Sex Differences in Androgen Receptors of the Human

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0021-972X/01/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2001 by The Endocrine Society
Vol. 86, No. 2
Printed in U.S.A.
Sex Differences in Androgen Receptors of the Human
Mamillary Bodies Are Related to Endocrine Status
Rather Than to Sexual Orientation or Transsexuality
FRANK P. M. KRUIJVER, ALONSO FERNÁNDEZ-GUASTI*, MARIANN FODOR†,
ELISE M. KRAAN, AND DICK F. SWAAB
Graduate School of Neurosciences, Netherlands Institute for Brain Research (F.P.M.K., A.F.-G., M.F.,
E.M.K., D.F.S.), 1105 Amsterdam, The Netherlands; Department of Pharmacobiology, CINVESTAV
(A.F.-G.), and División de Investigaciones en Neurociencias, IMP (A.F.-G.), México City, México; and
Research Institute for Endocrinology, Reproduction, and Metabolism, Department of Pediatrics,
Free University Amsterdam (M.F.), Amsterdam, The Netherlands
ABSTRACT
In a previous study we found androgen receptor (AR) sex differences in several regions throughout the human hypothalamus. Generally, men had stronger nuclear AR immunoreactivity (AR-ir) than
women. The strongest nuclear labeling was found in the caudal hypothalamus in the mamillary body complex (MBC), which is known
to be involved in aspects of cognition and sexual behavior. The present
study was carried out to investigate whether the sex difference in
AR-ir of the MBC is related to sexual orientation or gender identity
(i.e. the feeling of being male or female) or to circulating levels of
androgens, as nuclear AR-ir is known to be up-regulated by androgens. Therefore, we studied the MBC in postmortem brain material
from the following groups: young heterosexual men, young homosexual men, aged heterosexual castrated and noncastrated men, castrated and noncastrated transsexuals, young heterosexual women,
and a young virilized woman. Nuclear AR-ir did not differ significantly between heterosexual and homosexual men, but was signifi-
cantly stronger than that in women. A female-like pattern of AR-ir
(i.e. no to weak nuclear staining) was observed in 26- to 53-yr-old
castrated male-to-female transsexuals and in old castrated and noncastrated men, 67– 87 yr of age. In analogy with animal studies showing strong activational effects of androgens on nuclear AR-ir, the
present data suggest that nuclear AR-ir in the human MBC is dependent on the presence or absence of circulating levels of androgen.
The group data were, moreover, supported by the fact that a male-like
AR-ir (i.e. intense nuclear AR-ir) was found in a 36-yr-old bisexual
noncastrated male-to-female transsexual and in a heterosexual virilized woman, 46 yr of age, with high levels of circulating testosterone.
In conclusion, the sexually dimorphic AR-ir in the MBC seemed to be
clearly related to circulating levels of androgens and not to sexual
orientation or gender identity. The functional implications of these
alterations are discussed in relation to reproduction, cognition, and
neuroprotection. (J Clin Endocrinol Metab 86: 818 – 827, 2001)
I
N ANALOGY WITH the nonhuman vertebrate brain (1, 2),
it is thought that in the human also the interaction between
sex hormones and their receptors may play an important role
in brain development (organizing effects) and may in adulthood alter brain function (activating effects), and that these two
mechanisms lead to sex differences in behavior in adult life.
Structural and functional sex differences in the brain may be
related to reproduction, sexual orientation, gender identity (i.e.
the feeling of being male or female), cognition, and disease (3,
4). In a number of areas of the human hypothalamus, structural
and functional differences between the sexes and between homosexual and heterosexual men have been described (5–7). In
addition, our group has found that the central part of the bed
nucleus of the stria terminalis (BSTc) is sexually dimorphic, i.e.
smaller in women, with a female volume and neuron number
in male-to-female transsexuals (4, 8).
It has been shown that various areas of the preoptic area
(POA) (9, 10), BST (11) and suprachiasmatic nucleus (12, 13) are
larger in men than in women, whereas the opposite was found
for the anterior commissure (14). Moreover, hypothalamic differences in relation to sexual orientation have been observed.
The suprachiasmatic nucleus (15) and the anterior commissure
(16) are larger in homosexual than in heterosexual men,
whereas the interstitial nucleus of the anterior hypothalamus-3
is smaller in homosexual than in heterosexual subjects (17).
These data together with the abundant information showing
that sexual orientation and gender identity do not vary with
adult endocrine changes (18, 19) suggest that any possible clue
to understanding the biological basis of sex differences, sexual
orientation, or gender identity will require careful analysis of a
large number of brain areas.
