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date: 30 November 2022

An Overview of Sexual Differentiation of the Mammalian Nervous System and Behaviorfree

An Overview of Sexual Differentiation of the Mammalian Nervous System and Behaviorfree

  • Ashley MonksAshley MonksUniversity of Toronto

Summary

There is growing appreciation for the numerous and often dramatic differences in the nervous system of males and females and the importance of these sex differences for behavioral traits. Sex differences in the nervous system and behavior result from a process of sexual differentiation that is carried out by the interplay of genetic, hormonal, and environmental factors throughout the life span. Although the preponderance of mechanistic study of mammalian sexual differentiation has occurred in traditional laboratory rodents, this field of study has benefitted from comparative studies, which highlight the diversity in sexual polymorphism in vertebrates and also point to strongly conserved mechanisms whereby these sexually differentiated traits develop.

Subjects

  • Neuroendocrine and Autonomic Systems

Introduction

There is currently great interest in sex and gender issues in biomedicine in the context of personalized medicine and more broadly among the public, in which issues surrounding diversity and social justice are hotly debated. However, this area of research is fraught with controversy and cultural barriers. Cultural barriers manifest as challenges both to the legitimacy of this type of inquiry and, relatedly, also to cultural beliefs concerning gender. Only recently has there been widespread appreciation among neuroscientists that male bias in the literature is problematic. Good theories must account for sex and relying on studies performed only for one sex (usually males) leads to problems with generalizability and knowledge translation (e.g., Cahill, 2006; Klein et al., 2015). As such, understanding sex differences, how to evaluate them, and how to develop hypotheses concerning their cause have become essential skills for neuroscientists.

Although one might envision a long-term scientific goal of accounting for all variability related to sex and gender, this article focuses on sex differences in the nervous system and behavior (referred hereafter collectively as “neurobehavioral traits”) and the developmental mechanisms that cause them. The article considers separately the main identified factors mediating mammalian sexual differentiation (i.e., hormones, genetics, and environment). The interrelation between these three factors is complex and to some extent species specific, but a general hierarchical model can be successfully applied to vertebrates, as depicted in Figure 1. In this model, the central dogma holds that genetics (via sex chromosomes or environmental trigger) initiate sexual differentiation of the gonads into ovaries or testes. Sexually differentiated gonads then produce sexually differentiated hormones, which in turn initiate sexual differentiation of many aspects of phenotype, including neurobehavioral traits. The complexity of interactions becomes apparent when one considers that, for example, hormonal mechanisms of sexual differentiation occur mainly by altering gene expression, or that environmental cues can regulate hormone production. As a further complication, the interplay of these factors does not always generalize across species. One must also recognize the limitations of existing animal models, which are notably constrained in two of these three factors, having artificially limited genetic and environmental variability.

Figure 1. General hierarchical model of vertebrate sexual differentiation. In this model, genetic action either due to sex chromosomes or environmental conditions initiates sexual differentiation of the gonads into ovaries or testes. Sexually differentiated gonads then produce sexually differentiated hormones, which in turn initiate sexual differentiation of many aspects of phenotype, including the gonads themselves, as well as neurobehavioral traits. In addition to this canonical hierarchy, the model also includes direct genetic influence on sexually differentiated phenotypes other than the gonads, gonadal contributions to sexually differentiated phenotype other than hormonal sex differences, environmental effects on hormones and genetics, and sexually differentiated contributions of the individual to the environment.

An appreciation for variability in the degree of sexual differentiation between and within species is not only of fundamental interest but is also of great applied value when attempting to translate studies of nonhuman animal models to humans, which exhibit much more diversity in sexually differentiated traits and in which the degree of sexual differentiation is often less. A related point is the distinction between sexual dimorphism and sex difference, which essentially differ in the extent of the observed sex difference. Sexual dimorphism requires statistically discontinuity of traits, whereas sex difference is appropriate in describing traits that differ on average but overlap between sexes. When considering neurobehavioral traits, both terms have been used, but sexual dimorphism is relatively rare, especially when a strict definition is applied (e.g., Ball et al., 2014). Nonetheless, whereas sexual equivalence in neural traits was once assumed, and evidence of sexual dimorphism, let alone sex difference in neuroanatomy was surprising (e.g., Gorski et al., 1980), it is increasingly apparent that sex differences in the nervous system and behavior are found ubiquitously. It is fair to say that even in the most intensively studied organisms, there is incomplete knowledge of the range and scope of sexually differentiated traits (due largely to the lack of studies adequately testing for these differences), and often only speculative theories exist concerning the function or cause of observed sexual differences, some of which are defined and presented in Table 1.

