Professional Documents
Culture Documents
1093/brain/awn276
Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences,
The Netherlands and 2Departamento de Psicobiologia de la Universidad Nacional de Educacion a Distancia, Madrid, Spain
Correspondence to: Dick F. Swaab, MD, PhD, Professor of Neurobiology, Netherlands Institute for Neuroscience,
Meibergdreef 47, 1105 BA Amsterdam, The Netherlands
E-mail: d.f.swaab@nin.knaw.nl
Introduction
The human brain differentiates early in development both
structurally and functionally in a sexually dimorphic way
(Swaab, 2007). Clear structural sex differences in the central
nucleus of the bed nucleus of the stria terminalis (BSTc)
have previously been found by our group, both in its
volumeas delineated by its vasoactive intestinal polypeptide innervation (Zhou et al., 1995)and in its number of
somatostatin immunoreactive neurons (Kruijver et al., 2000).
The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Transsexuality is an individuals unshakable conviction of belonging to the opposite sex, resulting in a request for
sex-reassignment surgery.We have shown previously that the bed nucleus of the stria terminalis (BSTc) is female
in size and neuron number in male-to-female transsexual people. In the present study we investigated the
hypothalamic uncinate nucleus, which is composed of two subnuclei, namely interstitial nucleus of the anterior
hypothalamus (INAH) 3 and 4. Post-mortem brain material was used from 42 subjects: 14 control males, 11 control females, 11 male-to-female transsexual people, 1 female-to-male transsexual subject and 5 non-transsexual
subjects who were castrated because of prostate cancer.To identify and delineate the nuclei and determine their
volume and shape we used three different stainings throughout the nuclei in every 15th section, i.e. thionin,
neuropeptide Y and synaptophysin, using an image analysis system. The most pronounced differences were
found in the INAH3 subnucleus. Its volume in thionin sections was 1.9 times larger in control males than in
females (P _ 0.013) and contained 2.3 times as many cells (P _ 0.002). We showed for the first time that INAH3
volume and number of neurons of male-to-female transsexual people is similar to that of control females. The
female-to-male transsexual subject had an INAH3 volume and number of neurons within the male control
range, even though the treatment with testosterone had been stopped three years before death. The castrated
men had an INAH3 volume and neuron number that was intermediate between males (volume and number of
neurons P ` 0.117) and females (volume P ` 0.245 and number of neurons P ` 0.341). There was no difference in
INAH3 between pre-and post-menopausal women, either in the volume (P ` 0.84) or in the number of neurons
(P _ 0.439), indicating that the feminization of the INAH3 of male-to-female transsexuals was not due to estrogen treatment.We propose that the sex reversal of the INAH3 in transsexual people is at least partly a marker
of an early atypical sexual differentiation of the brain and that the changes in INAH3 and the BSTc may belong
to a complex network that may structurally and functionally be related to gender identity.
3133
3134
Age
Fixation
Length
Postmortem Clock time
time (days) Uncinate (mm) delay (h:min) at death
Control men
00 -131
25
97- 075
33
1500
1410
44
32
1350
1428
18:30
18:45
98 - 031
33
1588
72
1074
546:25
18:35
12
81- 014
98 - 013
98 -169
98 - 006
98 - 027
97-103
98 - 090
92- 042
98 -122
00 - 090
98 -189
