expression anddistribution of nNOS at the level of the periventricular preoptic area (PP) and medial POA (MPOA) in castrated Cnemidophrus inornatus. Left panel issection of blank-treated male. Right panel is section from a testosterone-treated male. Bottom Panel: Laser capture microdissection (LCM) of PP.Alternate sections were used for in situ hybridization and for LCM. Redrawn from Sanderson, N.S.R., Le, B.D., Zhou, Z., Crews, D., 2008. Preopticneuronal nitric oxide synthase induction by testosterone is consistent with a role in gating male copulatory behaviour. Eur. J. Neurosci. 27, 183–190.




conducted an experiment where male green anoles were cas-trated and given combinations of T and/or E2, and concludedthat while androgens may increase courtship displays andcopulations, estrogens seem to play a role in increasing mountattempts (Latham and Wade, 2010). Interestingly, in this andother green anole studies, steroidal manipulations exert muchgreater effects in the breeding than nonbreeding season (e.g.,Neal and Wade, 2007a,b). Hence, a series of studies hasrecently been conducted in the Wade lab to determine whatvariables facilitate the salience of these hormonal signals inthe breeding season. These investigators have looked forchanges in aromatase, 5a-reductase, AR, ER, steroid receptorcoactivator-1, and cAMP response element-binding protein(CREB) that may underlie the seasonal change in steroidhormone salience, rather than being themselves regulated todifferent levels by seasonal fluctuations in hormone levels(Beck and Wade, 2009c; Cohen and Wade, 2012; Kerver andWade, 2013, 2014, 2015). However, none of the variablesexamined to date seem to consistently vary in the POA acrossseasons, at standardized hormone levels, thus suggesting thatother, still unknown, variables may underlie the seasonalchange in male responsiveness to sex steroid hormones.

2.10.5 Regulation of Sex Steroid HormoneReceptors

A next step typically would be to study the receptor proteins.However, cross-reactivity of the antibodies available at the timemade this impossible and the focus changed to cloning andsequencing reptilian steroidhormone receptor genes. In sodoing,advancement in understanding hormone receptor gene expres-sion in reptiles co-occurred with advances in rodents. While it isimportant to note that mRNA abundances do not necessarilyreflect concentration of the corresponding protein, the ability tolocalize and compare relative abundance levels of the threemain gonadal steroid hormone receptors in whiptail lizards hasallowed a variety of questions related to sex and species differ-ences in their regulation to be addressed. Recent methods inmolecular techniques obviate this problem altogether.

Figure 31 The effect of androgen on behavior, neurochemistry andgene expression in the male whiptail lizard (Cnemidophorus inornatus).Panel A: Percentage of castrated males exhibiting male-typical

copulatory behavior after various lengths of time following implantationof testosterone-filled (gray bars) or blank (empty bars) Silasticcapsules. Panel B: Expression of nNOS in the periventricular preopticarea (PPA) as revealed by in situ hybridization. Presented are thenumbers of nNOS-expressing cells inferred from silver grain clusters inthe PPA. For comparison, the 95% confidence interval around theglobal mean of the blank treated animals is shown as a light gray band.Panel C: Relative nNOS transcript abundance in laser-microdissectedfragments of PPA. Raw measures of nNOS transcript abundance areexpressed relative to 18S ribosomal RNA abundance and normalized tothe mean value of the blank-implanted individuals. Panel D: Expressionof nitric oxide synthase as inferred from NADPH diaphorasehistochemistry. Presented are numbers of NADPHd þ cells in the PPA.For comparison, the 95% confidence interval around the global mean ofthe blank treated animals is shown as a light gray band. Numbers inparentheses above bars are group sizes. Modified from Sanderson,N.S.R., Le, B.D., Zhou, Z., Crews, D., 2008. Preoptic neuronal nitricoxide synthase induction by testosterone is consistent with a role ingating male copulatory behaviour. Eur. J. Neurosci. 27, 183–190.

