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published; 14 August 2014 
doi: 10.3389/fnins.2014.00242 

The mechanisms underlying sexual differentiation of 
behavior and physiology in mammals and birds: relative 
contributions of sex steroids and sex chromosomes 

Fumihiko Maekawa^*, Shinji Tsukahara^, Takaharu Kawashima^, Keiko Nohara^ and 
Hiroko Ohki-Hamazaki** 

' Molecular Toxicology Section, Center for Environmental Health Sciences, National Institute for Environmental Studies, Tsukuba, Japan 
' Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama, Japan 

^ Ecological Genetics Research Section, Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, Tsukuba, Japan 
* College of Liberal Arts and Sciences, KItasato University, Sagamihara, Japan 

Edited by: 

Sonoko Ogawa, University of 
Tsukuba, Japan 

Reviewed by: 

Gregoy Y. Bedecarrats, University of 
Guelph, Canada 
TakayoshI Ubuka, Waseda 
University, Japan 


Fumihiko Maekawa, Molecular 
Toxicology Section, Center for 
Environmental Health Sciences, 
National Institute for Environmental 
Studies, 16-2 Onogawa, Tsukuba, 
305-8506, Japan 
Hiroko Ohkl-HamazakI, College of 
Liberal Arts and Sciences, KItasato 
University, 1-15-1 KItasato, 
MInami-ku, Sagamihara, 
Kanagawa 252-0373, Japan 

From a classical viewpoint, sex-specific behavior and physiological functions as well as the 
brain structures of mammals such as rats and mice, have been thought to be influenced 
by perinatal sex steroids secreted by the gonads. Sex steroids have also been thought to 
affect the differentiation of the sex-typical behavior of a few members of the avian order 
Galliformes, including the Japanese quail and chickens, during their development in ovo. 
However, recent mammalian studies that focused on the artificial shuffling or knockout 
of the sex-determining gene, Sry, have revealed that sex chromosomal effects may be 
associated with particular types of sex-linked differences such as aggression levels, social 
interaction, and autoimmune diseases, independently of sex steroid-mediated effects. In 
addition, studies on naturally occurring, rare phenomena such as gynandromorphic birds 
and experimentally constructed chimeras in which the composition of sex chromosomes 
in the brain differs from that in the other parts of the body, indicated that sex chromosomes 
play certain direct roles in the sex-specific differentiation of the gonads and the brain. In 
this article, we review the relative contributions of sex steroids and sex chromosomes in 
the determination of brain functions related to sexual behavior and reproductive physiology 
in mammals and birds. 

Keywords: sexual differentiation of the brain, neurosteroids, birds, rodents, chimera 


In most eukaryotic species, sex differences exist within aspects 
of behavior as well as physiology. Since typical sexual behaviors 
and reproductive physiology are crucial to produce offspring, it 
is important to understand the mechanism of sexual differen- 
tiation in the brain in conjunction with sex-specific behaviors 
and physiology. From the standpoint of environmental science, 
several external factors from the industrialized world can impair 
reproduction. Studies have shown that the specific chemicals 
called endocrine disrupters can influence sexual differentiation 
in many species (Colborn et al., 1993). It has been reported that 
bisphenol-A and organotin compounds are examples of such 
endocrine disruptors that mimic and/or antagonize the functions 
of sex steroids and impair sexual differentiation in wildlife (Flint 
et al., 2012; Lewis and Ford, 2012). Therefore, research has been 
conducted on how such chemicals affect sex steroid-dependent 
organization of various organs, including the brain, during devel- 
opment, in order to protect wildlife and humans from potential 
endocrine disruptors (Frye et al., 2012). On the other hand, the 
knowledge regarding sexual differentiation has increased with 
progress in basic research (Arnold, 2004; Arnold and Chen, 
2009) and the endpoints for investigating how environmental 
chemicals impair normal sexual differentiation might be updated 

subsequently. In this article, we review the relative contribution 
of gonadal steroids and sex chromosomes to the differentiation 
of brain functions related to the sexual behavior and reproduc- 
tive physiology in mammals and birds by focusing on the recent 


The molecular mechanism of gonadal development in mammals 
and birds has been studied extensively. In most mammals, the 
males have one X chromosome and one Y chromosome whereas 
females have two X chromosomes. The gene Sry/SRY, located 
on the Y chromosome has been shown to be critical for testis 
development in most mammals (Gubbay et al., 1990; Sinclair 
et al, 1990; Koopman et al., 1991). The mechanism of testis 
determination in mammals is similar to that of the gonadal sex 
determination system of some teleost fish whose sex is determined 
by the specific gene DMY located on the Y chromosome (Matsuda 
et al., 2002). The sex chromosomes of birds are designated Z and 
W, where male birds have two Z chromosomes, while female birds 
have one Z chromosome and one W chromosome. The DMRTl 
gene on the Z chromosome, which encodes the doublesex and 
mab-3-related transcription factor 1 (DMRTl), is required for 

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Maekawa et al. 

