Hormones and Behavior 55 (2009) 655665

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Hormones and Behavior

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h

New tricks by an old dogma: Mechanisms of the Organizational / Activational

Hypothesis of steroid-mediated sexual differentiation of brain and behavior
Margaret M. McCarthy , Christopher L. Wright, Jaclyn M. Schwarz
Departments of Physiology and Psychiatry, Program in Neuroscience, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore MD 21201, USA

a r t i c l e i n f o a b s t r a c t

Article history: The hormonal regulation of sexual behavior has been the topic of study for over 50 years and yet
Received 14 January 2009 controversies persist regarding the importance of early versus late events and the identity of the critical
Revised 24 February 2009 neural and cellular substrates. We have taken a mechanistic approach toward the masculinizing actions of
Accepted 25 February 2009
the gonadal steroid estradiol, as a means to understand how organization of the neuroarchitechture during a
perinatal sensitive period exerts enduring inuences on adult behavior. We have identied important roles
Keywords:
Preoptic area
for prostaglandins, FAK and paxillin, PI3 kinase and glutamate, and determined that cell-to-cell signaling is a
Hypothalamus critical component of the early organizational events. We have further determined that the mechanisms
Lordosis mediating different components of sexual behavior are distinct and regionally specic. The multitude of
Prostaglandins mechanisms by which the steroid estradiol, exerts divergent effects on the developing nervous system
Glutamate provides for a multitude of phenotypes which can vary signicantly both within and between the sexes.
Estradiol 2009 Elsevier Inc. All rights reserved.
Testosterone
Development
Sex differences
Sexual behavior

Introduction detailed exploration of the cellular mechanisms of organizational

steroid effects as a means for understanding how early hormone action
It has been 50 years since the publication of the iconic Phoenix, Goy, could persist into adulthood and thereby regulate behavior.
Gerall and Young (Phoenix et al., 1959) manuscript proposing the
Organizational / Activational Hypothesis of hormone effects on sex Brief historical overview
differences in reproductive behavior and now it is the focus of this
celebratory special issue of Hormones and Behavior. Fifty years is There are several seminal events that are built on the original
either a long time or the blink-of-an-eye for the study of a single Phoenix report that have inuenced the course of the eld ever since.
research topic, depending on your point of view. In this time, the The original animal model, the guinea pig, is a species with a small
dogma that has emerged is, simply put, that developmental exposure litter size, long gestation and precocial young. The fact that the
to gonadal steroids acts on the brain to organize the neural substrate seminal organizational events occurred prenatally both confounded
which is then selectively activated in the adult to induce expression of an investigation by the hormones of pregnancy and precluded the
sex specic behavior. This elegant synthesis effectively explained a ability to manipulate males and females independently. The labora-
collection of disparate data and provided a framework against which tory rat soon replaced the guinea pig as the dominant species for study
future work could be tested. Evidence in support of the essential truths due to the large litter size, short gestation and the observation that the
of the hypothesis have steadily accumulated over the intervening sensitive period for sexual differentiation began prenatally but
50 years, but evidence challenging or refuting the hypothesis has piled extended well into the postnatal period. That estradiol is the mascu-
up in an equally compelling fashion. The majority of published reports linizing steroid in rodents was codied by the Aromatization
have focused on hormonal manipulations and quantication of Hypothesis, which reconciled the divergent observations that exo-
behavioral endpoints. The approach of this laboratory has been a genous administration of estradiol was as or more effective than
testosterone at masculinizing behavior, that the critical brain regions
expressed estrogen receptors and that the synthetic enzyme, estradiol
synthase, or aromatase, was highly active in the neonatal brain. An
Corresponding author. Fax: +1 410 706 8341. additional critical piece of the puzzle was determining that the steroid
E-mail address: mmccarth@umaryland.edu (M.M. McCarthy). binding globulin, alpha-fetoprotein, preferentially binds estrogens in

0018-506X/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.yhbeh.2009.02.012
656 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665