Median
Range
Mean SD
42
47
49
50
54
55
58
61
66
70
81
52
25^ 81
52 15
1510
1307
1730
1436
1350
1530
1528
2220
1461
1560
1276
1505
1276 ^2220
1529 231.4
42
28
35
41
59
70
72
52
49
34
33
43
28 ^72
47.3 15.5
672
1476
1104
708
2070
1218
876
726
1434
1332
1086
1182.4
672^2070
1161 378.7
21:50
33:45
09:50
8:30
508:00
541:45
541:00
13:50
541:00
07:45
5:20
11:10
23:45
6:25
11:00
9:00
23:15
NA
21:00
NA
8:00
22:00
4
1
11
1
12
6
4
4
6
7
12
975
1300
1500
1287
1348
1345
1197
991
1221
65
35
31
45
61
63
35
41
28
1074
1662
768
954
1842
1032
978
816
2520
19:35
510:00
517:00
541:00
571:30
592:00
10:25
6:15
6:45
16:55 11
07:00 11
00:00 - 03:00 5
NA 12
17:30 3
21:00 7
2:35 1
6:30 8
7:45 3
65:30
5:30
16:30 1
15:10 12
Control women
01- 009
21
84 - 025
23
01- 072
25
02- 006
32
91- 009
36
97-131
43
01- 011
46
98 -125
58
97- 037
58
98 - 034
72
NA
56
00 -142
82
1280
36
Median
43
1283.5
41
Range
21^ 82
975^1500
28 ^ 65
Mean SD 45.1 20.4 1244.4 160.5 45.1 13.7
1902
2052
1074
768 ^2520
1418.2 596
Month
Clinicopathological information
of death
15:00 10
14:15 5
Myocard infarction
Acute myeloid leukemia
Found dead
Severe pulmonary hypertension
Faecal peritonitis
Myocard infarction
Multiple organ failure
Multiple organ failure
Epileptic convulsions
following craniotomy
Bronchopneumonia
Myocardial infarction
Histology
Immunocytochemistry
NeuropeptideYstaining
The sections were pre-incubated for 1 h in TBS-milk [5% non-fat
dry milk (Elk, Campina, Melkune, Eindhoven, The Netherlands)]
at room temperature. Subsequently, a circle was drawn around the
sections with a Dakopen (Glostrup, Denmark) to prevent the
antibody diffusion. Primary antiserum [rabbit-anti-porcine-NPY,
polyclonal (Niepke 26/11/1988, Netherlands Institute for
Neuroscience)] diluted in Supermix-milk (a solution of 0.5%
Triton X-100, 0.25% gelatin, 5% milk powder in TBS, pH 7.6) was
added at a concentration of 1:1000 (NPY) for 1 h at room
temperature, followed by an overnight incubation at 4 C. The
next day, after rinsing in TBS-milk and TBS, sections were
incubated in goat antirabbit biotylinated IgG (Vector Laboratories,
Brain
weight (g)
NBB
number
Symbol Age
Brain
weight (g)
Fixation
Length
time (days) Uncinate
(mm)
Sexual
orientation
98 -137
93- 042
T7
T4
26
36
1500
1145
NA
31
636
1284
88 - 064
T3
43
1540
NA
1854
84 - 037
T2
44
1450
NA
846
95- 018
84 - 020
93- 070
T6
T1
T5
48
50
53
1198
1380
1500
36
30
34
636
840
1824
04 - 074
T8
55
1321
33
07- 021
T9
58
1358
98 -141
T10
74
83- 004
S7
98 -138
FMT
Age
castration
Postmorten Clinicopathological
delay (h:min) information
NA
21:43
Suicide
Pneumonia
35
26
Not
operated
39
NA
Neurosarcoma
36
37
NA
21
36
Non-homosexual Early
Non-homosexual Early
Non-homosexual Early
35
42
50
36
44
51
540:20
NA
5100
2478
Toward both
Toward both
Female
orientated
Not known
Cardiovascular
death
Cardiac arrest
Suicide
Acute fatty liver
Not known
55
05:25
Lung cancer
37
876
Not known
Not known
7:25
1118
33
1674
40:55
84
1433
38
2112
51
1171
32
1182
Female
orientated
Female
orientated
Female
orientated
Renal cell
carcinoma
Multiple cerebral
infarction
Lung carcinoma
50.5
1380
33
26 ^ 84
1118 ^1540 30 ^38
51.8 15.5 1341.5 158 33 2.7
Not
known
Non-homosexual Early
43
Not
operated
46
64
65
Non-homosexual Early
No treatment
Not
40:55
at all
operated
28 ^ 48 Androgen 29 Ovariectomy 4:15
treatment
Homosexual
Early
Early
1233
636 ^2478
1353 619.9
GID = gender identity disorder; Homosexual = sexual orientation towards the same (genetic) sex; NA = not available; FMT = female to male transsexual; NBB = Netherlands Brain
Bank.
