RESEARCH COMMUNICATION

I*iplications of estrogen-djpendent brain organization for

:Ygoder .dtffrences in hypothalamo-pituitary-adrenal

regulation
- . /i.)flfl ,K. I’&1CHZV SHINJI IL45Ifl,t CHITOSE ORIKASA,t AND OSBORNE F. X. ALMEIDA
‘Deasimesit of.Neu ndorisology, Max Planck Institute of Psychiatry, Clinical Institute, 80804 Munich, Germany;

and tDepartsn#{232}nt#{243}fAnatomy and Embryology, lbkyo Metropolitan Institute for Neuroscience Fuchu-city, Tokyo 183,
Japan

ABSTRACT Estrogens, derived from the aromatization to negative feedback actions of adrenal steroids. It is now
of testosterone in the brain, account for sex-specific or- widely held that hypothalamic peptidergic “drive” and the
gani.zation of neural circuits controlling gonadotropin efficacy of glucocorticoid-mediated feedback largely deter-
release and sexual behavior. This study examines the pos- mine the characteristics of basal and stress-related HPA ac-
sible organizing role of perinatal gonadal steroids in the tivity (1-3). In addition, down-regulation of hippocampal
manifestation of known, albeit unexplained, male-female corticosteroid receptors (in particular, of the so-called
differences in basal and stress-related adrenocortical glucocorticoid-preferring receptors, GR), due to chronic
secretion. We document here the existence of gender- adrenal hypersecretion and the resulting impairment of
specific differences in the gene expression of hypotha- glucocorticoid feedback on hypothalamic peptidergic neu-
lamic corticotropin-releasing hormone (CRH), and hip- rons, are presumed to be causally involved in symptoms of
pocampal and hypothalamic glucocorticoid receptors HPA hyperactivity associated with stress-related and affec-
(GR), diurnal corticosterone secretion, as well as in the tive disorders (4, 5).
responsiveness of CRH and GR mRNA levels to ex- The neuroendocrine response to stress displays significant
ogenous estradiol. In addition, we report that neonatal es- gender differences that appear to be associated with the
trogenization of female rats profoundly affects several presence of sex-specific gonadal steroids (6-9). Several sub-
regulatory substrates of the hypothalamo-pituitary- strates of the HPA axis, such as CRH, AVP, and cerebral
adrenal (HPA) axis, namely, the gene expression of CRH, corticosteroid receptors, are reportedly affected by altera-
arginine-vasopressin (AVP) and GR in the brain, and the tions in the gonadal steroid milieu (10-19). Although a role
responsiveness of these parameters to estrogen. The ne- for gonadal steroids in the regulation of the HPA axis in the
onatal treatment appeared to “defeminize” a number of adult rat has been recognized for at least 30 years, the impor-
neuroendocrine mechanisms related to HPA function; tance of sex hormone-dependent brain development in the
these changes were reminiscent of those observed in organization of neuroendocrine circuits that control HPA
earlier studies on sexual differentiation of reproductive function has not been addressed. Gonadal steroids play an
behavior and hormonal secretion. The results indicate a important role in brain development, and in the rat there is
pivotal role for estrogens during early development for some temporal coincidence between their organizing actions
the determination of gender-specific differences in HPA and the maturation of the neuroendocrine response to stress
function in the mature animal and demonstrate for the (c.f. refs 20, 21-24).
first time that the brain-organizing actions of gonadal The efficacy of gonadal steroids, particularly estrogens in
steroids may extend to nonreproductive neuroendocrine sex-specific organization of neuroendocrine and behavioral
axes.-Patchev, V. K., Hayashi, S., Orikasa, C., Al- regulatory circuits, has been extensively documented with
meida, 0. F. X. Implications of estrogen-dependent brain respect to reproductive functions. The temporal patterns of
organization for gender differences in hypothalamo- gonadal steroid secretion during development are markedly
pituitary-adrenal regulation. FASEB J. 9, 419-423 (1995) different between males and females, and it is testicular
testosterone, secreted during the critical period of brain de-
Key Woids: corticotropin-releosing hoimone vosopressin glucocor/icoid velopment, that causes gender-specific differentiation of the
receptor corticosterone mammalian brain. Paradoxically, however, this organizing
action of testosterone requires its aromatization to estrogen,
i.e., exposure to testosterone-derived estrogen is responsible
THE NEUROENDOCRINE RESPONSE TO STRESS involves a cascade for the “masculinization”/”defeminization” of the brain with
of secretory events that is initiated by the hypothalamic neu-
ropeptides corticotropin-releasing hormone (CRH)2 and
arginine-vasopressin (AVP), and culminates in the release of
glucocorticoids from the adrenal cortex. The latter elicit a ‘To whom correspondence and reprint requests should be ad-
dressed, at: Department of Neuroendocrinology, Max Planck Insti-
host of biological effects that allow the organism to meet the
tute of Psychiatry, Clinical Institute, Kraepelinstr. 2, 80804 Munich,
requirements imposed by stressful challenges. Circulating
Germany.
glucocorticoids regulate the hypothalamo-pituitary-adrenal
2Abbreviations: CRH, corticotropin-releasing hormone; AVP,
(HPA) axis through feedback effects on the synthesis and arginine-vasopressin; GR, glucocorticoid receptor; MR, mineralo-
secretion of hypothalamic and pituitary hormones, thus corticoid receptor; ACTH, adrenocorticotropic hormone; HPA,
resetting the activity of the system to basal levels. Cor- hypothalamo-pituitary-adrenal; PVN, hypothalamic paraventricular
ticosteroid receptors in the hippocampus are highly sensitive nucleus; ISHH, in situ hybridization histochemistry; E2, estradiol.