Table 1. Processes of Sexual Differentiation

Male-typical (masculine) outcome

Female-typical (feminine) outcome

Gain of trait

Masculinization

Feminization

Example

Organizational testosterone

Activational estradiol

↓ apoptosis of MPOA neurons

↑ dendritic spines in Hpc

M > F number of neurons

F > M density of spines

Gorski et al. (1980), Davis et al. (1996)

Woolley et al. (1990)

Loss of trait

Defeminization

Demasculinization

Example

Organizational testosterone

Organizational estradiol

↑ apoptosis of AVPV neurons

↓ mounting

M < F number of neurons

F < M mounting of females

Forger et al. (2004)

Brock et al. (2011)

Sexually dimorphic traits may come about by diverse processes of differentiation. These processes may involve gain of traits and/or loss of traits. Although hormonal effects are the focus of this table, these principles may be equally applied to genetic or environmental effects on neurobehavioral traits. In addition to gain and loss, another factor of sex-typicality (masculine or feminine) is used to categorize processes of sexual differentiation. According to this system of categorization, gain of masculine traits is referred to as masculinization, and gain of feminine traits as feminization. Conversely, loss of masculine traits is referred to as demasculinization, and loss of feminine traits as defeminization. Generally speaking, sexual differentiation therefore involves processes of masculinization and defeminization in males whereas in females it involves processes of demasculinization and feminization.

With that caveat in mind, there has been considerable progress made in the understanding of mechanisms of sexual differentiation of mammals, with some theoretical frameworks that have survived decades of empirical scrutiny. Fortunately, one may also safely make some assumptions when studying humans and traditional laboratory animals that simplify and facilitate mechanistic study of sexual differentiation. These assumptions include having two sexes, genetic initiation of sexual differentiation, a prominent role for gonadal hormones (and especially gonadal steroid hormones) in mediating sexual differentiation, and stability in sexual morphs. It is interesting to note that there are exceptional species that challenge these assumptions, especially in other classes of vertebrates (i.e., birds, fish, and reptiles), which are also intensively studied in relation to sexual differentiation of neurobehavioral traits.

Sexual Differentiation as a Developmental Process

Sexual differentiation is often conceptualized as a developmental process in which traits become more or less male-typical (masculine) and more or less female-typical (feminine). This is a useful framework, but it is important at the outset to avoid several tempting but false assumptions that can lead to an underestimation of diversity in sexual differentiation. The first false assumption is that there is a single developmental period during which sex differences originate. Although there is generally speaking a prominent early developmental period where many traits are sexually differentiated, sexual differentiation occurs during multiple developmental periods throughout the life span, and these periods vary by trait. The second false assumption is that there is little variability within the sexes, whereas considerable variability is generally observed. The third false assumption is that all sexually differentiated traits are necessarily linked such that individuals are globally more feminine or more masculine, whereas dissociation of sex typicality of traits is observed. Understanding diversity in sexual differentiation is therefore facilitated by conceptualizing it not as a single process, but rather as multiple processes across the life span, which vary to some extent dissociably between traits. Related to this, although the three main factors that guide this process (genetics, hormones, and environment) are all essential to sexual differentiation, their relative influence, timing, and mechanism of action vary considerably from one trait to another, in addition to intra-individual and intraspecies variability.

By definition, each sexually dimorphic trait has a form that is male-typical (masculine) and female-typical (feminine), and for traits in which sex differences are observed, this is also generally assumed. These traits may come to be through processes in which elements of the trait are gained or lost. Accordingly, sexual differentiation is conceptualized as four types of process: (a) feminization, in which elements of female-typical traits are gained; (b) masculinization, in which elements of male-typical traits are gained; (c) defeminization, in which elements of female-typical traits are lost; and (d) demasculinization, in which elements of male-typical traits are lost. In general terms, therefore, male-typical sexual differentiation emerges through processes of masculinization and defeminization and female-typical sexual differentiation emerges through processes of feminization and demasculinization (the scheme and representative processes are presented in Table 1). There are many sexually differentiated neuroanatomical traits, and without knowledge of the processes that led to the difference, it is not possible to conclude whether sex-typical forms are caused by loss of traits or gain of traits. For example, a greater number of neurons in a brain nucleus of females might be caused by increased neurogenesis in females (feminization) or increased apoptosis in males (defeminization). Nonetheless, some neuroanatomical sex differences have been characterized sufficiently that one may apply this framework to categorize the developmental processes at work (several of these are presented in Table 2).

Table 2. List of Sex Differences in Neuroanatomical Traits Discussed in This Article

Name

Description

Reference

Neuroanatomy

AVPV

F > M Overall cell number,

Simerly et al. (1985)

Anteroventral periventricular nucleus

tyrosine hydroxylase immunoreactive neurons

BNSTp

M > F in cell number,

De Vries et al. (1986)

Principal nucleus of the Bed nucleus of the stria terminalis

volume, number of arginine vasopressin neurons

Wang et al. (1993)

CALB-SDN

M > F in number of calbindin-

Edelmann et al. (2007)

sexually dimorphic region of the preoptic area of mice

immunoreactive neurons

MEPD

M > F in volume, cell size, astrocyte

Cooke et al. (1999)

Medial amygdala posterior Division

number

Pfau et al. (2016)

SDN-POA

M > F in volume, cell size, cell number

Gorski et al. (1980)

Sexually dimorphic nucleus of the preoptic area of the rat

SNB

M > F on many anatomical features

Breedlove & Arnold (1980)

Spinal nucleus of the Bulbocavernosus

VMHvl

F > M in number of estrogen receptor

Brown et al. (1988)