=




Estradiol increases ER mRNA abundance in discrete brainregions in the whiptail lizards. Young and coworkers docu-mented this using a 0.5 mg injection of estradiol benzoate(EB) and measuring ER mRNA abundance 24 h after adminis-tration. The EB effectively stimulates female-typical receptivebehavior in parthenogenetic whiptail lizards (Young et al.,1995a) as well as increases ER mRNA in some regions (torussemicircularis, VMH), decreases it in others (LS), and causesno change in still other nuclei (periventricular nuclei of thehypothalamus, periventricular nucleus of the POA, andthe dorsal hypothalamus). The increase seen in ER mRNA inthe VMH is particularly interesting for two reasons. First, aspreviously mentioned, this nucleus critically regulatesfemale-typical sexual behavior in both the sexual andunisexual parthenogenetic whiptail lizards. Second, thepattern of increased ER mRNA in the mediobasal hypothal-amus is opposite that seen in rats where estrogen downregu-lates its receptor. This difference between whiptail lizardsand rats may relate to differences in the nature of their ovariancycles (Young and Crews, 1995). Whiptail lizards haveelevated E2 levels for a relatively long period prior to ovula-tion and display receptive behavior for the duration of thisperiod while female rats are receptive for only a short windowfollowing ovulation. Young and Crews (1995) suggest thatprolongation of the length of time E2 levels are elevatedand of sexual receptivity may be quite common in mammals(e.g., cats and rabbits). Lastly, species comparisons indicatethat parthenogenetic whiptails have higher concentrationsof ER mRNA expression in the POA than do sexually repro-ducing female whiptails (Young et al., 1995b). This observa-tion led in turn to the sensitivity compensation hypothesis.That is, an inverse relationship exists between expression ofthe genes coding for sex steroid hormone receptors in thePOA and circulating concentrations of sex steroid hormone.The increased level of ER gene expression in the POA resultsin a greater sensitivity to the circulating concentrations of E2that, in turn, results in lower levels of circulating E2 throughfeedback effects.

Estradiol also stimulates increases in PR mRNA abundancein lizard brains, but again typically in a manner specific tospecies, sex, and region. Female green anoles show increasesin progestin-binding sites with estrogen treatment (Tokarzet al., 1981) as well as induction of sexual receptivity (Tokarzand Crews, 1980). Estradiol benzoate treatment stronglyinduces PR mRNA in the VMH of whiptail lizard females.The degree of this induction is tightly correlated with thedisplay of female-typical receptive behavior in C. inornatusand female-like pseudosexual behavior in the parthenoge-netic C. uniparens (Young et al., 1995b), with EB beingmore effective in the parthenogenetic C. uniparens (Figure 32).Estradiol benzoate also effectively stimulates increases in PRmRNA in the POA of female C. inornatus, again withsimilar dosages being more effective in the parthenogenC. uniparens than in females of the sexual ancestorC. inornatus (Figure 33; Godwin and Crews, 1999). Thisgreater estrogen stimulation of PR mRNA in the brain regionmediating male-like pseudosexual behavior in C. uniparensmay be related to the display of male-like pseudosexualbehavior by C. uniparens, but not by C. inornatus females(Godwin et al., 1996).

While E2 increases PR mRNA in both the VMH and POA offemale and parthenogenetic whiptail lizards, exogenous Pinhibits both female-typical receptive behavior and decreasesestrogen-stimulated ER and PR mRNA in the VMH (Godwin

Figure 32 Species differences in neuroendocrine controlling mecha-nism. Species differences in the induction of sexual receptivity (thinlines) and progesterone receptor mRNA expression (thick lines) byestradiol benzoate (EB) in ovariectomized whiptail lizards. Ovariecto-mized animals were given a single injection of EB and either tested dailyfor receptivity for 4 days following the injection or brains were removed24 h after treatment and analyzed using in situ hybridization. Verticalerror bars represent standard errors of the mean.