Median
Range
Mean SD
Blanchard
category
3135
3136
Symbol Age
Brain
weight (g)
Fixation
Length
Postmortem Clock time Month Age bilateral
time (days) Uncinate (m) delay (h:min) at death
of death orchidectomy/
hormone change
89-103
S3
67
1290
28
1542
24:00
12
97-157
S8
69
1475
45
1590
5:55
11:20
11
95- 062
S9
80
1400
24
1224
4:30
14:30
94 -109
S10
82
1110
32
1620
5:35
3:00
10
94 - 090
S5
86
1663
93
2208
3:00
2:00
Median
Range
Mean SD
80
1400
32
67^ 86
1110 ^1663
24 ^93
76.8 8.4 1387 206.3 44 28.3
Clinicopathological
information
67 (4 months
before death)
66
Carcinoma of pancreas
and prostate
Metastasis with
prostate carcinoma
75 (Radiotherapy) Renal insufficiency and
prostate carcinoma
None
Respiratory insufficiency
and prostate carcinoma
86 (20 months
Lung and prostate
before death)
carcinoma
1590
1224 ^2208
1636 356.4
SYN staining
The monoclonal SYN antibody (Chemicon International, clone
SY 38) was diluted in Supermix (SUMI, pH 7.6) (0.5% Triton
X-100 and 0.25% gelatin in TBS) at a concentration of 1:1000.
A circle was drawn around the sections with a Dakopen (Glostrup,
Denmark) to prevent the antibody diffusion and then the antibody was added for 1 h at room temperature and subsequently
incubated overnight at 4 C. The next day the sections were
washed in TBS and incubated with the secondary antimouse
biotinylated IgG (Vector Laboratories, Burlingame, CA, USA
cat.no. BA-2000) 1:400 in SUMI for 1 h at room temperature.
Then the sections were washed in TBS and incubated with avidin
biotin complex (Elite, ABC kit, Vector Laboratories), 1:800 in
SUMI for 1 h. For ABC reaction, slices were rinsed in TBS and
incubated in TrisHCL containing 0.5 mg/ml 3,30 -diaminobenzidine [(Sigma Chemical, St Louis, MO, USA), 0,01% H2O2 and
0.2% nickel ammonium sulphate)] for 20 min at room temperature. Finally the sections were rinsed, dehydrated in graded
ethanols, cleared in xylene and coverslipped with Entellan (Merck,
Darmstadt, Germany).
Thionin
The sections were immersed in a thionin solution (0.5% thionin
dissolved in aquadest containing 1% acetic acid, pH 2.4) for
1015 min. The Nissl-stained sections were rinsed, dehydrated in
graded ethanols, cleared in xylene and coverslipped with Entellan
(Merck, Darmstadt, Germany).
Morphometry
The volume of the uncinate nucleus was estimated unilaterally by
measuring cross-sectional areas (in mm2) delineated by thionin,
NPY and SYN innervations (Fig. 1) in every 15th section, that
were sampled in a randomized systematic way. The localization of
the nucleus and its borders was defined on the basis of the three
stainings. If the borders of the nucleus were not easily distinguishable in one section, the decision on the border was made by
comparing adjacent thionin and/or immunocytochemically stained
sections. The angle under which the hypothalamus was cut was
comparable among the individuals and not systematically different
between the various groups. Cross-sectional digital images were
made using a 5 objective (PlanNeofluar lenses) on a Zeiss
Axioscope microscope mounted with a black and white CDD
videocamera (Sony-XC77) and connected to an ImageProPlus
version 5.1 image analysis system (MediaCybernetics, Silver
Spring, MD, USA). All images were collected with exactly the
same settings of the camera microscope and the images were
optimized with the same brightness/contrast setting values. The
contour of the uncinate nucleus was manually outlined by one
investigator (AGF) with a cursor from rostral to caudal, in the
three different stainings. The subsequent processing and the
morphometric analysis of each staining volumes were done
without the investigator knowing to which group each patient
belonged. For every outlined section in each different staining the
area was measured and the total volume analysis of the uncinate
nucleus was determined according to the Cavalieri principle,
taking into account the area measured in each section, the
distance between sections and the thickness of the sections (6 mm)
(Gundersen et al., 1988). For these calculations a homemade
program (IC95) was used.
To determine the density of the neurons in thionin sections
(cells/mm3) a low-magnification image (2.5 objective of the
microscope) covering the INAH3 in thionin was obtained for each
section. Per subject we used thionin sections at three levels in the
INAH3, i.e. the central section that had the largest surface area
and the two adjacent sections. Pilot data, measuring neuronal
density from rostral to caudal throughout the INAH3, showed
that the density values were independent of the level in this
nucleus. In these experiments no difference was found in the
densities in the central sections (three sections) compared to the
3137
Fig. 1 Representative photomicrographs depicting the uncinate nucleus in a thionin (A), NPY (B) or SYN (C) staining. NBB (Netherlands
Brain Bank) # 98169, man 49 years old, INAH3 and 4: interstitial nuclei of the anterior hypothalamus 3 and 4, 3V: third ventricle.