rodents, thereby allowing testosterone to freely enter cells where it is expression of male sex behavior directed at sexually receptive females.
converted to estradiol (see for review (McCarthy, 2008). Decades later It is useful to divide male sexual behavior into an appetitive compo-
did it become apparent that androgens mediate sexual differentiation nent, meaning the motivation to seek out and pursue receptive
of the primate brain (see for review (Wallen, 2005), but this does not females, and a consummatory component, the physical act of
undermine the utility of the rodent model unless the cellular mecha- copulation with a female, which in male rodents is quantied as
nisms of estradiol and androgens to mediate masculinization are mounts, thrusts and ejaculations. Appropriate expression of both
different. Determining if this is true depends rst on determining the components of male sexual behavior requires a functioning medial
mechanisms in rodents before attempting a more focused approach in preoptic area (POA) and it is reasonable to assume that this is a
primates. principle site of organizational actions of steroids to masculinize
At the time of elucidation of the Aromatization Hypothesis it was behavior. This is also the brain region housing the SDN and much
self evident that the brain controlled behavior and was therefore the attention was given to the notion that the larger nucleus in males is
hormone responsive tissue referred to by Phoenix and colleagues, critical for male behavior (De Jonge et al., 1989; Jarzab et al., 1990;
who had limited their hypothesis to behavior. Despite overwhelming Houtsmuller et al., 1994), an idea proved false by some (Todd et al.,
condence that the brain was responsible, there was surprisingly little 2005), but still persisted by others (Jeong et al., 2008). There is some
evidence for sex differences in this structure. There were early hints of evidence that the SDN may be part of a network that suppresses female
hormonally-mediated changes in dendritic proles (Pfaff, 1966; sexual behavior (Hennessey et al., 1986), and therefore a substrate for
Raisman and Field, 1971), but the magnitude of the differences were defeminization, or that its function may be to direct sexual preference
small and of unknown signicance. It was the discovery of spectacular (Baum, 2006). Regardless, in addition to the SDN there is a marked sex
sex differences in the song control nuclei of birds (see for review difference in the morphology of neuronal dendrites and protoplasmic
(Arnold et al., 1996) that prompted a re-examination of the rodent astrocytes of the medial POA (Amateau and McCarthy, 2002a,b).
brain and led directly to the discovery of the sexually dimorphic Beginning in the rst few days of life, male rats have a higher density
nucleus of the preoptic area (Gorski et al., 1980). This small group of of spine synapses per unit dendrite, and male astrocytes have longer
Nissle dense cells located in the central heart of the medial preoptic and more frequently branching processes. The POA neuronal
nucleus is 57 times larger in males than females and has proved dendrites of male Macaques also have a higher density of spines
valuable both for establishing mechanisms by which the brain is and this is observed prior to puberty (Ayoub et al., 1983), conrming
organized and as a bell weather of estradiol-mediated masculiniza- that the neuronal architecture of primate brains is organized in a sex
tion. The realization that subregions of the brain could be substantially specic manner and suggesting that regardless of which hormone
larger in one sex led to the discovery of many more volumetric sex mediates the effect (i.e. estradiol versus testosterone), the endpoint
differences in the mammalian brain (Guillamon and Segovia, 1996; is the same.
Byne, 1998; Simerly, 2002), including humans (Swaab and Fliers, Estrogen receptors are members of a superfamily of transcription
1985; Allen et al., 1989; Allen and Gorski, 1990, 1992) The rst insight factor receptors that interact directly with DNA to either promote or
into the mechanism establishing the sex difference in SDN size came repress gene expression. Estrogen receptors also reside in the
from the spinal cord, in particular the spinal nucleus of the membrane where they interact directly with various protein kinases
bulbocavernosus, or SNB, which houses motor neurons innervating to directly activate signal transduction pathways. This duality of action
the penis. Not surprisingly this nucleus is substantially larger in males, provides for a startling level of complexity in potential steroid effects.
and Arnold and colleagues established that this is because the motor The same hormone can directly impact the expression of multiple
neurons in the female die early in development (Nordeen et al., 1985). genes, which can initiate a cascading prole of secondarily activated
The principle that males and females start with the same cohort of genes, while at the same time exerting rapid membrane initiated
cells but they preferentially die in one sex or the other has now been classic signal transduction pathways (see for review McCarthy 2008).
generalized to several reproductively relevant brain nuclei (see for Given this complexity, it was not terribly surprising that no one effector
review (Forger, 2006), most notably the SDN (Davis et al., 1996), and pathway had been identied to mediate estradiol-induced masculini-
the AVPV (Murakami and Arai, 1989). zation of sex behavior. Indeed, many had quite justiably concluded
While it is highly likely that size matters, there are many brain that instead of activating one gene 100-fold, it was more likely that
regions critical to the control of reproduction that are not noticeably estradiol activated ten genes 10-fold, or even 100 genes 1-fold or less.
larger in one sex. In the 1980s, after the extensive attention given to For many years the empirical data supported this view. Almost all the
SDN, was a re-examination of the potential for synaptic differences. major neurotransmitter systems had been implicated as important to
Exhaustive and conclusive studies by Arai and colleagues, most normal masculinization, including noradrenaline (Beyer and Feder,
notably Matsumoto, demonstrated profound differences between 1987), acetycholine (Dohler et al., 1991), dopamine (Hull et al., 1998),
males and females in the frequency of dendritic spine versus somatic serotonin (Ladosky and Gaziri, 1970) and even the amino acid GABA
synapses in the arcuate nucleus (Matsumoto and Arai, 1980), (Davis et al., 2000). However, the data implicating each system was
ventromedial nucleus (Matsumoto and Arai, 1986) and amygdala restricted to demonstrating that if neurotransmission was disrupted,
(Nishizuka and Arai, 1981). Their use of EM to identify and quantify so was masculinization. What was missing was the ability to induce
the different types of synapses left no room for doubt that the masculinization in females in the absence of exogenous steroid
synaptic prole was differentially weighted towards excitatory spine administration, in other words to substitute for the steroid by
synapses or inhibitory somatic synapses in one sex or the other in administering the downstream cellular process activated by the
specic brain regions. Moreover, the work of Matsumoto and Arai steroid.
demonstrated unambiguously the ability of steroid hormones to This goal was achieved with the surprising discovery that the agent
permanently organize this sex difference in synaptic patterning. of change in this case was not a traditional neurotransmitter at all, but
What remained to be determined was the cellular mechanism behind was instead a lipid membrane derived molecule, prostaglandin E2
the hormone action. (PGE2). Prostaglandins are derived from arachidonic acid following
cyclinization by the cyclooxygenase enzymes, COX-1 and COX-2
Mechanisms mediating masculinization of sexual behavior (Kaufmann et al., 1997). Usually associated with inammation, PGE2
is emerging as an important modulator of neural activity both by
Masculinization in this context is a steroid-hormone driven indirect effects on glia (Bezzi et al., 1998; Sanzgiri et al., 1999), and
developmental process that organizes the neural substrate so that it directly on neurons (Rage et al., 1997; Kasai and Mizumura, 2001;
is conducive to being activated by androgens in adulthood for the McCullough et al., 2004). In the mature POA, PGE2 is a potent
 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665 657