Figure 33 Evolution of a novel neuroendocrine controlling mecha-nism. Progesterone receptor mRNA levels in the PvPOA for femaleC. inornatus (open bars) and C. uniparens (black bars) given either blankor estradiol injections. Depicted is the abundance of progesteronereceptor mRNA measured as average number of silver grains percluster (mean � SEM) in the periventricular region of the preoptic areaof the ancestral sexual (C. inornatus) and descendant parthenogenetic(C. uniparens) whiptail lizards. From Godwin, J., Crews, D., 1999.Hormonal regulation of progesterone receptor mRNA expression in thehypothalamus of whiptail lizards: regional and species differences. J.Neurobiol. 39, 287–293.




et al., 1996). This effect of P on both receptivity and ER and PRmRNA abundance is similar to patterns in well-studied rodentmodels. In contrast, exogenous P has no effect on PR mRNAabundance in the periventricular POA in this experiment.

Neither the effective induction of female-typical receptivebehavior or increases in ER- and PR-mRNA in the VMH seenin female and parthenogenetic whiptail lizards occur in short-term castrate males (1 week) (Godwin and Crews, 1995).This lack of responsiveness to estrogen in the VMH of malewhiptail lizards parallels patterns in rats. In contrast, maleC. inornatus castrated for longer periods (6 weeks) showed PRmRNA responses to estrogen that were not different fromfemales (Wennstrom and Crews, 1998). Females implantedwith T, however, did not show an attenuation of the femalepattern of responsiveness. These results indicate that mainte-nance of the male-typical pattern of nonresponsivenessrequires the activational effects of T while the female-typicalpattern is less plastic.

The abundanceofPRmRNA is also correlated to the displayofmale-typical sexual behavior in male C. inornatus (Crews et al.,1996b). Male C. inornatus can be classified as either P-sensitiveor P-insensitive based on the effectiveness of exogenous Pdelivered in Silastic capsules implanted intraperitoneally inreinstating sexual behavior following castration (Lindzey andCrews, 1992). Males classified as P-sensitive are alsosignificantly more likely to respond to intracranial implants ofP (directed at the POA) than P-insensitive males (Crews et al.,1996b). Interestingly, there are also differences in both PR- andAR mRNA abundance between the two groups followingintracranial implantation of P. Progesterone-sensitive malesdisplay lower abundances of PR mRNA in both the medial andperiventricular portions of the POA, but higher abundances ofAR mRNA in the medial POA, external amygdala, and LS. Nodifferences are seen between P-sensitive and P-insensitive maleswithout an intracranial implant.

Sex and species differences are also found in androgenicregulation of ER-, PR- and AR mRNA. Implantation of gonad-ectomized male and female C. inornatus and parthenogenfemale C. uniparens with either T or DHT reveal a diversityof effects, suggesting that gonadal sex, aromatization, andgene dosage (ploidy) all influence steroid receptor mRNAresponse (Godwin et al., 2000) (see also Young et al.,1995a for PR mRNA). For example, males have higher ARmRNA in the medial POA than females of either speciesand these levels decrease with T treatment in males, but notin females. In contrast, ER and PR mRNA levels in the VMHare higher with androgen treatment, but these effects do notdiffer by sex. There also are species effects in that the triploidparthenogen shows higher steroid receptor mRNA abun-dances overall than the diploid sexual females. Finally,aromatization of T to estrogen is likely important in someregions. Progesterone receptor mRNA in the periventricularPOA is increased in both males and females by T, but notby nonaromatizable DHT.

Lastly, individual experiences might influence gene expres-sion in the brain directly rather than via modulation of theendocrine physiology of the partner. For example, in thehamster and the rat, exposure to sexual behavior of the oppo-site sex induces expression of the immediate-early gene c-fosin those brain regions that mediate sexual behavior. Using

ovariectomized hormone-primed parthenogenetic whiptaillizards, Hartman and Crews (1996) demonstrated that partici-pating either as a male or as a female during a pseudosexualencounter significantly alters the abundance of ER- and PRmRNA in the hypothalamus of whiptail lizards.