The mechanisms underlying sexual differentiation 

testis determination in birds of the order Galliformes (Smith et al., 
2009). Birds lack a global mechanism of dosage compensation to 
equalize the expression of genes between sexes on the Z chromo- 
some(s). Moreover, male-biased gene expression in gonads has 
been reported for most genes located on the Z chromosome, with 
variable levels of expression depending on the locus (Mank and 
Ellegren, 2009). DMRTl in the order Galliformes is an exam- 
ple of such a gene and the amount of DMRTl produced from 
expression of the single DMRTl gene on the single Z chromo- 
some in females is insufficient to induce the formation of a testis. 
It has also been reported that knockdown of DMRTl by RNA 
interference in the male chicken gonadal primordium results in 
feminization of the gonads (Smith et al., 2009). Therefore, the 
lower level of DMRTl expression presumably induces the gonadal 
primordium to develop into ovary. Dosage compensation seems 
to occur on the Z chromosome at a gene-by-gene level in avian 
organs besides the gonads, such as the brain (Mank and Ellegren, 
2009). This fact is the basis for the argument that sex chromo- 
somes autonomously regulate sexual physiology and function in 
somatic tissues. 


In contrast to gonadal development, evidence suggests that sexual 
behavior is masculinized and defeminized during the perinatal 
or postnatal period by androgen secreted by the testes, irre- 
spective of Sry expression in the brain. The action of testicular 
androgens in the developing brain is critical to the expression 
of the male-typical pattern of behaviors in mammals (Phoenix 
et al., 1959; Arnold and Gorski, 1984). In rodents, testosterone 
secreted by the testes is locally converted to 17P-estradiol (E2) 
by the cytochrome P450 enzyme, aromatase, in the brain, and 
E2 in turn, masculinizes/defeminizes the brain (MacLusky and 
Naftolin, 1981). 

In rat, which is one of the most popular mammalian mod- 
els for studying sexual differentiation in the brain, testosterone is 
first detected in the testes on embryonic day 15.5 and testosterone 
synthesis in the testes peaks at approximately, embryonic day 18.5 
(Warren et al, 1973). Testicular testosterone synthesis as well as 
plasma testosterone concentrations decrease sharply after birth. 
Since the plasma testosterone level in rats from day 18 of gestation 
to day 5 postpartum is higher in male than in female rats (Weisz 
and Ward, 1980), the male brain is exposed to higher levels of 
testosterone than female brain. Therefore, this perinatal period is 
considered to be a critical period for sexual differentiation in the 
rat brain (MacLusky and Naftolin, 1981) and this hypothesis was 
then supported by the experiments described below. 

Injection of female rats with either testosterone propionate 
(TP) or E2 during the perinatal period resulted in decreased abil- 
ity to display female sexual behaviors in adulthood (Phoenix et al, 
1959). In female mice, knockout of a-fetoprotein that binds to 
estrogen, thus protecting the developing female brain from expo- 
sure to estrogen, has been reported to cause reduction in lordosis, 
a manifestation of female sexual behavior, and an increase in 
expression of the male-typical sexual behavior, that is, mount- 
ing (Bakker et al., 2006). In addition, it has been reported that 
estrogen receptor (ER)- a-knockout males exhibited decreased 

frequency of intromission, another male-typical sexual behavior 
(Ogawa et al., 1997). Based on these results, the differentiation 
of stereotypic pattern of sexual behavior in rodents is thought to 
be, at least in part, due to the effects of sex steroids. Sex-specific 
copulatory behavior is assumed to be accompanied by structural 
changes in the brain. Various studies demonstrating morpho- 
logical sex differences in the brains of rats and mice discussed 