Recently we found that in a number of hypothalamic areas
men showed stronger androgen receptor (AR) immunoreactivity (AR-ir) than women. Interestingly, in the anterior
hypothalamus only moderate sex differences were found,
whereas a conspicuous sex difference occurred in the posterior hypothalamus, i.e. the medial mamillary nucleus
(MMN) and lateromamillary nucleus (LMN) of the mamillary body (MB) complex (MBC) (20). Such sexual dimorphisms may be related to gender differences in certain aspects of reproduction or sexual behavior, as various studies
Received February 8, 2000. Revision received August 29, 2000. Rerevision received October 9, 2000. Accepted October 16, 2000.
Address all correspondence and requests for reprints to: F. P. M.
Kruijver, M.D., or D. F. Swaab, M.D., Ph.D., Netherlands Institute for
Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]
* Sabbatical stay in The Netherlands partially supported by a grant
from CONACyT (Grant 971017).
† Recipient of a grant from the NWO (Grant 903-47004).
818
ANDROGEN RECEPTORS IN HUMAN HYPOTHALAMUS AND TESTOSTERONE
showed that lesions in the rat MB produced a complete loss
of sexual activity (21). Moreover, electrical stimulation of this
area in squirrel monkeys induces penile erection (22, 23). In
addition, Lisk (24) reported in 1967 that implanting testosterone into the MBC of male castrated rats restored sexual
excitability in the presence of receptive females (see also Refs.
25 and 26). These data suggest that this area participates in
the control of male sexual motivation. In contrast, in the
female rat, Galindo-Estaun (27) showed that lesioning the
MB did not alter the estrous cycle or alter sexual or maternal
behavior.
The present study was carried out to investigate whether
the sex difference in nuclear AR-ir of the human MBC is
related to sexual orientation, gender identity, or endocrine
status. In many species, castration strongly reduces or even
eliminates nuclear AR-ir, whereas testosterone, but not estrogen, injection restores such strong nuclear AR-ir (28 –30).
As nuclear AR-ir is up-regulated by androgens (28, 31, 32),
we would expect decreased AR-ir in castrated/aged men.
Therefore, we studied AR-ir in the MBC in groups of subjects
with different testosterone levels (33, 34), i.e. young heterosexual men/young homosexual men, young heterosexual
women, aged heterosexual castrated and noncastrated men,
and castrated and noncastrated transsexuals.
Subjects and Methods
Subjects
In the present study we included the area of the MBC from the
posterior hypothalamus of the following 47 patients: 1) young heterosexual men (n ⫽ 9), 2) young homosexual men (n ⫽ 10), 3) old heterosexual castrated men (n ⫽ 5), 4) castrated male-to-female transsexuals
(n ⫽ 6), 5) old heterosexual intact men (n ⫽ 5), 6) young heterosexual
women (n ⫽ 8), 7) a 36-yr-old noncastrated male-to-female transsexual,
8) a nontreated 84-yr-old male subject with strong cross-gender identity
feelings, 9) a 51-yr-old female-to-male transsexual, and 10) a 46-yr-old
woman with high levels of androgens.
Brains were obtained by autopsy (for clinicopathological information
and ages, see Table 3). Unless stated otherwise, patients had no primary
neurological or psychiatric diseases. The sexual orientation of the subjects was presumed to be heterosexual (15) unless stated otherwise,
whereas the sexual orientation of the homosexual group was documented in the clinical records (15). All homosexual patients died of
acquired immunodeficiency syndrome or related diseases. The patient
data have previously been reported (15). General pathology and neuropathology were performed either at the Free University of Amsterdam
(Dr. W. Kamphorst, Prof. F. C. Stam or Prof. P. van der Valk) or at the
Academic Medical Center of the University of Amsterdam (Dr. D.
Troost). The subjects had no primary endocrine illnesses, except for
those who had undergone orchidectomy, had been given hormonal
treatment, or had had abnormal hormone fluctuations that are mentioned in Table 2. The pathologically high levels of androgens in a
46-yr-old woman, [androstenedione, 48.0 ng/mL (normal values for
women, 0.4 –3.5 ng/mL); testosterone, 26.82 nmol/L (normal values for
women, 1.04 –3.30 nmol/L)] were due to an adrenal cortex carcinoma.
Histology and immunohistochemistry
After autopsy, the hypothalamus was fixed for about 1 month in 4%
formaldehyde at room temperature, dehydrated, and embedded in paraffin. Serial 6-␮m frontal sections were cut on a Leitz microtome (Rockleigh, NJ).