Ventrolateral division of the ventromedial hypothalamus

alpha-immunoreactive neurons

Many neuroanatomical sex differences are caused by apoptosis in early life. The importance of apoptosis in sexual differentiation of neuroanatomy is illustrated by studies of loss of Bax function mutant mice, which do not undergo apoptotic programmed cell death in the nervous system. Bax knockout (BaxKO) mice do not exhibit many of the well-characterized neuroanatomical sex differences (Forger et al., 2004; Jacob et al., 2005), including the spinal nucleus of the bulbocavernosus (SNB), principal nucleus of the bed nucleus of the stria terminalis (BNSTp), and anteroventral periventricular nucleus (AVPV). Interestingly, elimination of apoptosis prevents both sex differences favoring males (SNB and BNSTp), and favoring females (AVPV). Absence of neural sex differences in BaxKO mice is also associated with absence of sex differences in olfactory preference and sociability, but not in territorial aggression (Holmes et al., 2011). Notably, neural sex differences that are defined based on phenotypic markers are unaffected by BaxKO, including the number of calbindin immunoreactive neurons in the preoptic area (CALB-SDN), number of arginine vasopressin-immunoreactive neurons in the BNSTp, and number of tyrosine hydroxlase-immunoreactive neurons in the AVPV (see Forger, 2009, for review). These sexually differentiated traits may be caused by epigenetic modifications resulting from testosterone action in the hypothalamus (e.g., Ghahramani et al., 2014; McCarthy et al., 2009).

In addition to apoptosis and epigenetic modification, sexually differentiated neuroanatomical traits may come about by diverse cellular mechanisms, including cell proliferation, process outgrowth, and induction of gene expression. For example, pubertal cell proliferation contributes to volumetric sex differences in several brain structures, including the AVPV, sexually dimorphic nucleus of the preoptic area (SDN-POA), and medial amygdala posterior division (MEPD) (Ahmed et al., 2008). Another pubertal example of gain of features is estrogenic stimulation of SNB motoneuron dendrite outgrowth, which presumably relies on target-derived trophic factors (Nowacek & Sengelaub, 2006).

Behavioral traits can also be sexually dimorphic, as exemplified by copulatory behavior. Copulatory behaviors in many animals differ greatly between males and females, and in laboratory rodents, these have often been operationalized as the masculine pattern of mounting and the feminine pattern of receiving the mount through lordosis (arching of the back and associated tail deflection). Although these copulatory behaviors are not exclusive to one sex, their expression is statistically discontinuous between males and females. When considering behavioral traits, it is much more difficult to infer by what mechanism sexually dimorphic behaviors emerge. Nonetheless, the study of sexual differentiation of behavior preceded the study of sexual differentiation of neuroanatomy. The most influential pioneering study of sexual differentiation of behavior of mammals was Phoenix et al.’s (1959) investigation into the importance of both early testosterone and acute gonadal steroid hormones for sex-typical sexual behavior in guinea pigs. In brief, the study concluded that sex typicality of sexual responses requires both sex-typical early testosterone as well as acute sex-typical gonadal hormones (testosterone or estradiol). This study provided a theoretical framework that remains influential, coining the terms “organizational” to refer to early, permanent actions of hormones, and “activational” to refer to acute, reversable actions of hormones. The study was also influential in proposing that these behavioral effects of hormones were mediated via actions on the nervous system.

The organizational, or activational, framework and the central hypothesis that hormones affect behavior primarily via actions on the nervous system have been mainstays of the larger field of behavioral neuroendocrinology and not only drove initial inquiry into sexual differentiation of the nervous system but continue to do so, including applying the framework to other causal factors in sexual differentiation (e.g., genetics, see Arnold, 2009). Inherent to this framework is the concept of critical or sensitive periods (this distinction is reviewed in McCarthy et al., 2018), and of associated fixed developmental timing and permanence of developmental outcomes. In this case, these periods are defined primarily in relation to developmental response to gonadal steroid hormone exposure.

When considering neurobehavioral traits, organization by gonadal testosterone mainly occurs early in development. In traditional laboratory rodents, this corresponds to a perinatal period in which testosterone can produce masculine outcomes (e.g., promoting mounting during copulation, and attenuation of apoptosis in the sexually dimorphic nucleus of the preoptic area) and prevents feminine outcomes (e.g., inhibiting lordosis during copulation, and increased apoptosis in the AVPV nucleus). This critical period reflects normal timing of transiently increased testosterone production by testes of developing males, which in mice and rats begins a few days prenatally and continues into the first postnatal day. It is important to note that in less altricial mammals (e.g., guinea pigs and humans), this developmentally equivalent period is prenatal.

Testosterone powerfully organizes sexually differentiated neurobehavioral traits early in development and until recently was thought to subsequently have only activational action in sexual differentiation, as there was little empirical support for other temporally limited and relatively permanent effects of testosterone on sexually differentiated neurobehavioral traits. However, it has become clear that the pubertal gonadal hormone environment can have organizing effects on neuroanatomy, as may be seen in pubertal organization of dendritic length of SNB motoneurons (reviewed in Sengelaub & Forger, 2008) and socio-sexual behavior (e.g., Brock & Bakker, 2011; Brock et al., 2011; Schulz et al., 2004). Sexual dimorphism in the nervous system in these cases behaves mainly in a manner analogous to that of the more obvious external anatomic sexual dimorphisms in mammalian genitalia, in which development results from essentially irreversible hormone actions early in development and during puberty. When considering both genital and neural cases, it is interesting to note that early and pubertal critical periods share the characteristic of apparent permanence but are dissimilar in the bounds of the critical period, which appear more flexible in the pubertal case, reflecting greater variance in pubertal onset between individuals. An extreme example of the variability in pubertal onset is provided by naked mole-rats (H. glaber), which appear to have the potential for indefinite pubertal delay when living in colony (reviewed in Holmes et al., 2009; Swift-Gallant et al., 2015).