Certain brain nuclei exhibit sex differences, in terms of vol- 
ume and number of neurons and/or synapses and are generally 
referred to as sexually dimorphic nuclei. Difference in volume of 
the nuclei is attributable to the differences in number of neu- 
rons. Gorski et al. discovered a nucleus in the preoptic region 
that shows morphological sex differences in rats (Gorski et al., 
1978, 1980); this nucleus is now known as the sexually dimorphic 
nucleus of the preoptic area (SDN-POA), though its physiological 
functions are still unknown. The SDN-POA is significantly larger 
and contains more neurons in male than in female rats. Similarly, 
the volume of the calbindin-D28K-immunoreactive area in the 
SDN-POA is 2-4 times larger in male than in female rats (Simerly 
et al, 1997; Sickel and McCarthy, 2000; Orikasa et al., 2007). 
Moreover, following neonatal castration, the SDN-POA volume 
in adult male rat decreased (Gorski et al., 1978), whereas injection 
of TP in females during the early developmental period resulted 
in an increase in the SDN-POA volume in adult female rats to 
match that of adult male rats (Gorski et al., 1978; Dohler et al, 
1984). On the other hand, injection of TP in adult rats has no 
effect on the volume of the SDN-POA. These findings suggest 
that testosterone synthesized in the rat testes affects the brain 
during development and not during adulthood, to increase the 
volume of the SDN-POA. Injection of E2 instead of TP in post- 
natal female rats also increased the size of their SDN-POA during 
adulthood. Injection of an ER-a agonist, and not an ER-P ago- 
nist, mimicked the effect of E2 (Patchev et al., 2004). The effects of 
estrogen, which is converted from androgen by aromatization and 
then binds to ER-a during the perinatal period, may be essential 
to establish the sexual dimorphism in the SDN-POA in rats. 

During development, gonadal steroids also influence the sex- 
ually dimorphic formation of the principal nucleus of the bed 
nuclei of the stria terminalis (BNSTp). The BNSTp of the adult 
male rats is larger and contains more neurons than that of adult 
females (del Abril et al, 1987; Hines et al, 1992). Perinatal 
orchidectomy of males and perinatal androgenization of females 
by injection of TP prevent the occurrence of sexual dimor- 
phism in BNSTp (Guillamon et al, 1988; Chung et al, 2000). 
Inactivation of androgen receptor results in feminization of the 
testes, which then show an ovary-like phenotype, and also reduc- 
tion in the volume of the BNSTp in male rats (Durazzo et al, 
2007). Sex differences in the volume and number of neurons 
in BNSTp do not occur in mice deficient in either aromatase 
or the ER-a gene, because of feminization of BNSTp in males 
(Tsukahara et al, 201 1). On the other hand, mice deficient in the 
ER-P gene show sex differences in BNSTp (Tsukahara et al., 2011). 
These results suggest that estrogen is synthesized from androgen 

Frontiers in Neuroscience | Neuroendocrine Science 

August 2014 1 Volume 8 | Article 242 | 2 

Maekawa et al. 

The mechanisms underlying sexual differentiation 

and that its effects, exerted through binding to ER-a during the 
perinatal and/or aduk stage, appear to be involved in the male- 
typical formation of the BNSTp in mice. It has recently been 
reported that, in the BNST, gene expression of Brs3, Cckar and 
Sytl4, was sexually dimorphic, and regulated female and male sex- 
ual behaviors (Xu et al, 2012). However, since expression of these 
genes was altered by gonadectomy, sexual dimorphism was sus- 
pected to be attributable to the effect of sex steroids at the adult 
stage. More recently, it has been reported that the number of CRH 
neurons in the BNST (Fukushima et al., 2013) can be altered by 
TP injection in the perinatal period. 

In contrast to the effects of sex steroids on the SDN-POA and 
BNSTp, the size of the anteroventral periventricular nucleus of 
POA (AVPV) in rats was reduced by androgens or estrogens dur- 
ing the perinatal period (Ito et al., 1986; Patchev et al., 2004). 
Injection of an ER-a agonist or an ER-P agonist in the perina- 
tal period has been shown to decrease the neuronal cell density 
in the AVPV in female rats (Patchev et al, 2004), indicating 
that this reduction in neuronal cell density in the AVPV is an 
effect of estrogen mediated through binding to either ER-a or 
ER-p. The AVPV in female rats contains a greater numbers of 
tyrosine-hydroxylase (TH) mRNA-positive, dopaminergic neu- 
rons (Simerly et al, 1985) and Kissl mRNA-positive neurons 
(Kauffman et al, 2007) than the AVPV in male rats. Perinatal 
orchidectomy in males and perinatal treatment with testosterone 
in females resulted in an increase and decrease, respectively, 
in the number of TH mRNA-positive neurons in the AVPV 
(Simerly, 1989). In addition, perinatal treatment with TP in 
females decreased the number of Kissl mRNA-positive neurons 
(Kauffman et al, 2007). 