The immunohistochemical protocol followed for the AR staining has
been previously described in detail (20). Briefly, this protocol consisted of
mounting paraffin-embedded sections of the posterior hypothalamus onto
SuperFrost Plus (Menzel, Darmstadt, Germany) slides. The sections were
deparaffinized and rehydrated in a series of ethanol concentrations. To
retrieve antigenicity, sections were microwaved (10 min at 700 watts) in 0.1
819
mol/L citric acid monohydrate buffer (pH 6.0) (35, 36), after which they
were rinsed with TBS buffer (0.05 mol/L Tris-0.9% NaCl, pH 7.6). To
decrease background, the slides were preincubated for 1 h with TBS-milk
[5% milk-TBS solution with commercially available powdered milk (ELK,
Campina Melkunie, Eindhoven, The Netherlands)] before incubation with
the primary antibody PG21 (donated by Drs. Gail Prins and Geoffrey
Greene; 1:1000) for 1 h at room temperature and subsequently kept overnight at 4 C. After rinsing in TBS-milk buffer, sections were incubated for
1 h with a goat antirabbit biotinylated second antibody (1:200), followed by
another hour of incubation in the avidin-biotin complex (1:800). The subsequent signal amplification method consisted of an incubation in biotinylated tyramine (1:1000) and 0.01% peroxide (Merck & Co., Darmstadt,
Germany) for 20 min (37). Thereafter, sections were rinsed with TBS, and
the avidin-biotin complex procedure was repeated. After rinsing in 0.05
mol/L Tris-HCl (pH 7.6), slides were developed by incubation for 10 min
in 0.05 mol/L Tris-HCl containing 0.05% 3,3⬘-diaminobenzidine (Sigma, St.
Louis, MO), 0.01% hydrogen peroxide, and 0.3% nickel ammonium sulfate.
Developed sections were dehydrated in alcohol, cleared with xylene, and
coverslipped with Entallan (Merck & Co.).
Analysis of AR staining intensity
The sections were rated for staining intensity by three independent
investigators blind to the details of the patients. The few differences in
rating were concurred by settlement (20). The category assigned to the
MMN and LMN corresponded to the predominant cell type within that
area according to the following scale: 0 ⫽ no staining, 1 ⫽ staining
diffuse and transparent, and 2 ⫽ intense staining with individual granules of the reaction product distinguishable. The staining range was
established for both the cytoplasm and the nucleus. The estimates were
made at three different microscopic magnifications: ⫻2.5, ⫻10, and ⫻40
objectives (20). The identification of MMN and LMN was made with the
aid of maps of coronal sections of the human brain published by Mai et
al. (38) and using alternating thionine-stained sections for orientation.
Statistics
The assigned categories of AR-ir in the MMN and LMN were compared using the Kruskal-Wallis ANOVA, followed by the Mann-Whitney U test. The fixation time and postmortem delay were analyzed by
the Kruskal-Wallis test. Differences were considered statistically significant at P ⬍ 0.05 (two-tailed).
Results
Staining specificity
Negative controls, i.e. without the first antibody, and positive
control sections, i.e. tissue of mouse testes and human anterior
TABLE 1. Androgen receptor intensity staining in the medial
mamillary nucleus (MMN) and the lateromamillary nucleus (LMN)
of the mamillary body of heterosexual young men, heterosexual
young women, homosexual young men, and castrated transsexual
men
N
Heterosexual men
Heterosexual women
Homosexual men
Transsexual men
9
8
10
6
LMN
MMN
c
n
c
n
1
1
1
1
2
1
2
0
1
1
1
1
2
0
1
0
The data of these 33 subjects that were used for the statistical
analysis (see Results and Table 3) are summarized here with the
median values for nuclear and cytoplasmic AR-ir. The table shows
median values. The AR-ir intensity was assigned according to the
following scale: 0 ⫽ no staining; 1 ⫽ staining diffuse and transparent,
and 2 ⫽ intense staining with individual granules of the reaction
product still distinguishable. c, Cytoplasmic; n, nuclear. The KruskalWallis and Mann-Whitney U tests for nuclear staining indicated
statistically significant differences among the groups (see Results).
The cytoplasmic staining in both brain areas studied was relatively
weak for the four groups and did not differ statistically.
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KRUIJVER ET AL.
JCE & M • 2001
Vol. 86 • No. 2
FIG. 1. Photomicrographs showing AR-ir
in neurons of the MMN of the mamillary
body of a heterosexual man (A), a heterosexual woman (B), a homosexual man (C),
and a woman with high levels of androgens (D). Note that in the mamillary body
there is a clear sex difference in AR-ir (see
A and B), whereas there is no difference in
the intensity of AR staining between the
representative heterosexual man (A),
the homosexual man (C), and the virilized
(androgenized) woman (D). Scale bar,
150 ␮m.
pituitary, were run parallel with the hypothalamic sections.
Literature data have shown the specificity of the anti-AR antibody for brain immunohistochemical studies (29 –31, 39, 40).