stimulator of LHRH release and plays an essential role in maturation of tions from the amygdala relevant to olfactory stimuli, important
the pulse generator at puberty (Ma et al., 1997). This observation reciprocal connections with the ventromedial nucleus (VMN) of the
prompted a talented graduate student, Stuart Amateau, to ask hypothalamus, a critical brain region in the control of female sexual
whether PGE2 might also play a role in the developing POA, and behavior, and thalamic nuclei that convey information about genital
more specically in the process of masculinization. Through a series of stimulation (Simerly, 2002). We currently have no information as to
studies, Amateau established that during the sensitive period for whether any or all of these regions are contributing to the increased
sexual differentiation, activation of estrogen receptors in the POA excitatory input to the male POA.
induces expression of COX-2 (and COX-1), which leads to a 7-fold
increase in PGE2, but not the other prostanoids (Amateau and Mechanisms mediating defeminization and feminization of
McCarthy, 2004). PGE2 induces the formation of neuronal dendritic sexual behavior
spines via a mechanism that involves at least in part, the release of
glutamate and the activation of AMPA, but not NMDA, glutamate Defeminization is the active developmental process whereby the
receptors (Amateau and McCarthy, 2002a). Research by others reveals capacity to express female sexual behavior in adulthood is lost. It has
a calcium dependent release of glutamate from astrocytes following been known for some time that this is a separate physiological process
stimulation by PGE2 (Bezzi et al., 1998), and astrocytes of the POA are from masculinization (Vreeburg et al., 1977), and it was recently
responsive to estradiol and sexually dimorphic (Amateau and established that the mechanism of defeminization is entirely
McCarthy, 2002b). This has led us to propose a working model in independent of PGE2 action (Todd et al., 2005). As with the gonad
which PGE2 released from estradiol-responsive neurons in the male and gonadal duct systems, the female phenotype is the default, and for
promotes glutamate release from astrocytes which in turn promotes a male to develop properly, the female phenotype must be repressed
the induction and/or maturation of dendritic spines. Treating females or removed. That this is required for the reproductive organs seems
with either estradiol or PGE2 results in a male-like pattern of dendritic clear, but why it occurs in the brain is a mystery. Given that sexual
spines, and blocking COX-1 and COX-2 activation in males produces a behavior must be activated in adulthood by sex-typic hormones, the
female-like level of spines. Conrmation that this is truly an need to alter the neuronal substrate controlling female sex behavior in
organizational effect in the classic sense is conrmed by maintenance males is not obvious.
of the induced changes well into adulthood (Amateau and McCarthy, Regardless of why it occurs, advances into the mechanism of
2002a, 2004; Wright et al., 2008). defeminization have been made. Male mice with a null mutation for
Prostaglandins act via multiple receptors but for PGE2 the four the beta isoform of the estrogen receptor, known as BERKOs, have
principle ones are EP1-4. Each receptor has a different afnity for incomplete defeminization but normal masculinization (Kudwa
PGE2 and a unique signal transduction prole, yet the degree of et al., 2005), suggesting a differential role for each receptor subtype
cross-talk and overlap between the receptors is substantial (see for
review (Regan, 2003). There are no detectable sex differences in
the mRNA levels for each receptor during the sensitive period for
sexual differentiation or after (Burks et al., 2007), and it appears
instead that it is sex differences in the COX enzymes that drive the
process of masculinization (Amateau and McCarthy, 2004). All four
receptors for PGE2 are expressed in the neonatal POA, and all four
have been implicated as contributing to PGE2-induced masculini-
zation (Burks et al., 2007), but it does not seem that all four are
necessary. Convergent evidence generated by Christopher Wright
implicates EP2 and EP4 as the dominant receptors involved
(Wright et al., 2008). Activation of either EP2 or EP4 increases
protein kinase A (PKA) signaling, and recent ndings indicate that
disrupting PKA neonatally blocks masculinization (Wright and
McCarthy, unpublished observation Fig. 1). The activation of PKA
may also explain the apparently positively reinforcing effects of
PGE2. As little as a single intracerebroventricular injection of PGE2
to newborn females is sufcient to induce full masculinization of
dendritic spine density and adult sexual behavior (Wright et al.,
2008). The activation of PKA following binding of PGE2 to EP2 or
EP4 is capable of inducing more PGE2 production by up regulating
the synthesis and activity of the COX enzymes via a GSK mediated
pathway (Regan, 2003). This positive feed forward effect of PGE2
likely involves both COX-1 and COX-2, which can be found in
neurons, astrocytes and microglia. Thus distinguishing the precise
cellular site of estradiol action in this system is challenging and
may ultimately prove to be unimportant as the role for cell-to-cell
communication emerges as a critical component of the process of
masculinization.
The next frontier in understanding PGE2-induced masculinization
Fig. 1. Estradiol induces prostaglandin E2 (PGE2) synthesis to promote dendritic spine
is to place the ndings in the POA into the context of the entire formation and sexual differentiation in the POA. Estradiol aromatized from testicular
circuitry controlling male and female sexual behavior. The increase in androgens binds to ER alpha and induces transcription of the COX-1 and COX-2 genes,
dendritic spines on POA neurons must reect an increase in afferent the rate limiting enzymes in PGE2 synthesis. PGE2 activates multiple receptors, the
input, but from where is unknown. Does the increase in dendritic most important being EP2 and EP4, both of which are linked to activation of Protein
Kinase A (PKA). Through mechanisms that remain poorly understood, PKA enhances
spines reect more afferent inputs from the same source or the the actions of glutamate at AMPA, but not NMDA receptors, and the formation of new
establishment of a new source of input? There are multiple dendritic spines and the organization of a higher density of spine synapses on male POA
behaviorally relevant sources of input to the POA, including projec- neurons compared to female.
658 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665