There is accumulating evidence that a certain type of sexual dif- 
ferentiation in the brain is attributable to mechanisms that are 
independent of steroid hormones. The mouse model specific to 
this phenomenon called "four core genotypes" (FCG) was made 
possible by bioengineering techniques that created a mismatch 
between gonadal sex and chromosomal sex (XX vs. XY) . This FCG 
model provides a breakthrough in the understanding of how sex 
chromosomes affect sexual differentiation in the brain. Based on 
observations in the FCG mouse model, the investigators reported 
that the latency and frequency of copulatory behavior are some- 
what influenced by sex chromosomes (Park et al., 2008). However, 
sexual orientation seems to be mainly determined by differences 
in gonadal sex and subsequent secretion of sex steroids. This find- 
ing is generally consistent with the results of studies in which 
the sexual differentiation in the brain was examined after hor- 
monal manipulation during the critical period. Indeed, the FCG 
model revealed that expression of progesterone receptor, which 
is inducible under the control of estrogen, is also dependent on 
gonadal sex (Wagner et al, 2004). On the other hand, aggression 
manifested in the form of attacks against intruders and parenting 
studied in pup-retrieval tests, have been reported to be regulated 
by chromosomal sex as well as by gonadal sex steroids (Gatewood 

et al., 2006). The aggression score, based on the proportion of 
mice that attacked intruders and the latency of attacking on first 
trial, was reported to increase in the presence of either testes or Y 
chromosome. By contrast, parental behavior, scored by latency to 
retrieve pups and number of pups retrieved, was low in the pres- 
ence of either testes or Y chromosome. The sexual orientation of 
social behavior, including sniffing and play behavior in juvenile 
mice has been reported to be organized, at least in part, by the 
interaction between gonadal sex and chromosomal sex (Cox and 
Rissman, 2011). Therefore, neural circuits in the brain, responsi- 
ble for social communications such as aggression, sniffing, and 
play behavior, may be differentiated not only by gonadal hor- 
mones, but also by the sex chromosome complement. In addition, 
the differentiation of neural circuits related to nociception, drug 
abuse, and autoimmune disease is related to the chromosomal 
sex, although the precise mechanisms by which chromosomal sex 
affects the neuronal circuits are not yet known. Taken together, 
the differentiation of core sexual behavior might be predomi- 
nantly under the control of gonadal hormones, whereas the sex 
differentiation of various other aspects of physiology and behav- 
ior, including social communications might be determined, at 
least in part, by the interaction of gonadal hormones and sex 
chromosomes in mice. 


The sexual dimorphism of midbrain dopaminergic neurons in 
rodents is reported to be controlled directly by chromosomal sex. 
Mice that have a Y chromosome, irrespective of their gonadal sex, 
have more dopaminergic neurons in their midbrain than mice 
that have only X chromosomes (Carruth et al., 2002). Expression 
of Sry, located on the Y chromosome in the male brain, has been 
reported to directly affect TH expression in the dopaminergic 
neurons of the substantia nigra (Dewing et al, 2006). In the lateral 
septum, on the other hand, both testosterone and the presence of 
a Y chromosome in male mice have been reported to increase the 
number of vasopressin neural fibers (De Vries et al., 2002). De 
Vries et al. revealed that XY males whose Sry gene was lost from 
the Y chromosome but had the heterotopic Sry transgene show a 
higher density of vasopressin fibers in the lateral septum than XX 
"males" with a heterotopic Sry transgene, whereas XY "females" 
whose Sry gene was lost from Y chromosome showed a higher 
density of vasopressin fibers in the area compared to the XX 
females (De Vries et al., 2002). These reports suggest that certain 
genes that are located on the Y chromosome, other than Sry affect 
the sexually dimorphic structure of the brain in mice. In associa- 
tion with such structural differences caused by sex chromosomes, 
expression of the genes located on X and Y chromosomes is also 
sexually dimorphic in the mouse brain (Xu et al, 2002; Dewing 
et al, 2003). 

In addition, substantial differences in expression between 
sex-specific parental alleles on the X chromosome have been 
reported in the mouse brain (Gregg et al, 2010a). One study 
demonstrated that sex-specific imprinted genes whose expres- 
sion differs between paternal and maternal alleles are mostly 
found in the hypothalamic area in the female brain (Gregg et al, 
2010a), although early studies showed that maternal and paternal 

August 2014 | Volume 8 | Article 242 | 3 

Maekawa et al. 