The specificity data we have added (20) are briefly described
below (data not shown). Omitting the AR antibody PG21 totally
prevented staining. Paraffin-embedded sections of formalinfixed mouse and human testes showed clear AR staining in the
peritubular cells as previously reported (41, 42). In the human
anterior pituitary isolated groups of cells with moderate to
strong AR-ir were observed, as has also been reported in the
Brazilian opossum (43) and rat (42). An adsorption test, including an immunoblotting analysis for PG21 with a peptide that
consisted of the first 20 amino acids of the peptide that is
recognized by PG21, showed the expected concentration gra-
ANDROGEN RECEPTORS IN HUMAN HYPOTHALAMUS AND TESTOSTERONE
dient on nitrocellulose paper [the technique has been described
by Van der Sluis et al. (44)]. After adsorption of the PG21 antibody with its corresponding peptide, nuclear and cytoplasmic
stainings were completely eliminated (20).
Staining pattern
ANOVAs for fixation time and postmortem delay among the
heterosexual men, heterosexual women, homosexual men, and
castrated transsexual men did not exhibit statistically significant differences (P ⬎ 0.1 and P ⬎ 0.9, respectively).
The immunohistochemical staining for AR in the MMN
and LMN revealed cells with nuclear or cytoplasmic labeling
or with both types of staining (Tables 1 and 3 and Figs. 1-3).
The heterosexual men showed strong nuclear AR-ir in
both brain regions (Tables 1 and 3 and Fig. 1A). In contrast,
the women revealed much less intense labeling in the nucleus
of neurons of the LMN and MMN (Tables 1 and 3 and Fig.
2B). This sex difference was statistically significant for nuclear staining in both areas (P ⬍ 0.05). The homosexual men
showed a similar staining to that of the heterosexual men for
both areas (P ⬎ 0.2) with a more moderate staining in the
MMN (Tables 1 and 3 and Fig. 1C). Women differed significantly from homosexual men in nuclear AR-ir in both the
MMN and LMN (P ⬍ 0.05). The castrated male-to-female
transsexual group had a lack of nuclear staining in both brain
areas, but had cytoplasmic labeling in the LMN and MNN
(Tables 1 and 3 and Fig. 2B). This group was statistically
different in the LMN from the heterosexual and homosexual
men group (P ⬍ 0.05) and similar to that in women (P ⬎ 0.5).
The castrated male-to-female transsexual group had significantly less nuclear AR-ir in the MMN than the heterosexual
male group (P ⬍ 0.05). This difference showed only a trend
when compared with homosexual men (P ⫽ 0.10). When
FIG. 2. Illustration of the staining intensity of nuclear AR-ir in neurons of a
noncastrated 36-yr-old male-to-female
transsexual (A) compared with the lack
of such staining in a 26-yr-old castrated
male-to-female transsexual. Scale bar,
150 ␮m.
821
compared with women, the castrated male-to-female transsexual group did clearly not differ from women in the MMN
(P ⬎ 0.7).
In the 36-yr-old noncastrated male-to-female transsexual,
strong nuclear and cytoplasmic staining was observed in
both areas (Fig. 2A). Similarly, the 46-yr-old woman with
high levels of androgens revealed strong nuclear labeling
and weak to intermediate cytoplasmic labeling in the LMN
and MMN (Fig. 1D). The female-to-male transsexual who did
not receive androgen replacement therapy during the last 3
yr before death (Table 2) showed less intense nuclear and
weak to intermediate cytoplasmic staining in both the LMN
and MMN.
The results of staining in the posterior hypothalamus of
old patients are illustrated in Fig. 3 (A and B). In old castrated
heterosexual men almost no nuclear and weak cytoplasmic
AR-ir were found (Table 3 and Fig. 3B). Thus, in this group
of five, two subjects had very weak nuclear and cytoplasmic
AR-ir in both areas, whereas three of five had no nuclear but
weak cytoplasmic AR-ir (Table 3 and Fig. 3B). A similar trend
of weak AR staining with more, but less intense, nuclear
AR-ir was observed in five old intact men (Table 3 and Fig.
3A). In this group four of five of the individuals had very
weak nuclear as well as cytoplasmic AR-ir (Table 3). Between
the castrated old men and the intact old men no statistical
significant differences were found.
Discussion
The present study confirms the clear sex differences in
nuclear AR-ir expression in neurons of the MBC (20) and
shows, for the first time, that this sex difference is related to
circulating levels of testosterone rather than to sexual orientation or gender identity.
822
KRUIJVER ET AL.