As has been shown, sexual differentiation of many neurobehavioral traits occurs in a short time span and is orchestrated in large part by a single endocrine signal, testosterone. These pleotropic actions of testosterone occurring in a finite critical period are sufficiently explanatory that they can lead to the assumptions of common developmental dynamics and molecular mechanisms shared by all sexually differentiated traits. However, neither assumption is warranted in light of the demonstration of organization of neurobehavioral traits pubertally and in the existence of multiple signaling pathways for testosterone. As discussed next in the “Hormones” section, testosterone action in producing masculine outcomes is mediated to a variable extent by androgenic and estrogenic mechanisms.

Although one must adopt a more complex model to account for multiple mechanisms for sexual differentiation, even when accounting only for testosterone action, this more complex model allows us to begin to explain intrasexual diversity in sexually differentiated traits. That is, although it may be tempting to hypothesize that, for example, variation in the amount of testosterone produced on the day of birth will lead to corresponding global variation in masculine or feminine traits, sexual differentiation of traits is at least to some extent dissociable. Individuals may be masculine in one trait but feminine in another, for example. Instead, one might account for intrasexual variation in sexual differentiation by considering not only the amount of testosterone production but also the developmental profile of its production, together with individual differences in efficacy in response to the several mechanisms whereby testosterone produces sexual differentiation (e.g., androgenic vs. estrogenic mechanisms). This complexity brings a more realistic view of individuals manifesting a relatively unique mosaic of sexually differentiated traits that differ between males and females at the population level (see Figure 2). Specific mechanisms that share, for example, critical periods or estrogenic mediation would be expected to cluster at the population level. Indeed, there is evidence for several distinct clusters of developmental processes being associated with androphilia in men (e.g., Swift-Gallant et al., 2019).

Figure 2. Graphical illustration of variation in sexual differentiation between individuals due to complex interactions of causal factors across development. This figure illustrates variation in sexual differentiation abstractly and is not intended to accurately depict this process. To the left is a circle representing an individual with only sexually undifferentiated traits, represented by smaller white circles. 2 alternative developmental trajectories are presented in which causal factors such as genetics, hormones and environment (represented by red, blue, and yellow, respectively) organize and activate sexually differentiated traits (represented by colored dots) at different developmental stages (represented by two sets of arrows). Each trait might vary in the relative importance of each causal factor, with some traits being more influential than others (represented by the color of dots reflecting relative influence of these factors), and in the timing of critical periods of influence (represented by the width of the arrows in the developmental periods). The causal factors and outcome for individual traits, represented by the size and color of the dots, vary predictably between females and males at the population level. However, due to individual variability in the timing and strength of causal influences, each individual represents a mosaic of developmental outcomes in sexually differentiated traits, represented on the right by Ishihara charts for testing color blindness (Wikicommons), which are circles composed of many dots of variable size and color (incidentally, there is a sex difference in incidence of color blindness).

Hormones

The central dogma of mammalian sexual differentiation of the nervous system and behavior holds that sexually differentiated traits are caused primarily by sexually differentiated gonadal hormone production (Figure 1). Furthermore, this dogma holds that a female developmental pathway is followed by default without the requirement of gonadal hormones, and that early hormone action in sexual differentiation is mediated primarily by testicular hormones that can promote masculine outcomes (masculinize) and inhibit feminine outcomes (defeminize). The organizational-activational framework was developed through developmental study of early testosterone action on future sexual behavior, but at least one more testicular hormone contributes to sexual differentiation. Anti-Mullerian hormone (AMH) is produced by Sertoli cells early in development and mediates regression of the Mullerian system early in development (Behringer et al., 1994). Beyond the absence of internal structures of Mullerian origin plausibly affecting sexual and reproductive behavior, there is some evidence that AMH has more direct actions on the development of neurobehavioral traits. For example, Amh-KO affects neuroanatomical sexual differentiation of prepubertal mice (Wittmann & McLennan, 2011, 2013a, 2013b).

Before shifting to discussing the roles of testicular testosterone in sexual differentiation, it is important to consider the assumption that early development of feminine neurobehavioral traits is independent of hormone action. There is reason to suspect that this assumption is not warranted. For example, female mice with loss of aromatase function mutations, and which therefore lack the ability to synthesize estradiol, have reduced lordosis response (Bakker et al., 2003) and female-atypical partner preference and mounting (Brock & Bakker, 2011; Brock et al., 2011). This phenotype is rescued by prepubertal (postnatal day PND10–15) estradiol treatment, but not by estradiol treatment in adulthood or early postnatally (PND1–5). These observations provide an example of feminine behavioral development requiring hormonal organization.