The mechanisms underlying sexual differentiation 

influence occurs in the cortex and in the hypothalamus, respec- 
tively (Men et al, 1995; Keverne et al, 1996). On the other hand, 
paternal bias of autosomal genes in the brain was also reported 
(Gregg et al., 2010b). Understanding the epigenetic process that 
underlies the mechanism of parental bias would open up a new 
avenue of research on sex chromosomal effects in the brain. 


The Japanese quail is an animal model that has been used to 
examine how sex steroids determine the sexual differentiation in 
the brains of birds (Balthazart et al., 1983). The mating behav- 
ior of Japanese quail is sexually dimorphic: males strut and crow 
in front of the females and mount them (Adkins and Pniewski, 
1978), whereas females never exhibit mounting behavior, even 
when injected with testosterone at adulthood (Adkins, 1975; 
Balthazart et al., 1983). It has been reported that in the adult 
male quaQ, estradiol produced by aromatization of testosterone 
in the brain induces male mounting behavior and that the testos- 
terone metabolite 5 -hydro testosterone in the adult male induces 
strutting and crowing (Adkins and Pniewski, 1978; Balthazart 
et al., 1985). In contrast, exposure of male Japanese quail embryos 
to either testosterone or estrogen prior to day 12 of incuba- 
tion resulted in significant reduction of mounting behavior at 
adult stage, indicating that actions of testosterone and estrogen 
at embryonic stage demasculinize male-type copulatory behavior 
at adulthood (Adkins-Regan, 1987). On the other hand, admin- 
istration of an anti-estrogen agent to females prior to day 9 
of incubation masculinizes their copulatory behavior (Adkins- 
Regan and Garcia, 1986). These results suggest that the order 
Galliformes and mammals are different in terms of the devel- 
opmental effects of steroids. More specifically, the neuronal cir- 
cuit related to copulatory behavior is masculinized in mammals 
by estrogen produced from testosterone in the brain, whereas 
it is feminized and de-masculinized in Galliformes by estrogen 
secreted from the ovary. As for plasma steroids in Japanese quail, 
it has been reported that from day 10 of incubation to hatch- 
ing, estrogen concentrations are higher in females than in males, 
and conversely, testosterone concentrations are higher in males 
than in females (Ottinger et al., 2001). Therefore, the mechanism 
protecting the male quail brain from testosterone exposure has 
been postulated but is still under debate. Since a high activity 
of SP-reductase has been reported in the embryonic male brain 
(Balthazart and Ottinger, 1984), this enzyme possibly metabolizes 
testosterone into SP-dihydrotestosterone instead of E2 and pro- 
tects the male brain from being de-masculinized by testosterone 
exposure (Balthazart and Ottinger, 1984). Taken together, these 
results suggest that endogenous estrogen secreted by the ovary 
is sufficient to differentiate the neuronal circuits related to cop- 
ulatory behavior into the female-type in quail, but the precise 
mechanism by which the male brain escapes from feminization 
remains unclear. 


Song performance of the zebra finch is observed only in males and 
song-related nuclei including the nucleus hyperstriatum ventrale, 

pars caudale (HVc), and the nucleus robustus archistriatalis (RA) 
have been reported to be sexually dimorphic. The song perfor- 
mance is affected by developmental exposure of steroids. Double 
treatment, consisting of either estrogen or testosterone during 
hatching and testosterone at the adult stage, enables females to 
sing (Gurney and Konishi, 1980), indicating that the presence 
of estrogen in the brain during development can masculin- 
ize the song-related nuclei. Thus, there may be a difference 
between the actions of steroids on sexual behavior in Galliformes 
and on song performance of Passeriformes during development. 
Although the source of estradiol in the song control system dur- 
ing development of the zebra finch was long unknown, male 
brain slices containing the HVc and the RA regions have been 
found to produce more estrogen than corresponding female brain 
slices (HoUoway and Clayton, 2001). This suggests that estro- 
gen produced locally in the HVc and/or the RA contributes to 
masculinization of song-related nuclei. Indeed, various steroid- 
synthesizing enzymes are expressed in the zebra finch brain 
(Schlinger and Remage-Healey, 2012) and the same is true for 
the Japanese quaQ brain (Tsutsui, 2011). Studies performed by 
Remage-Healey et al. (2010, 2013) suggested that neurosteroids 
rapidly produced in the brain are important for social interaction 
via modulation of song production and perception of acoustic 