JCE & M • 2001
Vol. 86 • No. 2
TABLE 2. Patient data
Male to female transsexuals (n ⫽ 7 with 6 orchiectomized subjects)
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (84020)
50
42
44
Hormone treatment
Age 42: stilbestrol (5 mg 1 dd); after 2 months to 5 mg 2 dd); age 44: CPA (50 mg 2 dd; treatment lasted 4 yr; stopped 2
yr before death); ethinyloestradiol (50 ␮g 2 dd; treatment lasted 8 yr until death)
Cause of death: suicide
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (88064)
43
36
39
Hormone treatment
Age 36: received standard CPA treatment (50 mg 2 dd) until 2 yr before death; at age 39 received standard ethinyl
estradiol treatment (50 ␮g 2 dd) that stopped 3 months before death
Cause of death: sarcoma, right side temporal
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (93042)
36
NA
no orchiectomy, testes atrophy
Hormone treatment
CPA (50 mg 1 dd) at least the last 10 months before death; the patient did receive estradiol in combination with
hydroxyprogesterone in therapeutic dosages. Exact period of treatment is not known but based on the significant
testes atrophy she was probably treated for about 5 yr or more.
Cause of death: AIDS, pneumonia, pericarditis, cytomegaly in brain
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (93070)
53
40
50
Hormone treatment
Age 40: stilbestrol treatment (stopped after 1 yr); at age 43– 47: premarin (0.625 mg dd); at age 47–50: Premarin (3.75
mg dd); at age 50 –53: Premarin (2.5 mg 3 dd); CPA (50 mg 1 dd); topical estrogen cream (estrogen treatment stopped
3 months before death)
Cause of death: acute fatty liver due to alcohol abuse
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (95018)
48
35
36
Hormone treatment
Age 35: spironolactone (100 mg 2 dd); CPA (50 mg 2 dd); ethinyl estradiol (50 ␮g 2 dd); at age 36 – 40: CPA (50 mg 2 dd);
ethinyl estradiol (50 ␮g 2 dd); at age 40 – 48: aldoctone (100 mg 1 dd); ethinyl estradiol (50 ␮g 1 dd; treatment lasted
until death)
Cause of death: Cardiovascular death
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (98334)
26
23
24
Hormone treatment
Age 23: received standard CPA treatment (50 mg 2 dd) and ethinyl estradiol (50 ␮g 2 dd) treatment until death
Cause of death: Suicide
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
Transsexual (98141)
74
64
64
Hormone treatment
Age 64: received standard CPA treatment (50 mg 2 dd) and ethinyl estradiol (50 ␮g 2 dd) treatment; at age 67: received
estraderm (100 mg 1 dd); at age 74 received spironolacton (50 mg 1 dd) and estraderm (100 mg 1 dd)
Cause of death: coma after appendicitis, pneumonia, lung embolism, and occipital cerebral infarction
Nontreated male with cross-gender identity feelings (n ⫽ 1)
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(96088)
84
—
no orchiectomy or sex reassignment therapy
Hormone treatment
Male patient with strong cross-gender identity feelings who did not receive sex hormone replacement therapy
Cause of death: lung carcinoma
Female to male transsexual (n ⫽ 1)
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-ovariectomy
(98138)
51
27
28
Hormone treatment
At age 27 testosterone, Sustanon (250 mg), twice a month injections; at age 30 testosterone undecanoate (40 mg 3 dd); at
age 34 testosterone undecanoate (40 mg 2 dd); at age 36 testosterone undecanoate (40 mg 4 dd); at age 44
testosterone, Sustanon (250 mg) twice a month injections; at age 47– 48 testosterone, Sustanon (250 mg) every 3 weeks
From age 48 until death (51) no testosterone replacement therapy
Cause of death: cachexia
Castrated males (n ⫽ 5)
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(94090)
86
85
85
Hormone treatment
Male patient with prostate cancer; orchiectomy 20 months before death; patient received an additional antiandrogen
therapy, Androcur (50 mg 4 dd) during the first 14 months, 50 mg 2 dd during the last 6 months)
ANDROGEN RECEPTORS IN HUMAN HYPOTHALAMUS AND TESTOSTERONE
823
TABLE 2. Patient data
Cause of death: septic shock with lung and prostate carcinoma
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(94109)
82
—
82
Hormone treatment
Male patient with prostate cancer; orchiectomy 20 days before death; patient did not receive additional antiandrogen
therapy
Cause of death: respiratory insufficiency, prostate carcinoma, renal insufficiency
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(95062)
80
—
75
Hormone treatment
Male patient with prostate cancer; orchiectomy 5 yr before death; patient did not receive additional antiandrogen
therapy
Cause of death: renal insufficiency with metabolic
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(97157)
69
67
67
Hormone treatment
Male patient with prostate cancer; orchiectomy 3 yr before death; patient received anandron (150 mg 1 dd) during the
last 3 yr before death.
Cause of death: prostate cancer with metastases
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(89103)
67
—
67
Hormone treatment
Male patient with prostate cancer; orchiectomy 3 months before death; patient did not receive additional antiandrogen
therapy
Cause of death: carcinoma of pancreas with multiple metastases; cachexia
Virilized syndrome
Patient no. (NBB)
Age (yr)
Age of hormonal treatment/-orchiectomy
(83004)
46
—
44
Hormone treatment
Female patient with a virilizing adrenocortical carcinoma for ⬎1 yr that produced high levels of cortisol, androstendione,
and testosterone levels; latest androstendione serum level before death was 48.0 ng/mL (normal range for women 0.4 –
3.5 ng/mL); the latest serum testosterone level before death, 26.82 nm/L (normal range for women is 1.04 –3.30 nm/L).