0892-6638/95/0009-0419/$01.50. © FASEB 419

RESEARCH COMMUNICATION
regard to the regulation of gonadotropin release and sexual Services Branch, NIMH). Four measurements of the optical density of ana-
tomical regions of interest (hypothalamic PVN, hippocampal subfield CA,,
behavior (21-24). Administration of testosterone or estradiol
and the dentate gyrus) were made in two adjacent sections from each animal
to genetic females during a critical early postnatal period and averaged to obtain individual means. Nonspecific signals were assessed
results in similar defeminization, thus modifying “brain sex” in sections hybridized with the corresponding sense probe.
irreversibly (25, 26). The question of whether these organiz- Data are presented as mean ± SEM. was used to com-
One-way ANOVA

ing effects of gonadal steroids also extend to other (non- pare multiple group means, and the single-tailed Student’s #{163}
test was applied
to assess differences between pairs of treatment groups. The level of
reproductive) brain functions remains to be examined.
significance was preset at P < 0.05.
On the assumption of a major role for estrogens in the for-
mation of gender-specific patterns in neuroendocrine regula-
tion, the objectives of this study were 1) to characterize
RESULTS
gender differences in the gene expression and responsiveness
to estrogen of hypothalamic CRH and AVP, and hippocam- Resting corticosterone levels in the morning did not
pal and hypothalamic GR in the adult rat, and 2) to explore significantly differ between males and diestrous females;
the significance of estrogen-dependent organization of the brain however, they were significantly elevated in neonatally es-
for the emergence of sex-specific differences in these parameters. trogenized females (efficacy of treatment verified by ovarian
acyclicity and uterine involution). The same relationship was
observed between plasma levels of ACTH in males (60 ± 6
MATERIALS AND METHODS pg/ml), intact females (71 ± 9 pg/ml), and neonatally es-
trogenized females (110± 10 pg/mI; n = 5-6 per group).
Animals The nocturnal increase in adrenocortical secretion in dies-
Male, female, and neonatally estrogenized female Wistar rats (JCL, Tokyo) trous female rats was significantly higher than that observed
were used in these studies. They were housed under specific pathogen-free in males. In neonatally estrogenized females, the magnitude
conditions, with food and water available ad libitum, and a 12 h illumination
of the nighttime rise in corticosterone secretion was
cycle (lights on from 7:00 AM to 7:00 PM). Neonatal rats were obtained from
time-pregnant dams that delivered in the animal colony. All experiments significantly lower than in intact females, but about 25%
were performed in compliance with national regulations and the National greater than in males (Fig. 1). Thymus weights in female
Institutes of Health Guidelines on Animal welfare. rats (200 ± 9 mg/i00 g body wt) were significantly higher
All data were collected when animals were aged 60-68 days. Neonatally
than those in males (163 ± 10 mg/100 g) and neonatally es-
estrogenized females were prepared using a well-established protocol (27).
Briefly, newborn litters were adjusted to 5-6 female pups each and received
trogenized females (142 ± 12 mg/100 g); the difference be-
daily subcutaneous injections of either 10 j.g estradiol-benzoate (Sigma, St. tween the latter two groups was insignificant.
Louis, Mo.) or vehicle (corn oil) from the first postnatal day onward for 7