Testicular testosterone has many actions in sexual differentiation of neurobehavioral traits, but does not necessarily do so by activating its receptor, the androgen receptor (AR). Rather, testicular testosterone functions to a large extent as a prohormone, which is locally converted within the nervous system into other active steroid hormones, including a more potent androgen (dihydrotestosterone), and 17β‎ estradiol within brain through a process of aromatization. When considering mechanisms of testosterone action in sexual differentiation, it is therefore important to consider the extent to which androgenic and/or estrogenic mechanisms mediate outcomes of interest. Various pharmacological and genetic approaches have been taken to studying this question and have led to a hypothesis that many of testosterone’s actions within the brain are primarily estrogenic. This aromatization hypothesis has proven explanatory especially in rodent models, in which estrogenic mechanisms appear both necessary and sufficient for masculinization and defeminization of behavior, and seem to mediate sexual dimorphic neuroanatomical outcomes at least partly (see Roselli et al., 2009; Tsukahara & Morishita, 2020, for review). Curiously, the aromatization hypothesis has proven less explanatory for primate models, in which non-aromatizable androgens, but not estrogens, are effective in sexually differentiating neurobehavioral traits (see Thornton et al., 2009, for review).

Whereas AR has a single type encoded at a single genetic locus, there are multiple subtypes of estrogen receptor (ER), which are encoded on separate genetic loci. The function of two subtypes (ERα‎ and ERβ‎) in sexual differentiation of neurobehavioral traits has been characterized in genetic and pharmacological studies, which indicate additive roles in some cases (e.g., Bodo et al., 2006) and dissociable roles in other cases (e.g., Kudwa et al., 2006). As further complication, the cellular and molecular mechanisms whereby ERs affect sexual differentiation vary. Although canonical “genomic” actions, in which ERs act as ligand-dependent transcription factors, are generally thought to underlie estrogenic action on sexual differentiation of neurobehavioral traits, alternative estrogenic mechanisms can operate in a neurobehavioral context (e.g., see Micevych et al., 2017, for a review).

Androgenic mechanisms are centrally important for many masculine outcomes in sexual differentiation. Genetically male loss of AR function mutants exhibit an intersex phenotype characterized by diverse outcomes for sexually differentiated traits, which are variously masculine, feminine, intermediate, or atypical for either sex. For example, XY spontaneous loss of AR function mutants, known as Tfm in laboratory animals and cAIS in humans, have external genitalia, which is morphologically feminine, but lack associated internal structures derived from the Mullerian system, including the vagina, which is atypical for females, while also lacking internal structures derived from the Wolffian system, which is likewise atypical for males. These individuals are intermediate in body size. This complex set of outcomes reflects partial or complete androgenic mediation of sexually differentiated traits. In this case, masculine outcomes in genitalia and the Wolffian system are mediated mainly by androgenic mechanisms; however, body size is determined by a variety of signaling systems, and Mullerian differentiation is prevented by AMH, whose function is preserved in loss of AR function mutants. Similarly, neurobehavioral traits are similarly variable in XY Tfm rats, which neither mount nor lordose, and exhibit masculine (e.g., SDN-POA volume), feminine (e.g., SNB), and intermediate (e.g., MEPD) neuroanatomical outcomes (see Zuloaga et al., 2008, for a review).

Experiments with global loss of Ar function mutants have indicated complex phenotypes in which sexually differentiated traits of XY mutants are variously feminine, masculine, intermediate, or atypical, reflecting variable androgenic mediation of sexually differentiated traits (reviewed in Monks & Swift-Gallant, 2018; Swift-Gallant & Monks, 2017). In light of the strong evidence in support of the aromatization hypothesis, androgenic contributions to sexual differentiation of neurobehavioral traits in laboratory rodents were historically considered as secondary to estrogenic mechanisms, with some important exceptions (e.g., SNB motoneurons) that could be ascribed to non-neural androgenic mediation (i.e., SNB motoneuron rescue via target musculature). Nonetheless, more recent experiments with neural-specific AR function mutants have revealed a dependence of masculine sociosexual behavior on neural AR function (Juntti et al., 2010; Raskin et al., 2009; Swift-Gallant et al., 2016). Furthermore, experiments with AR overexpression mutants have indicated that the relationship between androgenic dose and masculine outcomes is complex, varying by site of AR action (neural or global) and according to trait. One caveat when interpreting studies that attempt to dissociate androgenic and estrogenic signaling is that their interpretation is complicated by their cross regulation. For example, AR has been implicated in regulating aromatase expression in rat brain (Roselli et al., 1987), and estrogenic mechanisms have been implicated in initiating Ar expression in mouse brain (Juntti et al., 2010).