In contrast to the conservation of steroid receptor distribu-tion in the brain in reptiles and other vertebrates, patterns ofsteroid receptor regulation vary greatly. This regulation showsvariation across brain nuclei, both within and between thesexes, between closely related species, and with social interac-tions. Some of the patterns found are strikingly similar tothose seen in well-studied rodent models, but there are alsodifferences that appear to be related to differences in thenature of reproductive cycles. Most of the studies examiningsteroid receptor regulation have either shown behavioraleffects or used behaviorally relevant dosages of hormone, sup-porting a role for this regulation in behavioral display.Remaining challenges in this area include determining thedegree of colocalization of receptor types within neuronsand crosstalk between signaling systems, exploring the influ-ences of other mediators (e.g., corticosteroids, thyroidhormones, neurotransmitters) on receptor regulation, andcharacterizing the downstream effects of steroid receptoractivation.

2.10.6 Conclusions and Future Directions

Reptiles enable study of the neuroendocrine mechanismsunderlying of sociosexual behaviors in ways not possiblewith conventional animal model systems. This work has hadtwo important impacts on our understanding of sociosexualbehavior. First, it has revealed that great diversity exists amongvertebrates in reproductive behaviors and the neuroendocrinemechanisms underlying these behaviors. In this manner, studyof species with dissociated reproductive tactics (including non-reptilian species such as birds and mammals) and unisexualspecies suggests three factors which may explain species differ-ences in endocrine physiology and behavior: (1) sensitivity tosex steroid hormones, (2) hormone-dependent regulation ofsex steroid hormone receptor gene expression, and (3) neuro-anatomical distribution of steroid receptor gene expression,especially in nonlimbic structures.

The second major impact arises from explorations of thisdiversity within and across major taxa. These explorationsallow us to begin defining which mechanisms show strongconservation and which are evolutionarily more labile. Reptilesand mammals diverged approximately 350 million years ago,yet research in reptiles has revealed apparent conservation ofmany behavioral controlling mechanisms between thesegroups. For example, research with reptiles has led us to re-examine certain assumptions in behavioral neuroendocri-nology. One such example concerns the idea that P isa ‘female-specific’ hormone with no function in males. Experi-ments with four lizard species have demonstrated that P is vitalto the display of male copulatory behavior in lizards and,further, that androgen and P synergize in males, much likeE2 and P synergize in females to facilitate sexual receptivity;subsequent studies with mice and rats have revealed similarroles for P and its receptor in male sexual behavior in mammals




(Phelps et al., 1998; Witt et al., 1994, 1995). Continuing toidentify those mechanisms that are fundamentally importantin all vertebrates and those that represent axes along whichevolutionary change may take place will lead to a morecomplete understanding of the diversity we see and how thisdiversity arose.

Research in reptiles has also contributed and continues tocontribute to our understanding of animal sexuality and thenature of individual variation. For example, study of animalsthat lack sex-linked sex-determining genes has reinforced theconclusion that the same genes are involved in the develop-ment of testes (males) and ovaries (females) and are containedin each individual. That is, the species may differ in theirpatterns of regulation, but the genes associated with sex deter-mination are conserved. What differs is the trigger; in some it issex chromosomes at fertilization, in others it is environmentalfactors during embryogenesis, and in still others it is the socialcontext in which the animal finds itself. This understanding ischanging the classic paradigm idea of an ‘organized’ anda ‘default’ sex; rather, we now regard both sexes as organizedand the question now becomes why the activation of onecascade (e.g., the ovary-determining cascade) activelysuppresses the complementary sex-determining cascade. Thisunderstanding, and the obvious fact that many reptiles lacksex chromosomes, requires that the construction of a new para-digm take the place of the organized-default concept as gener-alizing this canonical concept to all vertebrates appearsdebatable. Species with environmental sex-determining mech-anisms are a case in point (Crews, 1993). In those with TSD,gonadal sex is determined by incubation temperature duringthe middle of development. In hermaphroditic species suchas sex-changing fish, sex-change occurs in the adult as a resultof changes in the social environment.