Cause of death: adrenocorticocarcinoma; postoperative hemorrhage
NBB, patient number of The Netherlands Brain Bank; T, male-to-female transsexual; CPA, cyproterone acetate; NA, not available; AIDS,
acquired immune deficiency syndrome.
Heterosexual and homosexual men had the most intense
nuclear AR-ir in the LMN and MMN which were statistically
not different from each other but showed significantly more
AR-ir than women. A similar male-like staining intensity was
also found in a 36-yr-old bisexual noncastrated male-to-female transsexual and in a 46-yr-old heterosexual virilized
woman with high circulating levels of testosterone. In all
cases studied, a close relationship was found between the
endocrine status and the intensity of AR staining. High levels
of testosterone went together with high nuclear AR-ir and
low levels of testosterone with weak or no nuclear AR-ir. The
fact that homosexual men showed more variability in their
nuclear AR-ir profiles compared with heterosexual men,
whereas in the MMN the AR-ir did not differ statistically
from the transsexual group might be due to their acquired
immunodeficiency syndrome status, as some of these patients have subnormal testosterone levels (45– 47). The strong
decrease in nuclear MBC AR-ir in five old male heterosexual
intact subjects also fit the idea of an androgen-dependent
nuclear expression of the AR, as decreased circulating levels
of androgens occur with aging (48, 49). The weak AR-ir in an
84-yr-old man, who was gynecophilic and who had well
documented strong cross-gender identity feelings but never
received hormonal treatment or sex reassignment therapy,
fits with his age. The fact that the 51-yr-old female-to-male
transsexual who did not receive testosterone replacement
during the last 3 yr before death still had a female-like pattern
of AR-ir is also fully in agreement with the assumption that
circulating testosterone is crucial for nuclear AR-ir.
From our data there appeared no relationship between
AR-ir and sexual orientation or gender identity. Regardless
of sexual orientation or gender identity, a female pattern of
AR-ir, i.e. low nuclear staining in the LMN and MMN, was
observed in women, castrated male-to-female transsexuals,
a female-to-male transsexual, and old men.
Animal studies show that castration induces a shift from
strong nuclear to weak cytoplasmic AR-ir in hypothalamic
neurons, which can be reversed by treatment with androgens
(29, 30). Regarding this point it seems of particular interest
to note that ongoing testosterone treatment in female-to-male
transsexuals was accompanied by up-regulation of AR in
peripheral ectocervix tissue, resulting in increased nuclear
AR-ir (32). In addition to the specificity tests (Refs. 29, 36, 39,
and 43 and our additional specificity data), the analogy between data on ARs in animals and humans under different
levels of testosterone (33, 34) now also seems to provide
biological evidence that in neurons of the human brain, PG21
indeed recognizes ARs. Our data from postmortem tissue are
in agreement with the experimental data reported by Wood
and Newman (29, 30), as we also show that gonadectomy in
transsexuals (34) goes together with cytoplasmic AR-ir, in
contrast with the mainly nuclear AR-ir pattern in the male
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KRUIJVER ET AL.
JCE & M • 2001
Vol. 86 • No. 2
FIG. 3. Illustration of the almost complete lack of nuclear AR-ir in a noncastrated old man (A) and in a castrated old
man (B). Scale bar, 50 ␮m.
heterosexual and homosexual group, the noncastrated
young transsexual subject, and the virilized woman who also
had strong nuclear AR-ir. These observations support the
concept that the AR occupied by androgens is mainly present
in the nucleus, where it can alter gene expression and regulate cell activity, whereas the unoccupied AR is for the most
part displaced to the cytoplasm (29, 30). The strikingly complete lack of nuclear AR-ir in the MBC of one male control
patient (no. 97101) and in the MMN of two homosexual
patients (no. 87084 and 88087) may be due to their antemortem status with a severely compromised immune system that
may be accompanied by a strong down-regulation of testosterone levels (45– 47). In aged males the decreased levels of
testosterone (50) do not seem to have a strong effect on the
AR in the MBC, as after castration no significant change in
AR distribution was noticed.