consecutive days. All rats were handled daily for at least a week before ex-
perimentation. Ovarian cycles in intact and neonatally estrogenized females
were monitored by daily vaginal smears; only intact females in diestrus I and
800
permanently anestrous, neonatally estrogenized females were used for neu-
roendocrine assessment.
Three independent studies were performed using different sets of animals.
To examine differences in basal activity of the HPA axis, small blood sam-
ples (50 to 100 tl) were collected for corticOsterone determination by
venepuncture of the tail vein at 8:00 AM and 7:00 PM. Subsequently, basal
adrenocorticotropic hormone (ACTH) measurements were performed in
plasma from trunk blood collected between 10:00 AM and noon. 600
For comparisons of steady-state levels of mRNAs coding for CRH, AVP,
and GR in intact males, diestrous females, and neonatally estrogenized fe-
E
males, rats were decapitated between 10:00 AM and noon. Brains were
rapidly removed, frozen by brief immersion in prechilled isopentane, and 0)
C
stored at - 70#{176}C until sectioning. Ovaries were inspected at autopsy for ma-
ture follicles; uteri and thymus glands were weighed to confirm the efficacy
of neonatal estrogenization and to provide an additional index of adrenocor- C

tical activity, respectively. 2 400

0
The responsiveness of neuropeptide and GR gene expression to estradiol 4-.
U)
was assessed in subgroups of rats that had been gonadectomized under ether
0
anesthesia at the age of 58-60 days. Five days after surgery, the animals C)
received a single subcutaneous injection of 10 g estradiol-benzoate or vehi- -a
I-
cle at 10:00 AM and were killed 48 h later. 0
C)
Methods 200

Plasma ACT!-! and corticosterone concentrations were measured by radi-

oimmunoassay (ICN, Costa Mesa, Calif.). Steady-state levels of mRNAs
coding for CRH and AVP in the hypothalamic paraventricular nucleus
(PVN), and for GR in the hippocampus and PVN, were determined in
coronal brain sections of the corresponding region by semiquantitative in
situ hybridization histochemistry (ISHH) as previously described (28, 29).
Oligonucleotide 48-mer probes complementary to bases 496-543 of the rat 0
CRH gene (30) and 1009-1056 of the pro-AVP gene (31) were commercially day night
synthesized (MWG-Biotech, Ebersberg, Germany). They were labeled with
Figure 1. Diurnal changes in serum corticosterone levels in dies-
[“S]dATP (NEN DuPont, Boston, Mass.) by terminal deoxynucleotidyl
transferase. The plasmid, containing nucleotides 81-528 of the rat OR se- trous females (open bars), males (solid bars), and neonatally es-
quence (32), was a kind gift of Drs. L. S. Brady and M. Herkenham trogenized females (hatched bars). Blood samples were collected at
(NIMH, Bethesda, Md.). After linearization, [55S]dUTP-labeled cRNA the circadian nadir (day) and zenith (night) of adrenal activity.
probes were generated by transcription from the SP6 (antisense) or T7
Each point represents the mean ± SEM from six individuals.
(sense) promoter, respectively. Autoradiographs from hybridized sections
Asterisks indicate significant differences from the time-matched
were obtained by exposure to Hyperfilm a-max (Amersham, Little Chal-
font, U.K.). Optical densities of hybridization signals were digitized and value in diestrous females; crosses denote significant differences as
quantified using the image analysis program NIH IMAGE 1.52 (Research compared to males.