in mice. In rats, early work suggested a distinction between the An additional group of signaling molecules of interest is focal
relative importance of androgens versus estrogens in defeminization adhesion kinase (FAK) and its associated proteins, including paxillin
versus masculinization, respectively (Vreeburg et al., 1977), but it is (Ren et al., 2004). These molecules were rst of interest in the study
clear that defeminization can be induced by estradiol just as of metastatic cancers but have since been found to be integral to
effectively (Todd et al., 2005). An important limitation in studies neuronal growth and branching (Yang et al., 2003; Rico et al., 2004;
of defeminization is the lack of an identiable neuroanatomical Robles and Gomez, 2006). In a search for sex differences in signaling
substrate. The POA is the critical brain region controlling male sexual molecules, Debra Speert found FAK and paxillin were both
behavior, but as noted above, the SDN-POA may act to suppress signicantly higher in neonatal female hypothalamus and were
female sexual behavior and therefore serve as a substrate for defe- down regulated by estradiol (Speert et al., 2007). The decrease in
minization. The VMN of the hypothalamus is the essential region for FAK and paxillin induced by estradiol was correlated with an
expression of female sexual behavior and it is also possible that this increase in neurite complexity, suggesting a plausible mechanism
nucleus is a substrate for defeminization, either alone or in con- establishing the longer and more frequently branching dendrites of
nection with the SDN. males. The observation that estradiol decreases FAK and paxillin is
Exploration of the VMN as a potential site subject to defeminiza- unusual since most, if not all, other molecules implicated in sexual
tion begins with a search for sex differences in the neuroanatomy. differentiation involve an increase in expression in response to
Similar to the POA, male VMN neurons have more dendritic spine estrogens or androgens. Given that defeminization is the active
synapses than females (Schwarz et al., 2008). However, unlike the removal of the capacity for female sexual behavior, it is plausible that
POA, the greater number of spines in males is not due to an increased suppression of the endogenously high levels of FAK and paxillin by
spine density per unit dendrite but is instead the result of a more estradiol is a mechanism to achieve that end. In this instance, higher
complex dendritic tree in males. Male VMN dendrites are signi- levels of FAK and paxillin in females could be a component of the
cantly longer and branch more frequently than those of females default process of feminization. Distinguishing between the two
(Mong et al., 1999). Neurite growth and branching is a complex possibilities may be difcult. If defeminization is simply the sup-
process that involves intrinsic and extrinsic signals, including inter- pression of mechanisms inducing feminization, then they are two
action with the substrate as well as other cells. Membrane to sides of the same coin and cannot be separated. Conversely, if
membrane signaling via the ephrins (Penzes et al., 2003; Gamble defeminization were a process that simply overrides feminization,
et al., 2005) and membrane to substrate signaling via the integrins they would indeed be two separable processes (Fig. 2). In both
(Robles and Gomez, 2006) are viable targets for hormone action but scenarios the behavioral endpoint would be the same, the loss of the
have not been investigated to-date. capacity to respond to adult activational steroids with expression of