Hormones and their receptors in relation to sexual
orientation and transsexuality
The possible role of steroid hormones in the development
of sexual orientation has been studied in various animal
models (51) and in humans (52). The animal data show that
steroid hormones during the neonatal period contribute to
the organization of the brain and influence sexual preference
(53). In contrast, the exposure to sex steroids during adulthood stimulates sexual behavior, but does not modify sexual
orientation in animals or humans (18, 54). In the present
study no statistical difference was found in AR-ir between
heterosexual and homosexual men. The lack of differences in
the AR-ir in the posterior hypothalamus and in the sequence
variation in the AR gene (55) between homosexual and heterosexual men suggests that neither the intensity of AR-ir nor
a variation in AR structure is related to the expression of
homosexuality. Although possible differences between homosexual and heterosexual men in the AR-ir in other brain
areas have not yet been systematically studied, the data
obtained to date reinforce the idea that homosexuality does
not depend on differences in activational effects of testosterone in adulthood (18).
In the present study we found no clear relationship between MBC AR-ir and gender identity as we did, for instance,
find for the size of the BSTc and gender identity (4, 8). Recent
studies (56 –59) and our observation that the volume and
neuron number of the BSTc in male-to-female transsexuals in
adulthood is independent of sex hormone levels (4, 8) support the idea that steroids do not act in adulthood but, rather,
earlier during development to establish gender identity.
Functional implications
The results obtained in the present study fully agree with
the idea that the AR-ir sex differences in the MBC of the
posterior hypothalamus are due to differences in circulating
levels of androgens and give additional support to the paradigm that endocrine features during adulthood do not contribute to sexual orientation or gender identity.
Anatomical and functional studies in rats have shown that
the MMN and LMN are larger in males than in females (60),
a sex difference that is accompanied by an increase in the rate
of global protein synthesis (61). In addition, experimental
data in animals show a sex-specific involvement of the MB
in aspects of sexual behavior such as sexual motivation,
penile erection, and sexual activity (21–24, 27). Also in humans, the MBC has been implicated in the regulation of
reproduction, possibly by inhibiting the release of gonadotropins, as lesions in the posterior hypothalamus go together
with precocious puberty (62). Our findings of gonadal hor-
ANDROGEN RECEPTORS IN HUMAN HYPOTHALAMUS AND TESTOSTERONE
825
TABLE 3. Brain material showing nuclear and cytoplasmic androgen receptor labeling (AR-ir) in the MBC
NBB
Sex
Age
Pmd
Fix
Young heterosexual men
81-009
m 28
23:00
82-020
m 27
41:00
84-023
m 37
39
88-017
m 31
96:00
88-035
m 23
65:00
94-040
m 20
08:00
97-075
m 33
18:45
97-083
m 22
16:29
97-101
m 43
09:15
32
40
35
24
39
82
—
—
—
Mean ⫾ SEM 29.3
2.7
35:10
10:19
42.0
9.13
Young homosexual men
86-038
m 37
86-043
m 42
86-046
m 32
87-030
m 41
87-084
m 40
88-087
m 41
88-121
m 42
89-031
m 25
89-084
m 39
89-109
m 32
5
—
49:00
—
24:00
12:00
19:00
23:00
—
49:00
—
24
11
40
28
34
30
28
—
11
Mean ⫾ SEM 37.1
1.8
25:51
6:59
25.75
3.88
Young heterosexual women
80-008
f
35
08:00
84-002
f
36
86:00
84-026
f
33
41:00
85-027
f
29
13:00
85-041
f
28
05:00
86-032
f
33
⬍41:00
92-037
f
32
30:00
96-410
f
38
53:00
Mean ⫾ SEM 33.0
1.3
Transsexuals
84-020
mtf
88-064
mtf
93-042
mtf
93-070
mtf
95-018
mtf
98-137
mtf
98-138
ftm
98-141
mtf
50
43
36
53
48
26
51
74
Mean ⫾ SEM 47.6
5.3
26
51
20
60
44
20
45
—
34:38
10:14
38.0
6.52
—
—
21:00
96:00
24:00
—
04:15
06:35
30
—
31
34
36
40
32
33
30:22
18:51
33.7
1.4
Old heterosexual, castrated men
89-103
m 67
24:00 28
94-090
m 86
03:00 93
94-109
m 82
05:35 32
95-062
m 80
04:30 24
97-157
m 69
05:55 45
Mean ⫾ SEM 76.8
4.2
Old heterosexual
80-005
m
82-005
m
93-019
m
93-039
m
97-039
m
men
70
68
78
79
87
Mean ⫾ SEM 76.4
3.8
Untreated transsexual
96-088
m 84
Virilized syndrome
83-004
f
46
8:02
4:31
44.4
14.1
17:00
05:45
—
3:00
4:00
—
30
70
53
45
7:26
3:44
49.5
9.6
MMN
LMN
Cause of death and clinicopathological information
N
C
N
C
2
2
2
1
1
2
1
2
0
1
1
0