It has been suspected that genes located on the sex chromosome 
act in a cell-autonomous manner in brain cells to differentiate 
song-related circuits. Findings from a naturally-occurring gynan- 
dromorphic finch whose right and left sides show different sex 
genetics, demonstrated that the sexually dimorphic neural circuit 
for the song system is differentiated in part due to chromosomal 
sex. Most interestingly, the expression level of androgen receptor 
in the one half showed a masculine phenotype as compared to 
the other half, despite the identical influence of steroid hormones 
on both sides (Agate et al., 2003). Similar chromosomal sex influ- 
ences on the sex difference of somatic tissues may also apply to 
Galliformes. The coloration, wattle, and leg spur of gynandromor- 
phic chicken were observed to be different on the right and left, 
indicating that cell-autonomous sex determination in somatic 
cells occurs in Galliformes as well as in Passeriformes (Zhao et al, 


To determine whether the sex of the brain affects brain function 
and behavior, chimeras were constructed, in which the brains of 
two embryos were switched. The pioneering work by Nicole Le 
Douarin showed that developmental fate of cells can be moni- 
tored by creating quail-chick chimeras (Le Douarin and Jotereau, 
1975). By a surgical method using a microscalpel, the brain pri- 
mordium of a chick embryo at 1.5 days after incubation of the 
egg could be replaced by that of a quail embryo (Balaban et al, 
1988; Teillet et al, 1991). By applying a similar method, the male 

Frontiers in Neuroscience | Neuroendocrine Science 

August 2014 1 Volume 8 | Article 242 | 4 

Maekawa et al. 

The mechanisms underlying sexual differentiation 

(female) brain could be replaced by the female (male) brain of 
conspecies in Galliformes. 

The first study with chimeras that have a brain with differ- 
ent chromosomal sex was conducted in Japanese quail (Gahr, 
2003). In this study, the forebrain primordia were switched 
between two embryos before gonadal differentiation. The results 
showed that the chimeras with female karyotype in the forebrain 
but a male karyotype in other tissues did not exhibit mount- 
ing and exhibited only rudimentary crowing behavior. Since the 
adult chimeras showed low plasma level of testosterone which is 
required for male-type copulatory behavior, their impaired repro- 
ductive behaviors were attributable to their lower testosterone 
level. The volume of the preoptic area (POA) which is known 
to be dependent on plasma testosterone level (Panzica et al, 
1987) was also reported to be feminized in the chimeras (Gahr, 
2003). Therefore, it is speculated that male-typical chromoso- 
mal set complement is required in the forebrain of the quail, so 
that gonadotropin regulation can maintain testicular function in 

On the other hand, we recently analyzed chicken chimeras 
with a brain of different chromosomal sex (Maekawa et al., 
2013). However, in contrast to the previously observed results 
in Japanese quaU chimeras, we did not observe any abnormali- 
ties in male-typical copulatory behavior and spermatogenesis in 
the chicken chimeras that had a male karyotype in gonads and a 
female karyotype in their brain. Rather, sexual maturation was 
delayed and an irregular ovulatory cycle was exhibited in the 
chimeras that had a female karyotype in their gonads and male 
karyotype in their brains. This abnormality in sexual matura- 
tion and ovulatory cycle was not reported in the previous study 
conducted with Japanese quail. Since the baseline gonadotropin 
level in chicken chimeras that had a female karyotype in their 
gonads and male karyotype in their brains was comparable to that 
in female chickens, we hypothesized that irregular oviposition in 
the chimeras is caused by a timing mismatch of the gonadotropin 
surge due to the male-type chromosomal sex in the brain. 

Meanwhile, we also found that overall sexually dimorphic 
behavior, judged based on the results of the open field test and 
tests of sexual behavior, was not influenced by brain chromoso- 
mal sex. This suggests that gonadal steroids may determine brain 
function related to overall sexual dimorphic behavior. In addi- 
tion, the blood steroid level of the chicken chimeras was not 
affected by the sex of the brain, which was different from the sex 
of the remaining tissues; the sexual dimorphism of the BNSTp, a 
nucleus that is thought to be related to sexual identity in humans, 
was dependent on gonadal hormones. 