Fig. 2. Feminization versus defeminization. We propose three theoretical conceptualizations of feminization versus defeminization. The rst option is that the two processes are
inseparable and estradiol-induced defeminization erases feminization. A prediction of this option would be the inability to recover the capacity for female sex behavior in adulthood.
The second option proposes distinct mechanisms for defeminization and feminization, both of which occur but the former is induced by estradiol and suppresses the latter which is
independent of estrogen action during development. An example would be the formation of a larger SDN in order to suppress the female sexual behavior neural template. The third
option involves two separate mechanisms induced by estradiol, one to suppress the process of feminization and the other to initiate defeminization. The suppression of FAK and
paxillin and the induction of glutamate release by estradiol in the developing VMN would t this model.
 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665 659

female sexual behavior, but in the rst instance the mechanistic and rapidly promotes the release of glutamate from presynaptic
process of feminization would be blocked, whereas in the second terminals, which in turn activates postsynaptic NMDA and AMPA
instance the cellular events of feminization would occur but would receptors, leading to calcium inux, activation of MAP kinase and
be negated by the cellular events of defeminization (Fig. 2). The only promotion of dendritic spine formation (Schwarz et al., 2008). The
hope for resolving the conundrum is to identify the cellular mecha- enhanced glutamate release requires estradiol-induced activation of
nisms of defeminization and feminization. PI3 Kinase, a kinase involved in a wide range of cellular functions, and
The development of the testes and the ovary provides an apt this occurs as rapidly as 1 h after steroid exposure. Neither the
analogy in that both begin from an undifferentiated gonadal anlage activation of PI3 Kinase nor the enhancement of glutamate release by
which will by default become an ovary but will differentiate into a estradiol requires protein synthesis, but they do require the estrogen
testis following expression of a single gene, SRY (Harley and receptor since the effects are blocked by pretreatment with the ER
Goodfellow, 1994). The undifferentiated gonad has two tissue types antagonist, ICI 182,780. In a series of elegant experiments involving
and the one, the cortex, develops in the female, and the other, the latrotoxin-induced glutamate release, Jaclyn Schwarz further demon-
medulla, develops in the male. This primordium is bipotential and strated that construction and maintenance of new spines in the post-
thus producing male characteristics necessarily reduces female synaptic cell does require protein synthesis, but not the activation of
characters. Thus for testis development there is a single critical ER (Schwarz et al., 2008). Thus there is an essential requirement for
event that provides a wedge point for exploration (Sekido and Lovell- cell-to-cell communication for establishment of sexually dimorphic
Badge, 2009). The current state of understanding of how the testis synaptic patterning in the VMN (Fig. 3). This is similar to the POA
develops reects this advantage as it is highly detailed and advanced where cell-to-cell communication is also critical, and yet different in
and far exceeds that of the ovary, about which much less is known that there is no apparent role for astrocytes in the VMN but there is in
(Lofer and Koopman, 2002). This is likely only a matter of time, the POA.
however, given that the ovary is a complex well-characterized The observation that estradiol promotes glutamate release to
endocrine organ about which experimental predictions can be made. induce dendritic spines in the hypothalamus predicts that neonatal
In the brain, we do not even know what a female brain is, other than glutamate administration would substitute for estradiol in inducing
it is not male. Thus elucidating the mechanism(s) of feminization is a defeminization. Conversely, blocking glutamate transmission neona-
daunting challenge. tally should disrupt estradiol-induced defeminization. Both of these
Returning to the question of defeminization and the plausible predictions have been proven true when assessed behaviorally or
assumption that a good place to begin looking is the VMN of the anatomically (Schwarz et al., 2008; Schwarz and McCarthy, 2008).
mediobasal hypothalamus, a surprising role for non-genomic actions However, there is also a positive effect of glutamate receptor
of estradiol has recently emerged. As discussed above, steroid activation on organization of male sexual behavior, suggesting a
receptors are members of a transcription factor super-family and functional connection between the cellular events of masculinization
their principle action is at the genome. However, steroid receptors can and defeminization.
also interact with and initiate signal transduction pathways originat-
ing at the membrane (see for review McCarthy 2008). These effects Why so many mechanisms?
tend to be rapid and transient. Therefore, when considering steroid-
induced sexual differentiation of the brain it seemed unlikely that Given that there appears to be a functional connection between
membrane-initiated events would be an important contributor. But the cellular processes of masculinization and defeminization, it is
the facts have proved otherwise. Jaclyn Schwarz has demonstrated reasonable to ask, why they are not achieved by the same cellular
that in immature VMN neurons estradiol binds to its cogent receptor mechanism. If normal male development requires a 2-step process in