420 Vol. 9 March 1995 The FASEB journal PAICHEV El AL.

 RESEARCH COMMUNICATION

Steady-state levels of CRH mRNA in the PVN were A) Ovariectomized females

significantly higher, and the hybridization signal for GR 210
mRNA in the hippocampus (dentate gyrus and subfield
CA1) and PVN was significantly compared
lower, to
in male
diestrous female rats. The expression of AVP in the PVN of
intact animals did not display any gender differences. When
compared to males and diestrous females, neonatally es-
trogenized female rats displayed significantly higher levels of
CRH and AVP mRNAs in the PVN; there was no distin-
140
guishable treatment-associated redistribution of the AVP
mRNA signal between magno- and parvocellular divisions
of the PVN (data not shown). The GR mRNA hybridization
signal in the hippocampus of estrogenized rats was
significantly lower than that in intact females, and indistin-
guishable from that measured in males. In the PVN, GR
mRNA levels in males were significantly lower than those in
diestrous and neonatally estrogenized females (Fig. 2). 70
Administration of estradiol to ovariectomized control fe- CRH AVP GR/HIPP GR/PVN
males significantly increased CRH and AVP mRNA levels in
the PVN and decreased GR expression in the hippocampus
B) Gonadectomized males
and hypothalamic PVN (Fig. 3A). In castrated male rats
neither CRH nor AVP mRNAs responded to estradiol treat-
210
ment, although hippocampal GR mRNA levels were
significantly increased (Fig. 3B). Similarly, hypothalamic
CRH and AVP expression were not altered, and GR mRNA
levels in the hippocampus were significantly increased, after C
3
estradiol administration in ovariectomized, neonatally es-
>.
trogenized rats (Fig. 3C).
.0
140

DISCUSSION 0
0
The present data indicate that, in addition to well-known
gender differences in basal and stress-related pituitary
adrenal secretion (6-8), the gene expression of two major
regulators of HPA function, hypothalamic CRH, and 70
cerebral OR display characteristic sex-dependent patterns. CRH AVP GR/HIPP GR/PVN
Thus, adult male rats have higher CRH, and lower GR,

C) Ovariectomized neonatally #{149}strog.nlzed females

210
200

‘4

C 150
3
140
.0
V

100
0

70
50 CR11 AVP GR/HIPP GR/PVN
CRHIPYN AYP/PVN QR/HIPP GVPVN

Figure 2. Comparison of steady-state levels of mRNAs coding for

CRH and AVP in the hypothalamic PVN, and GR in the dentate
gyrus (HIPP) and PVN, in diestrous females (open bars), intact Figure 3. Effect of a single injection of 10 sg estradiol-benzoate on
males (solid bars), and neonatally estrogenzed females (hatched CRH, AVP, and GR mRNA levels in previously gonadectomized
bars). Asterisks indicate significant differences from diestrous fe- females (A), males (B), and neonatally estrogenized females (C).
males; crosses denote values that are significantly different from Asterisks indicate significant differences as compared to the parallel
those in males; each bar represents mean ± SEM from four or five vehicle-treated group; data represent mean ± SEM of 4-5 animals
individuals. per treatment group.