Only two studies of brain chimeras, one in Japanese quaU 
and another in chicken, have ever been conducted. Both studies 
showed that brain chromosomal sex directly affects reproduc- 
tive physiology, albeit with substantial discrepancies when com- 
pared in the details. We speculated the following three possible 
explanations for these discrepancies of pathology, resulting from 

1) Species difference: A comparison between the genome of quaU 
and chicken revealed that chromosome rearrangements may 
have occurred between these two Galliformes species over 35 

million years ago (Kayang et al, 2006). A draft Japanese quail 
genome sequence was assembled by means of next-generation 
sequencing technology (Kawahara-Miki et al, 2013). The 
results suggested that the genomes of Japanese quaU and 
chicken were closely related while being more distantly related 
to the genome of the zebra finch (Kawahara-Miki et al, 2013). 
However, genomic variation (Kawahara-Miki et al, 2013) 
and differences in reproductive physiology (Yoshimura, 2013) 
between Japanese quail and chicken have been found and such 
differences may affect pathology. 

2) Differences between the methods of transplantation: In the study 
conducted on Japanese quail chimera, only the forebrain 
was transplanted, whereas the total brain primordium was 
transplanted in our study of chicken chimera. Indeed, our pre- 
liminary results in quaU-chick transplantation revealed that 
the midbrain in which the dopaminergic neurons are known 
to show sexual dimorphism under sex chromosomal control 
in mammals was not exchanged by forebrain transplantation. 

3) Rejection: In our study of chicken chimera, the female tissue 
transplanted into male bodies was rejected at the time of sex- 
ual maturation (Maekawa et al, 2013). Since the rejection 
was strictly sex combination-dependent, we speculated that 
the rejection was attributable to the expression of a female- 
specific antigen coded by gene(s) on the W chromosome. 
Daily injection of an immunosuppressant was necessary to 
evaluate behavior and physiology of chicken chimeras that had 
a female karyotype in the brain and male karyotype in the rest 
of their body. On the other hand, no rejection was reported in 
the study on Japanese quail. It is possible that a mild rejection 
occurred in Japanese quail chimeras that had a female kary- 
otype in the forebrain and male karyotype in the rest of their 
body. In fact, we experienced that the partial transplantation 
of the brain primordium led to a mild rejection, which did not 
cause the death of the chicken chimera. 

Additional studies of Japanese quail and chicken chimeras that 
are created by the same experimental protocol would be necessary 
to fuUy understand whether the fundamental rule of brain sexual 
differentiation exists in Galliformes. 


Finally, we describe possible similarities in sexual differentiation 
of behavior and physiology in mammals and birds (Table 1). 


The sexual orientation of murine copulatory behavior is mainly 
determined by exposure of mice to sex steroids during devel- 
opment. Similarly, the sexual orientation of chicken copulatory 
behavior, including mounting in males and adopting a receptive 
posture in females, has been found in our study to be deter- 
mined by gonadal sex. Taken together, the above results suggest 
that overall sexual orientation of the copulatory behavior of both 
mammals and birds may be regulated by gonadal hormones. The 
brain nuclei related to sexual functions, namely the SDN-POA 
and BNST in mammals and the BNST in the chicken, are reported 
to be differentiated in a sexual dimorphic manner mainly by 

August 2014 | Volume 8 | Article 242 | 5 

Maekawa et al. 

The mechanisms underlying sexual differentiation 

Table 1 | Possible similarities of sex differences in brain, physiology and behavior in mammals and birds. 

Similarities Typical literatures 

1 Sex differentiation of core sexual behavior is mainly induced by the exposure of gonadal hormones Arnold and Chen, 2009; Maekawa et al., 2013 
during development (also, 2-5) 

2 The structure of BNST (M > F) is sexually differentiated mainly by the exposure of gonadal Tsukahara et al., 2011 
hormones during development 

3 Sex chromosomes in brain affects sexual dimorphism observed in communication such as song Agate et al., 2003; Cox and Rissman, 2011 
production in bird and social behavior in mouse 

4 Sex-biased local synthesis of neuroestrogen (M > F) Hojo et al., 2009, 2014 

5 Sexual differentiation of neuronal structures related to circadian timing could be affected by sex Kuljis et al., 2013 
chromosomes in the brain 

gonadal hormones. On the other hand, song performance in male 
zebra finches and sexual dimorphism observed in mice social 
behavior were reported to be affected by the sex chromosomes 
in the brain. Studies on a variety of species of mammals and 
birds may lead to a clear understanding of the precise interaction 
between such behaviors and the effect of sex chromosome in the 
brain or gonadal hormones. 