Genetics

It is uncontroversial that sexually dimorphic genetics play a central role in sexual differentiation of mammals, notably being the principal determinant of gonadal sexual differentiation. Studies of gene function in transgenic mice have led to the conclusion that a single Y-chromosome gene, SRY, is both necessary and largely sufficient for testicular development in this species at least (see Koopman et al., 2016, for review of this line of research). Because genetic studies of sex reversal in humans lead to the identification of the SRY gene, it is likely that SRY plays a similar role in human sexual differentiation. However, genetic contributions to sexual differentiation other than initiating testicular development have been increasingly documented. A classic example of genetic contribution to mammalian sexual differentiation is provided by X‑chromosome inactivation, which results in phenotypic mosaicism for traits influenced by X‑chromosome genes. Although a full accounting of the role of genetics in mediating sexual differentiation in mammals is lacking, it may safely be concluded that relative to invertebrates, as exemplified by fruit flies (D. melanogaster) and nematodes (C elegans), in which sexual differentiation is mediated largely via direct genetic mechanisms (reviewed in Forger & De Vries, 2010); or even of other vertebrate classes as exemplified by birds, which have sex chromosomes but do not exhibit genetic dosage compensation for the homogametic sex (males) and in which sex reversal of the gonads of zebra finches (T. guttata) has only limited effects on the sexually differentiated song system (e.g., Agate et al., 2003; Wade et al., 1999), studies of laboratory rodents have thus far indicated a lesser role for direct genetic mechanisms in sexual differentiation.

Several experimental paradigms have been employed to study direct genetic effects on sexual differentiation. These approaches mainly focus on dissociating genetic sex from gonadal sex and include (a) examining sex differences that precede sexual differentiation of the gonads, (b) examining sex differences in mice genetically incapable of producing steroid hormones (e.g., Sf1 knockout mice), and (c) examining sex differences in mice whose sex chromosomes are discordant with their gonadal sex (notably the “four core-genotype” mice). Taken together, these studies point to significant effects of sex chromosomes on sexually differentiated traits independently from or synergistically with gonadal hormones. However, it should be acknowledged that these studies have largely focused on sex differences that have a known or suspected hormonal basis, such as neuroanatomic dimorphism, or differences in socio-sexual behavior. When considering other notable sex differences, including sex limited human diseases, genetics are the primary cause (e.g., X‑linked recessive genetic diseases such as Duchenne’s muscular dystrophy). Furthermore, studies have taken place largely in model organisms with limited genetic variability, which may underrepresent the broader importance of genetic mediation of sexual differentiation in vertebrates (e.g., see Renfree et al., 2014). Regardless of whether the foregoing are fair tests of the broader role of genetics in mammalian sexual differentiation, attempts to disentangle genetic, hormonal, and environmental determinants of sexually differentiated traits should not be expected to yield results indicating “purely” genetic, hormonal or environmental causes, but rather that these factors should be expected to be integrated and interrelated.

Environment

The third major factor influencing sexual differentiation is environment, broadly construed. This term is often synonymous with sociocultural environment in discussions of gender, where it is also sometimes credited as the most influential casual factor (e.g., Maccoby & Jacklin, 1974). However, other types of environmental influences operate on sexually differentiated outcomes, and also environmental influences often operate via, as well as interact with, both hormonal and genetic mechanisms.

For example, environmental exposure to extrinsic hormones influences sexual differentiation in multiple contexts. In birds, there is a considerable literature on the significance of variation in maternal deposition of testosterone in the yolk of male embryos, with the idea that this variation serves to “calibrate” the degree of sexual differentiation to ecological conditions as articulated in the life-history trade-off theory (e.g., Hau, 2007). In this framework, testosterone-mediated investment in reproductive functions trades off against competing fitness demands. In laboratory animal models, demands such as parasites and food availability are generally not studied, although there is evidence that manipulation of diet can affect sexually differentiated neuroendocrine outcomes (e.g., Nätt et al., 2017).

In mammals, there has been considerable study of the significance of in utero androgen transfer from male fetuses to female fetuses, which produces variation in the degree of sexual differentiation of females that gestate with male siblings, and influences sexually differentiated somatic and behavioral traits (e.g., Clemens et al., 1978). An additional source of environmental hormone exposure comes from maternal estrogens, which is attenuated by alpha-feto protein (Afp). An example of this has been described in mice, where loss of Afp function mutation reduces lordosis and increases masculine mounting in female mice (Bakker et al., 2006). It is unclear to what extent this variation in environmental hormone exposure in development contributes to observed sex differences in many neurobehavioral traits.

Environmental sensory stimuli also play a key role in the development of sexually differentiated traits. Perhaps unsurprisingly given the central importance of these traits to reproduction, the main source of these sensory and experiential cues is in the context of social interactions. It is important to recognize that environmental guidance of development is a common theme in neural development. When considering the importance of environmental factors in sexual differentiation, it can be useful to distinguish between classes of environmentally mediated developmental events that are dysfunctional and result from deprivation and those that produce normal and likely adaptive variability in sexual differentiation. For example, songbirds with sexually differentiated courtship songs require minimal exposure to species-typical song to acquire species-typical characteristics, and also use parental “tutoring” to acquire individual song characteristics (e.g., Chen et al., 2016).