Fig. 3. Estradiol enhances glutamate release to promote cell-to-cell communication and sexual differentiation in the VMN. In the developing male VMN dendrites are longer and
branch more frequently than in the females, resulting in a greater number of dendritic spines. This permanent organizational effect begins with the nongenomic activation of PI3
kinase by ligand-activated ER alpha. The increased released glutamate binds to receptors on the post-synaptic neuron, inducing activation of MAP kinase, protein synthesis and the
formation of new dendritic spines (Schwarz et al., 2008).
660 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665

which the male phenotype is induced and the female phenotype is genetically or environmentally induced variation. Polymorphisms in
erased, it would seem much more efcient to simply achieve both alleles for COX-2, GAD, PGEsynthase, NR2, mGluR's, FAK, paxillin,
endpoints with one mechanism. In other words, the process of BDNF and trkB receptors could have profound inuences on the
masculinization would effectively erase the female neural substrate. actions of estradiol in discrete brain regions without compromising
That this is not the case is most denitively demonstrated by the function in other brain regions. Moreover, epigenetic changes in
behavior of animals in which masculinization has been prevented or associated genes in response to variance in maternal care (Cameron
induced via the prostaglandin pathways. Males that were treated et al., 2008; Champagne, 2008), diet (McGowan et al., 2008) or
neonatally with a COX1 and 2 inhibitor show very low levels of stress (McEwen, 2008) could impact estrogen action during the
masculine sex behavior as adults, and if the same animals are treated critical period, resulting in enduring variation in the organizational
with a hormonal milieu conducive to female sexual receptivity they substrate on which activational hormones will modulate adult
show no response. Thus these animals are essentially asexual as physiology and behavior.
adults. Conversely, females masculinized by PGE2 as neonates
respond perfectly well as adults when treated with sequential estra- Challenges to the dogma
diol and progesterone and exhibit normal female sexual receptivity
(Todd et al., 2005). Thus it is apparent that PGE2 mediated mascu- The value of the Organizational / Activational Hypothesis has
linization is entirely distinct from defeminization, but why? What is been the ability to clearly frame questions and thereby challenge the
the advantage of having an entirely separate mechanism for predictions stemming from this construct. The primary challenge was
defeminization? Is there a cost to retaining the capacity to show made even prior to the formulation of the hypothesis and persists to
female sexual behavior when a male would never see the appropriate this day, which is that under some circumstances males can be
hormonal milieu anyway? This question remains unanswered, but induced to show female typical behavior in adulthood, and likewise,
when exploring the role of glutamate in defeminization we found normal females will mount other females or even mount males on
some animals with both incomplete masculinization and incomplete occasion (Beach, 1948). The degree of behavioral plasticity and in
defeminization. This resulted in animals with a phenotype some- which sex it predominates varies by species and strain (Goy and
where in between normal males and females and revealed the Goldfoot, 1975).(Goy and Goldfoot, 1975) In our laboratory, male
capacity for a far greater variability in behavioral responding than Sprague Dawley rats treated with female hormones rarely if ever
would be possible if a single mechanism mediated both processes show lordosis behavior and likewise, females only occasionally
(Fig. 4). The potential for variability is even more pronounced when exhibit male mounting behavior. Nonetheless, that there is plasticity
one considers that during the same developmental time frame in adult sexual behavior is not in question, but whether it challenges
estradiol is preventing cell death in the SDN (Arai et al., 1996), or even negates the Organizational / Activational Hypothesis is a
inducing cell death in the anteroperiventricular (AVPV) nucleus topic of debate. A strict interpretation of the hypothesis insists that
(Murakami and Arai, 1989; Forger et al., 2004), increasing BDNF organizational events are permanent and therefore immutable,
levels in the hippocampus (Solum and Handa, 2002), inducing PR in whereas a more liberal interpretation allows for organizational
the POA (Quadros et al., 2002), increasing GABA and it's rate-limiting events to be enduring but ultimately reversible. Both interpretations
enzyme, GAD, in the arcuate nucleus (Mong et al., 2002) and nume- have been demonstrated for behavior. Male guinea pigs can be
rous others including some that may not yet have been discovered. induced to show lordosis if given pulsatile estradiol treatments over
Each one of these mechanisms provides multiple nodes for an extended period and tested repeatedly. However, these males