What can replace the heterogamety hypothesis and stillaccount for the evidence at hand? There can be little doubtthat the original vertebrate was a ‘female’ and that malesevolved only after the evolution of self-replicating (¼ female)organisms. Males have been gained (or lost), but femalesremain. Considering the female as the fundamental sex andthe male as the derived sex would account for the above obser-vations. This framework suggests the intriguing possibility thatmales may be more like females than females are like males. Inrodents the relative ease of masculinizing individualscompared to the difficulty in defeminizing individuals suggeststhis to be the case.

An example of the evolution of genetic control of sexualbehavior comes from studies of parthenogenetic, or all-female, whiptail lizards. These unique animals arose from thehybridization of sexual reproducing species and sex chromo-somes appear to exist in the ancestral sexual species withmale heterogametic (XY). The expected sexual dimorphismsare present in morphology, physiology, brain anatomy, andbehavior, all of which are under testicular hormone control.In the descendant unisexual species, however, no males existand all individuals have a female phenotype. Remarkably,these parthenogens reliably and regularly exhibit both male-like and female-like pseudosexual behaviors during the courseof their reproductive cycle. Although males do not exist, thegene(s) for male development have not been lost but, instead,appear to be repressed. Although the genetic (Y) trigger for

male development is absent, the male-determining cascadecan be activated by treating embryos with aromatase inhibitor,producing fully functional males (Wennstrom and Crews,1995; Wibbels and Crews, 1994; Hillsman et al., 2007). Suchanimals exhibit only male-like copulatory behavior. However,their brain anatomy remains similar to that of normal parthe-nogens who, despite the bisexual nature of their behavior, havestrictly female-like brain morphology. Thus, the expression of Ychromosome gene products appears in whiptail lizards notonly to influence brain anatomy but also to suppress thedisplay of female-like behavior and sensitivity to exogenousestrogen.

Many challenges remain in the study of hormones, brain,and behavior in reptiles. Nearly all the information availableregarding the hormonal and neural bases of behavior in reptilescomes from studies of lizards and snakes. While this givesinsight into these mechanisms in this the most speciose groupof reptiles, little is still known about hormone�brain�behav-ior relationships in the other major lineages of reptiles, theturtles and crocodilians. Modern birds represent the mostderived forms in the archosauromorph lineage with crocodil-ians being the most primitive and the extinct dinosaurs fallingin between. Our understanding of behavioral mechanisms inbirds would benefit from a more thorough understanding ofthese mechanisms in primitive members of the lineage, thecrocodilians.

The lack of correspondence between structure of thenervous system and behavioral phenotype highlights theneed for more comparisons of a functional nature. Insightsfrommeasurements of neural activity, neurotransmitter metab-olism and influences, and the regulation and actions of steroidhormone receptors all show the value of these approaches. Notonly are such investigations important to further our under-standing of species and sex differences but also of individualdifferences. The mechanisms that generate individual variationare an important focus across modern biology in general.Understanding these mechanisms is of fundamental impor-tance for understanding a broad range of phenomena, fromthe very basic question of how evolutionary change takes place,to the very applied problems in human health.

The diversity of patterns in sex determination and differen-tiation seen in reptiles has provided important evidence thatfactors other than gonadal steroid hormones can have criticalinfluences on the differentiation of the neural substrates ofbehavior. Elucidating these influences and the interplay offactors such as temperature and social interactions withgonadal steroids in shaping the function of the adult nervoussystem is an important research direction.

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edition hormones, brain, and behavior, 3rd author's...

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