Mammalian examples of environmental influence in development of sexually differentiated traits have also been described. For example, isolating infants from caregivers in a variety of mammalian species results in gross sociosexual, neuroendocrine, cognitive, and motivational deficits (reviewed in Brett et al., 2015). In this context of total parental deprivation, deficits may be prevented by simulating sensory stimulation normally provided by the parent, including tactile stimulation. A more discrete example is provided by the importance of genital tactile stimulation provided by grooming on sexual function in rats. Rat dams groom male pups more than female pups, which is attributed to androgen-dependent pheromone production by male pups (reviewed in Moore, 1992) Deprivation of anogenital tactile stimulation normally provided to male rat pups by dams prior to weaning results in less masculine copulatory behavior, morphology of SNB motoneurons and results in deficits in ex copula reflexive erections in adult male rats so deprived (Lenz et al., 2008; Moore et al., 1992). Similarly, prevention of auto-grooming of pubescent male rats results in pubertal delay (Moore & Rogers, 1984). As further example, social isolation of pubertal rats reduces sexual dimorphism in the MEPD and causes deficits in social behavior (Cooke et al., 2000).

These are clearly cases of deprivation of normal social environmental cues resulting in dysfunctional development of sexually differentiated traits, and may be distinguished from individual differences in developmental outcomes which reflect normal variation in experience. Several example of the latter form of environmental influence on sexually differentiated traits have been described, including studies of intergenerational transmission of maternal behavior (e.g., see Curley & Champagne, 2016, for review), and to some extent sexual behavior (e.g., Cameron et al., 2011). There is also evidence in rats that developmental and adult social experience can result in preference for arbitrary olfactory cues in sexual partners (Kippin & Pfaus, 2001; Moore et al., 1996). Additionally, copulatory experience itself, which increases efficiency of and therefore differentiation of these sexual behaviors, can induce plasticity in sexually differentiated neuroanatomy (e.g., Breedlove, 1997; Jean et al., 2017).

Whereas much focus has been given to interactions with parents as a source of developmental plasticity, siblings and other conspecifics likely also provide a source of necessary and variable social experience. This is increasingly true as individuals become more reproductively mature, and social feedback in puberty provides another identified source of individual differences in sexually differentiated neurobehavioral traits. Social exposure provides feedback that serves to adjust sexual differentiation of traits. Cavies (guinea pigs) provide an example of social environment at puberty influencing sexually differentiated neurobehavioral traits. In cavies, pubertal rearing of males in low density (i.e., pair housed) increases expression of male-typical sociosexual behaviors in relation to colony-reared males, including aggression to males and courtship of females (e.g., Zimmermann, Kaiser, Hennessy, et al., 2017). This is thought to reflect adaptive response to the social environment such that males with few competitors engage in potentially costly behaviors and endocrine responses that are likely to increase reproductive success, whereas males facing more competition avoid these potential costs given a lower probability of success (Zimmermann, Kaiser, & Sachser, 2017).

A more extreme illustration of this social forecasting can be found in species in which reproductive activation is induced through social cues. For example, reproduction can be delayed or accelerated by social housing conditions in a variety of species, including traditional laboratory rodents (reviewed in Liberles, 2014). Social regulation of reproduction and sexual differentiation is dramatic in naked mole-rats, in which most colony members are reproductively suppressed and exhibit remarkably few sexually differentiated traits, to the point of having virtual sexual monomorphism in genitalia (Seney et al., 2006), but also when considering neurobehavioral traits. In this species, removal from the colony induces reproductive activation and initiates sexual differentiation of copulatory responses and endocrinology as well as sex differences in neural morphology (e.g., Swift-Gallant et al., 2015; Zhou et al., 2013).

Developing Hypotheses Concerning Sexual Differentiation of Observed Sex Differences in Neurobehavioral Traits

Studying mechanisms of sexual differentiation of neurobehavioral traits is complex and requires an integrative approach. Not only must genetic, hormonal, and environmental factors be considered and integrated, but one also cannot assume that changes in neuroanatomy or behavior are caused by primary action of these factors on neurons, or even within the nervous system (e.g., De Vries & Forger, 2015; Swift-Gallant et al., 2012). The SNB was the first neuroanatomic dimorphism in which site of action of androgens could be evaluated, and it has mainly non-cell autonomous, non-neural, and non-nervous system mediation of sexual differentiation of many of its anatomic features (reviewed in Sengelaub & Forger, 2008; Swift-Gallant & Monks, 2017). To the extent to which this question has been asked in other model neurobehavioral sexual dimorphisms, it is similarly doubtful that sexual differentiation of the brain is solely or even mainly mediated by cell-autonomous or neural action. For example, recent work on sexual differentiation of rat preoptic area and associated copulatory behavior has largely focused on non-neural cells, including microglia and other immune cells (e.g., Lenz et al., 2018; see VanRyzin et al., 2018, for review). In this way, yet another parallel can be drawn between sexual differentiation of neurobehavioral traits and that of the urogenital system, in which primary hormone action appears to be mediated by cells of mesenchymal origin, which then indirectly induce epithelial differentiation (see Swift-Gallant & Monks, 2017, for discussion).

Nonetheless, one may safely conclude that steroid hormone action within neurons at least partially mediates sexual differentiation of neurobehavioral traits. This conclusion is reached by several observations: (a) Neurons express steroid hormone receptors in a spatiotemporal pattern consistent with direct mediation and (b) experiments that localize hormone action to the brain (e.g. through local implants of hormone within the hypothalamus), are neural-specific (e.g. through neural-specific promoters in transgenic or transfection approaches), or both.