Fig. 4. Multiple phenotypes due to multiple mechanisms. Estradiol simultaneously activates multiple cellular mechanisms in discrete brain regions during the sensitive period of
sexual differentiation for feminization, defeminization and masculinization. The ultimate impact is a far greater number of potential phenotypes than would be achieved by a single
unitary mechanism that mediated sex differences in physiology and behavior.
 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665 661

never show proceptive behavior, an additional critical component of behavior. But these two nuclei are intimately connected with each
sexual behavior (Olster and Blaustein, 1989). Thus the sexual other as well as being part of a larger network of nuclei integrating
differentiation of lordosis is enduring but ultimately reversible sensory stimuli and motivational cues to coordinate a coherent
whereas the sexual differentiation of proceptivity is permanent and behavioral response. Careful students of anatomy such as Newman,
apparently immutable. Simerly and De Vries have long suggested that there is only one
Central to observations of dual sexuality in adult animals is the neural network controlling sex behavior but it is differentially
nature of the neural circuits controlling male versus female sexual weighted toward expression of either male or female motor patterns
behavior. Because male and female sexual behaviors are so different in response to specic stimuli (De Vries and Simerly, 2002; Simerly,
in motor patterns and hormonal dependence, it is often assumed 2002). That this issue remains unresolved 50 years after the
that there are two distinct neural circuits that independently control codifying of the Organizational / Activational Hypothesis is a
each behavioral repertoire. And in an appealing analogy to the testament to the complexity of the neural and hormonal control of
Mullerian and Wolfan duct systems that give rise to the female reproduction.
versus male reproductive tracts, respectively, masculinization could Can studies on the mechanism of sexual differentiation provide
be seen as the retention of the male circuit and defeminization as any insight into this issue? At rst blush the observation of clearly
the active degeneration (i.e. Mullerian Inhibiting Hormone) of the separate processes for masculinization and feminization favors the
female circuit. The shortcoming of this analogy is the inability to view that there are distinct circuitries for male versus female sexual
actually identify separate male and female neural circuits. There are behavior. For example, PGE2-induced masculinization of females
clear nodes that are more critical to one sex than the other, such results in adult animals that show perfectly normal male sex
that an intact and functioning POA is critical for male sexual behavior under conditions of high testosterone, and perfectly normal
behavior and the same is true for the VMN and female sexual female sex behavior following sequential estradiol and progesterone

Fig. 5. Synaptic proles predict male behavior. Increases in spinophilin protein correlated with the density of dendritic spines on POA neurons and measures of adult male sexual
behavior correlated with adult POA spinophilin protein. A) Stylized representation of spinophilin in the necks of dendritic spines. B) Image of POA cultured neuron visualized by
immunoctyochemistry for spinophillin which is concentrated in spine-like protrusions (Amateau and McCarthy, 2004). C) Effect of neonatal treatment with EP receptor agonists on
adult POA spinophilin protein assayed by western blot. Each lane represents the POA of one animal in a typical western immunoblot for spinophilin (above) and GAPDH loading
control (below). POA spinophilin protein levels incrased 24 fold above vehicle (VEH) treated animals and equivalent to PGE2 treated animals (Wright et al., 2008). D) Regression of
adult POA spinophilin protein levels and number of mounts in individual animals shows a signicant correlation (p b 0.001) between the two endpoints.
662 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665

(Todd et al., 2005). The converse is also true, if normal masculiniza- sexually dimorphic). This leaves the sexual differentiation of synaptic
tion in males is blocked by inhibiting COX-2 activity, those males patterning as a potential mediator of male versus female sex
will show no sex behavior in adulthood, either male or female. Thus, behavior. As reviewed above, in the POA males have a 2- to 3-fold
if one uses the Mullerian / Wolfan duct analogy, PGE2-masculinized greater density of dendritic spine synapses, while in the VMN males
females have both systems, whereas males treated neonatally with have 2- to 3-fold more dendritic spines because the dendrites are
a COX inhibitor have neither system. But, as mentioned above, it is longer and branch more frequently. These hormonally induced
also possible to generate animals with a phenotype half-way be- differences are enduring and levels of a marker of dendritic spines
tween fully masculinized and fully feminized (Schwarz and in the POA correlate with measures of masculine sex behavior in
McCarthy, 2008), and under some circumstances there is tremen- adults (Fig. 5). This correlation is intriguing in that it suggests a
dous plasticity in adult sexual behavior. Does this mean that early direct relationship between the number of dendritic spines and the
organization of the neural substrate is not occurring? To answer this magnitude of the expression of male sexual behavior. The more POA
question, we must rst identify what organization of the neural spine synapses, which are excitatory, the more intense the behavior.
substrate means. In other words, the system is weighted towards male behavior by
The purest examples of early organizational actions of gonadal increased excitatory input onto the critical POA neurons. Females
steroids are arguably those involving differential cell death, and the still have plenty of POA dendritic spine synapses but simply not
two best examples of that are the larger SDN and smaller AVPV in enough to put them over a threshold necessary for expressing male
males. Each of these subnuclei begin as a similarly sized collection of sex behavior (Fig. 6). Steroid-induced neuronal plasticity in the adult
neurons in both sexes, and then neurons die selectively in one sex frequently involves induction of dendritic spine synapses (Woolley,
due to the lack or presence of estradiol, resulting in a signicantly 1999), and an important component of organization is likely a
smaller nuclear volume. Cell death is permanent, and this is reduction in sensitivity of the adult brain to the sex-typic hormonal
therefore a permanent organizational difference. Although even prole of the opposite sex and a retention of sensitivity to your own
this seemingly irrefutable statement is challenged by the recent sex-typic hormonal prole. The idea that steroid sensitivity is
observation that continuing cell genesis around puberty contributes organized is not new (Sodersten, 1984), but past focus has been
to the maintenance of the volumetric sex difference in both the SDN on changes in steroid receptor levels and we are now proposing that
and AVPV (Ahmed et al., 2008). More importantly from the point of perhaps the sensitivity is at the level of the neural plasticity, as
view of sexual differentiation of behavior, however, is that neither of opposed to the steroid receptors themselves. The threshold for
these volumetric sex differences appears to have any relevance to activation of behavior is thereby determined by the previously
sex behavior (the SDN may play a role in partner preference (Baum, organized sensitivity of neuronal plasticity and the threshold can be
2006), whereas the AVPV is critical to the control of gonadotropin shifted by the intensity and duration of activational hormone
secretion from the anterior pituitary (Simerly, 2002), which is treatment to induce plasticity. This interpretation could also help