1
1
1
1
1
0
2
2
2
2
2
2
2
2
0
2
1
0
1
2
0
1
2
0
Medial cerebral artery aneurysm; lung emboli
Drug addiction; sepsis (S. aureus); cerebral edema
Bronchopneumonia
Heart failure due to coronary anomaly (birth defect)
Cardiac arrest
Heart failure and acute fibronous hemorrhagic pneumonia
Brain damage after motor accident
Hypertrophic cardiomyopathy
Aspergillus pneumonia
2
2
1
1
0
0
2
2
1
1
1
1
1
2
0
1
1
1
1
1
2
1
1
2
1
2
2
2
1
2
1
1
1
2
1
2
1
1
2
2
AIDS
AIDS, disseminated Karposi sarcoma and generalized mycobacterium avium infections
AIDS, pneumocystic carinii pneumonia
Respiratory insufficiency
Cerebral (lymfome, fungal infection)
AIDS, bronchopneumonia, cytomegalic infections and toxoplasmosis
AIDS, cytomegalic meningoencephalitis
AIDS, pneumonia
AIDS, kaposisarcoma, suicide
AIDS, HIV encephalopathy
0
0
0
2
0
0
0
1
1
0
1
1
1
0
0
0
1
0
0
2
2
0
0
1
0
0
1
1
1
0
0
1
Acute lymphoblastic leukemia
Multiple fractures; rupture of thoratic aorta
Anoxia, status after resuscitation after progressive bronchial asthma
Hepatic coma
Cardiogenic shock
Adenocarcinoma with metastases
Bronchopneumonia/bronchitis
Pneumonia, respiratory insufficiency
0
1
2
0
2
0
1
0
1
2
1
0
1
0
2
1
0
1
2
0
2
0
1
0
1
2
1
0
1
0
2
1
Suicide
Sarcoma, right side temporal
Pneumonia after CMV and pseudomonas aeruinosa infection
Acute fatty liver (due to alcohol abuse)
Cardiac arrest
Suicide: XTC overdose
Cachexia
Recent multiple cerebral infarction, cardiac failure, pneumonia
0/1
0/1
0
0
0
0/1 0/1 0/1 Carcinoma of pancreas with multiple metastases; cachexia
0/1 0/1 0/1 Septic shock with lung and prostate carcinoma
0/1 0 0/1 Respiratory insufficiency: prostate carcinoma; orchiectomy; renal insufficiency
0/1 0 0/1 Renal insufficiency with metabolic acidosis and hyperkalemia
0/1 0 0/1 Serious prostate cancer with metastasis
0/1
0/1
0/1
0
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0
0/1
41:00
38
0
0/1
0
6:00
34
2
1
2
0/1
0/1
0/1
0/1
0/1
Pneumonia
Myocardial infarction; glomerulosclerosis of kidneys
Cardiopulmonary insufficiency; bronchopneumonia
Internal bleeding; decompensatio cordis
Myocardial infarction
0/1 Small cell carcinoma of the lungs with metastasis to the liver
1
Adrenocortical carcinoma; postoperative hemorrhage
m, Male; f, female; mtf, male-to-female transsexual; ftm, female to male transsexual.
826
KRUIJVER ET AL.
mone receptors in neurons of the MBC underline the possibility of its involvement in reproduction.
In addition to reproduction, the MBC plays a crucial role
in memory function (63). Mamillary bodies atrophy with age
(64) and even more so in Alzheimer’s disease (65) and are
damaged in alcohol-associated Wernicke-Korsakoff’s disease (66). The decline with age in nuclear AR-ir in the male
MBC as found in the present study may also be reflected in
functional changes. Whether the observed changes in the
MBC play a role in the relationship between low levels of sex
hormones and impairment in sexual and cognitive functioning (48, 67, 68) or in the increased prevalence of nonfamiliar
Alzheimer’s disease in the elderly (69, 70) should be further
investigated. Protective actions of androgens on neurons (71)
and memory loss (72, 73) have been described. It may in this
connection also be of interest to investigate the possible neuroprotective effects of androgens in age-related diseases in
men, in a similar way as is done for estrogen replacement
therapy in postmenopausal women with reported beneficial
effects on physical status, mood, cognition, and the prevention of Alzheimer’s disease (67, 74, 75), although the latter
certainly requires more investigation.
In conclusion, here we show for the first time that the sex
differences in nuclear AR-ir in the MBC of the posterior
hypothalamus reflect differences in circulating levels of androgens rather than differences in sexual orientation or gender identity. The functional implications of these alterations
should be studied in the future.
Acknowledgments
Brain material was obtained from The Netherlands Brain Bank (Anne
Holtrop, Michiel Kooreman, and José Wouda; coordinator Dr. R. Ravid).
We thank Bart Fisser for his technical assistance, Dr. F. W. van Leeuwen
for the antibody PG21, Dr. S. Kaiser for critically reading the manuscript,
G. van der Meulen for photography, and W. Verweij for secretarial help.
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