On the first point concerning the spatiotemporal expression pattern of steroid hormone receptors, early histological studies using steroid hormone autoradiography revealed that androgen receptors, estrogen receptors, and progesterone receptors are expressed at relatively high levels within sexually dimorphic neural structures (e.g., see McEwen et al., 1979, for early review) and subsequent molecular biological and immunological methods confirmed that these receptors are within neurons and within the critical periods for hormone action. For example, in the rat SNB, in which perinatal androgenic organization of SNB cell number is not mediated in a cell-autonomous manner within SNB neurons, AR is accordingly not expressed in these cells, but is expressed in the target muscles, which are the site of action. Similarly, AR in SNB motoneurons is expressed later in development, when cell-autonomous androgenic actions are present, providing evidence that the association between receptor and response is consistent in both positive and negative cases (Jordan et al., 1997).

On the second point concerning local manipulations of hormone action, direct delivery of drugs, RNAi oligonucleotides, and hormones to the neonatal and adult hypothalamus have been used to test for local steroid hormone mediation of sexual differentiation of associated neurobehavioral traits (e.g., Auger et al., 2000; McGinnis et al., 2002; Moralí et al., 1986). These manipulations are sufficient to reproduce or prevent many of the actions of systemic gonadal hormones. More recently, this route of administration has been coupled with viral transfection of neural-specific promoters to test functions of genes mediating steroid hormone signaling, including steroid hormone receptors and cofactors (Yang et al., 2013). Similarly, neural-specific transgenesis and gene-targeting experiments testing gene function of steroid hormone receptors have generally supported the hypothesis that steroid hormone action in sexual differentiation of the hypothalamus and associated sexual behavior is mediated at least in part via local actions within neurons.

A final caution is offered to readers when developing hypotheses considering the function of sexual dimorphisms in neuroanatomy. Although it is tempting to assume that sex differences in neuroanatomy cause sex differences in behavior, this does not logically follow. Like other correlations, the causal relationship between structure and function requires testing, and correlations between structure and function could very well be unrelated or have the opposite direction of causality. Consider, for example, the case of naked mole-rats, which begin to show sex differences in neuroanatomy only after a change in their social behavior (Holmes et al., 2007). Furthermore, even if sexually differentiated neuroanatomy affects behavior, it could be that it prevents a sex difference in behavior, rather than causing one in the same manner that X‑chromosome inactivation compensates for gene dosage between males and females (De Vries, 2004).

Conclusion

Sex differences in nervous system structure and in behavior are ubiquitous and can be dramatic. These sex differences in neurobehavioral traits can significantly inform neuroscience research or, if ignored, can result in problems with generalizability of results and with knowledge translation. Sex differences have multiple and variable causes, and are generally thought to result from interacting genetic, hormonal, and environmental factors throughout the life span. Sexually differentiated neurobehavioral traits tend to be linked but can be dissociated, indicative of distinct developmental processes that share some underlying causality. In mammals, sexual differentiation is initiated by genetic signals on the sex chromosomes and is powerfully mediated by gonadal hormones. Environmental conditions, notably including the uterine environment, interactions with parents, and other social interactions intersect extensively with hormonal mechanisms to regulate sexual differentiation. Sexual differentiation of neurobehavioral traits is most prominent during an early critical period corresponding to the perinatal period in rats and mice, but other critical periods exist, including puberty. The developmental processes whereby neurobehavioral traits become sexually differentiated have been most intensively studied for a few sexually dimorphic neuroanatomic structures and reproductive behaviors, including sexually dimorphic hypothalamic and spinal nuclei and copulatory behavior. In these cases, the major developmental process mediating sexual differentiation is thought to be organizational action of gonadal testosterone in permanently masculinizing and defeminizing traits coupled with sex-typical activational gonadal hormone action. Gonadal hormones have pleiotropic actions on diverse sites of action, and assumptions that sexual differentiation of neural structures is mediated by cell autonomous actions are not warranted. Taken together, sexual differentiation is best studied in an integrative manner that considers the interaction of genetic, hormonal, and environmental factors acting at multiple sites through development to cause sex differences in neurobehavioral traits.

Further Reading

  • Oliva M, Muñoz-Aguirre M, Kim-Hellmuth S, Wucher V, Gewirtz ADH, Cotter DJ, Parsana P, Kasela S, Balliu B, Viñuela A, Castel SE, Mohammadi P, Aguet F, Zou Y, Khramtsova EA, Skol AD, Garrido-Martín D, Reverter F, Brown A, Evans P, Gamazon ER, Payne A, Bonazzola R, Barbeira AN, Hamel AR, Martinez-Perez A, Soria JM; GTEx Consortium, Pierce BL, Stephens M, Eskin E, Dermitzakis ET, Segrè AV, Im HK, Engelhardt BE, Ardlie KG, Montgomery SB, Battle AJ, Lappalainen T, Guigó R, Stranger BE. The impact of sex on gene expression across human tissues. Science. 2020 Sep 11;369(6509):eaba3066. doi: 10.1126/science.aba3066. PMID: 32913072; PMCID: PMC8136152.
  • Holmes MM, Monks DA. Bridging Sex and Gender in Neuroscience by Shedding a priori Assumptions of Causality. Front Neurosci. 2019 May 9;13:475. doi: 10.3389/fnins.2019.00475. PMID: 31143099; PMCID: PMC6521798.

References