Fig. 6. Neural circuit(s) controlling sex behavior. The question of whether there are two distinct neural circuits that separately control expression of male versus female sexual
behavior, or a single neural circuit that is preferentially weighted towards one versus the other, remains unresolved. The observation of increased density of dendritic spines on POA
neurons correlating with male sexual behavior is consistent with a single circuit that is weighted in favor of males in response to salient olfactory and somatosensory input. Similarly,
the observation of decreased dendritic branching and fewer dendritic spine synapses in the VMN suggests a weighting in favor of female sexual behavior. But in both instances, the
potential for synaptic plasticity and expression of opposite sex behavior still exists.
 M.M. McCarthy et al. / Hormones and Behavior 55 (2009) 655665 663

explain the anomalous behavior of some mutant mouse strains, such denitive conclusions from being reached. There is still a great deal
as that lacking the Trp2c channel (Kimchi et al., 2007). This mouse to learn regarding the specic mechanisms of organizational actions
has altered vomeronasal organ function and females show very high of hormones on the developing brain and the consequences of this
levels of indiscriminant male sexual behavior. The threshold for organization for adult function. But on a more global level there are
expression of male sexual behavior appears to be much lower in two basic questions which constitute large gaps in our knowledge;
mice to begin with, compared to rats, and it is possible that the 1) how are the organizational actions of steroids maintained across
distortion of vomeronasal organ function induces a brainstorm of the lifespan?, and 2) how are the distinct cellular mechanisms of
electrical activity that reduces the threshold still lower by inducing steroids, in particular estradiol, coordinated across multiple brain
excessive excitatory input to the existing POA dendritic spine regions to generate a coherent neural network? Both of these are
synapses. Unfortunately there is currently no information available tractable experimentally but will require different approaches than
regarding the dendritic morphology of the mouse POA, making those currently being used. Epigenetic modications of critical
studies on sexual differentiation of the mouse brain and behavior genes is a reasonable candidate for organizing an enduring effect on
difcult to interpret. a neural circuit that could, under specic circumstances, be at least
Studies of mice have also constituted another source of challenge partly un-done, and this is likely to be a fruitful future area of
to the dogma, the importance of genetic mediators as opposed to research. The coordination across brain regions requires a more
gonadal or steroidal mediators of sexual differentiation. The ability to integrated approach. This has already been achieved for the sexually
generate mice that are genetically female (XX), but express the SRY dimorphic projection of the pBNST to the AVPV and the discovery of
gene on an autosome and thereby have testis and comparing them to a target-derived diffusible factor from the former that draws in the
genetic males (XY) lacking the SRY gene and thereby have ovaries, axons of the latter (Ibanez et al., 2001). Similar things may be
allows for discriminating between these two sources of biological occurring between other sub-nuclei, but there is more than one way
determinism (Arnold, 2004). Many interesting behavioral and to wire a brain and it cannot be assumed the same principle applies
neuroanatomic endpoints are impacted by genetic sex, but when it broadly. Finally, the use of live imaging to monitor the process of
comes to the differentiation of reproductive behavior, hormones still hormonally-induced synaptogenesis in real time may further our
carry the day (De Vries et al., 2002). So in this instance, a ability to understand cell-to-cell communication during these
wonderfully crafted challenge to the dogma was highly effective at critical developmental windows and provide evidence of more
demonstrating the validity of the dogma, while simultaneously new tricks for an old, but still very lively dogma.
highlighting that there are multiple other endpoints that exhibit sex
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