GENDER DIFFERENCES IN HPA REGULATION 421

 RESEARCH COMMUNICATION
mRNA levels than diestrous females. Although the differ- and cellular mechanisms underlying the organizing effects of
ences in CRH gene expression might be attributed to estrogens on hypothalamic CRH- and AVP-producing neu-
differential responses to GR-mediated feedback regulation, rons and hippocampal corticosteroid-sensitive neurons are
the lack of correlation between these parameters and the far from understood. Although estrogen receptors are found
peripheral concentrations of corticosterone (in particular, in only moderate numbers in regions of the adult brain in-
higher nocturnal secretion in females) cannot be readily ex- volved in HPA regulation (36, 37), the possibility that estro-
plained. In this context, the possibility that a stronger in- gen receptors might be transiently expressed in the PVN and
crease in hypothalamic peptidergic drive occurs in females at hippocampus during early brain development must be consi-
the time of peak circadian HPA activity requires further in- dered. The plausibility of this is suggested by recent reports
vestigation. Whatever the causes of this discrepancy might on the occurrence of this phenomenon in brain nuclei that
be, our observations led to the assumption that the “mascu- are unrelated to neuroendocrine functions (38). On the other
line” pattern of the parameters studied consists of increased hand, organizing and activating effects of estrogens on PVN
CRH mRNA levels in the PVN, decreased transcription of and hippocampal neurons might be transsynaptically medi-
the GR gene in the hippocampus and PVN, and relatively ated, i.e., through estrogen-sensitive pathways projecting to
flat diurnal oscillations of serum corticosterone. Miner- these regions. Oonadal steroid-sensitive brain sites that par-
alocorticoid receptors (MR), which have greater affinity for ticipate in the limbic control of the HPA axis (such as the bed
corticosterone than GR, have also been implied in the regu- nucleus of stria terminalis and amygdala) also deserve atten-
lation of HPA activity (2); in pilot studies we observed tion (39-42). The hypotheses outlined are currently under
gender differences in MR gene expression in the hip- investigation.
pocampus that closely resemble those seen in OR (Patchev Taken together, the results of this study demonstrate the
et al., unpublished results). In addition, substantial gender existence of gender differences in the gene expression of
differences exist with respect to the effects of exogenous es- CRH, AVP, and OR in the brain as well as in the regulation
tradiol on CRH, AVP, and GR mRNA levels. The changes of these parameters by estradiol. These differences are prob-
observed in female rats confirm previous presumptions that ably determined by the organizing effects of estrogens during
these parameters are potential targets for the activational early postnatal development. Thus, in addition to the well-
effects of estrogens (10-14, 33, 34). However, in the male rat known effects on neuroendocrine circuits involved in
neither of the neuropeptide mRNAs investigated responded reproduction, gonadal steroid-dependent brain organization
to estradiol, and OR mRNA levels in the hippocampus were may also influence neural mechanisms that control the func-
affected in a fashion opposite to that seen in females. Taken tion of the HPA axis.
together, these data suggest that the responsiveness of these
parameters to estrogen also belong to the gender-specific fea- This study was supported by the Max Planck Society Program
tures of HPA regulation. for Cooperation with Middle and Eastern Europe, a visiting fellow-
These results show for the first time that chronic exposure ship of the Tokyo Metropolitan Institute for Neuroscience, and the
of female neonates to estradiol profoundly affects several Deutsche Forschungsgemeinschaft (SFB 220/C8).
aspects of HPA regulation. Estrogen-induced alterations can
be summarily described as a conversion to male-like patterns
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29. Whitfield, H. J., Jr., Brady, L. S., Smith, M. A., Mamalaki, E., Fox, Accepted for publication November 14, 1994.

Serial Review

Flavoprotein Structure and Mechanism

Coordinated by V. Massey
Beghmlng April 1995

Scheduled articles

T. 0. Baldwin. Bacterial luciferase structure and I. Krauth-Siegel. Tiypanathione reductase and

mechanism lipoamide dehydrogenase as targets for a
D. P. Ballou. Phthalate dioxygenase and phthalate structure-based drug design
dioxygenase reductase M. A. Marietta. Nitric oxide synthase
B. Entsch. Structure and mechanism of para- E. Paz. Overview of flavoprotein crystal structures
hydroxybenzoate hydroxylase T. P. Singer. Monoamine oxidase: mechanistic and
K Hille. Xanthine oxidase and xanthine medical aspects
dehvdmeenaae:interconvertible forms of C. Thorpe. Structure and mechanism of action of
the same enzyme the acyl-CoA dehydrogenases
A. Karplus. Old yellow enzyme: structure and C. H. Williams. Structure and mechanism of
function thioredoxin reductase

GENDER DIFFERENCES IN HPA REGULATION 423