There is direct and indirect evidence that estrogen is locally pro- 
duced in the songbird brain and that its concentration is higher 
in the specific brain nuclei in males. The estradiol level in whole 
brain, at embryonic day 21 and at 13 months of age has been 
found to be significantly higher in males than in females, even 
though the plasma estradiol level in females was higher (Maekawa 
et al., 2013). Moreover, chicken chimeras that had a female kary- 
otype in their gonads and male karyotype in their brains had 
higher estradiol level in the brain relevant to male phenotype. 
Conversely, chimeras that had a female karyotype in their brains 
and male karyotype in their gonads had lower estradiol level 
relevant to female phenotype. These findings provide the first 
clear evidence that local estradiol production in the chicken brain 
is regulated by the chromosomal sex in the brain (Figure 1). 
Neuroestrogen has been reported to regulate the socio-sexual 
behavior in Japanese quail (Ubuka et al, 2014), although it 
remains uncertain whether sex difference of neuroestrogen is 
related to certain behavioral and physiological functions. In rat, 
the hippocampus has been shown to synthesize steroid hormones, 
which show sexual dimorphic concentrations (Hojo et al., 2004, 
2014). The estradiol level of the female hippocampus at all stages 
of the estrous cycle was much lower than in the male (Hojo 
et al., 2009), and this finding is consistent with the results show- 
ing higher estradiol level in the brain of male zebra finch and 
chickens. The common mechanism underlying the sex-biased 
local synthesis of neuroestrogen in mammals and birds could 
be further elucidated by focused research on the expression of 
steroid-synthesizing enzymes. 


Chicken chimeras that have a male karyotype in the brain and 
female karyotype in their gonads have an irregular ovulatory cycle 
(Maekawa et al., 2013). The circadian timing of ovulation was 
delayed in the chimera, indicating that the neural circuit respon- 
sible for the timing of ovulation may be differentiated under the 

Sexual differentiation 
of chicken brain 

chicken brain 



FIGURE 1 I Schematic diagram of the factors related to sexual 
differentiation of chicken brain. 

influence of chromosomal sex of the brain. It has been reported 
that mice having the XX chromosomal complement have longer 
activity duration than mice having the XY chromosomal com- 
plement irrespective of their gonadal sex (Kuljis et al., 2013), 
suggesting that sex chromosomal effect in mouse brain affects the 
circadian biological clock. Thus, the sex differences of neuronal 
structures related to determination of or mediating circadian tim- 
ing may provide an alternative target to elucidate sex- related brain 
function. Elucidation of the defect in circadian timing of ovula- 
tion found in chicken chimeras may provide new insights into the 
relationship between the biological clock and sex differences. 


In this review, we summarized the mechanisms underlying sexual 
differentiation of the behavior and physiology in mammals and 
birds. From a classical viewpoint, perinatal sex steroids secreted 
by the gonads differentiate the sex-specific behavioral and phys- 
iological functions as well as brain structures both in mammals 
and birds. However, recent studies suggest that brain sex chro- 
mosomes directly affect the sexual differentiation of certain types 
of behavior and physiology. Especially, our study using chicken 
chimeras revealed that brain sex chromosomes directly influence 
neurosteroid synthesis. 

In the context of environmental sciences examining harm- 
ful effects of endocrine disruptors, several studies have been 

Frontiers in Neuroscience | Neuroendocrine Science 

August 2014 1 Volume 8 | Article 242 | 6 

Maekawa et al. 

The mechanisms underlying sexual differentiation 

conducted to investigate how endocrine disrupters affect the 
production and secretion of steroid hormones, how they impair 
ligand-binding of steroid hormones to their receptors, and their 
effects on tissues. Based on the findings described in this review, 
the effects of endocrine disruptors on local steroid produc- 
tion in the brain should be considered as the focus for further 
investigation of the harmful effects of endocrine disruptors. 


This work was supported by Grant-in-Aid for Scientific Research 
(B) (23310043), (C) (24590307) from Japan Society for the 
Promotion of Science (JSPS) and by the National Institute for 
Environmental Studies [14309] [14013] to Fumihiko Maekawa. 


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Conflict of Interest Statement: The authors declare that the research was con- 
ducted in the absence of any commercial or financial relationships that could be 
construed as a potential conflict of interest. 

Received: 30 April 2014; accepted: 22 July 2014; published online: 14 August 2014. 
Citation: Maekawa -F, Tsukahara S, Kawashima T, Nohara K and Ohki-Hamazaki H 
(2014) The mechanisms underlying sexual differentiation of behavior and physiology 
in mammals and birds: relative contributions of sex steroids and sex chromosomes. 
Front. Neurosci. 8:242. doi: 10.3389/fnins.2014.00242 

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers 
in Neuroscience. 

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