Scale bar = 500 mm. The insert in (A) is indicating the INAH3 and INAH4 subdivisions (shown by dotted line) that are characterized
by a denser NPY and SYN innervation in (B) and (C). Scale bar = 63 mm.
Statistics
Differences among groups were statistically evaluated by the nonparametric KruskalWallis multiple comparison test, because the
data were not all normally distributed. Differences between groups
were analysed two-tailed using the MannWhitney U-test.
The shape of the nuclei was estimated by the correlation
between the length of the nucleus and the maximum crosssectional area (mm2) in the central section using a Spearman
correlation coefficient. Differences in clock time of death and
month of death (circular parameters) were tested with the
MardiaWatsonWheeler Test (Batschelet, 1991). P50.05 was
considered significant.
Results
The uncinate nucleus was localized in the preoptic area
of the human hypothalamus, close to the third ventricle at
3138
Fig. 2 Representative immunocytochemical staining of the NPY innervation of the uncinate nucleus showing the three different types:
(A) typical horse-shoe shape, in which fornix fibers do not completely bisect the nucleus, (B) the two subdivisions of the Un clearly
divided by fornix fibers, originally called INAH3 and INAH4 and (C) the nucleus as just one part, considered to be the INAH4
(A NBB # 98031 man 33 years old, B NBB # 98006 man 50 years old, C NBB # 91009 woman 36 years old. INAH3 and 4:
interstitial nuclei of the anterior hypothalamus 3 and 4, 3V: third ventricle. Scale bar = 250 mm.
Brain weight
3139
NPY-stained sections
A statistically significant difference was found in the NPYstained INAH3 subdivision among the four experimental
groups (KruskalWallis analyses50.05). For INAH4 and
uncinate nucleus no differences were found (INAH4:
P 4 0.681; uncinate: P 4 0.189).
The NPY delineated volume of the INAH3 subdivision was 2.6 times larger, and that of the entire uncinate
nucleus was 1.6 times larger in males than in females (INAH3:
M 4 F: P50.014; uncinate: P50.049). For INAH4 no
sex differences were observed (M = F: P 4 0.411). Maleto-female transsexual people had an intermediate value for
the NPY-stained values and for the INAH3 subdivision and
no significant difference was found when the group was
3140
Men
Women
LeVay, 1991
0.132 mm3
N = 11
0.047 mm3
N = 11
Homosexual
men
Castrated
men
Transsexual
m-f
10000
0.30
S5
0.25
0.20
S7
S3
S5
0.15
FMT
T4
T2
T1
0.10
S9
T3
T7
6000
FMT
T2
T4
4000
S7
T1
T7
S10
S8
T6
T6
MtF
S8
S9
T3
T5 T8T9 T10
S3
2000
0.05
0.00
8000
CAS
Pre-men Post-men
0
M
MtF
CAS
Pre-men Post-men
60000
S5
3141
70000
50000
T2
30000
T4
T1
S8
20000
T6
S3
S7
S9
10000
T3
S10
T5 T8T9 T10
0
F
MtF
CASPre-men
Post-men
SYN-stained sections
KruskalWallis analyses of SYN-stained material revealed
no statistically significant difference among the four experimental groups for the uncinate nucleus (P 4 0.197).
In 91% of the cases the double shape of the uncinate
nucleus in SYN-stained material in the female group could
not be distinguished, while such a lack of SYN staining for
Discussion
The present study shows, for the first time, a sex reversal
in male-to-female transsexual people for the volume and
number of neurons in the INAH3 subdivision of the
uncinate nucleus. The lack of testosterone in male-tofemale transsexual people in adulthood, due to the surgical
sex-reassignment, does not fully explain this sex reversal,
since the INAH3 volume and number of neurons of the
castrated group was intermediate between the male and the
female group. Moreover, the female-to-male transsexual
subject showed male values, in spite of the fact that no
testosterone treatment was given for the last 3 years. Adult
circulating estrogens did not have an effect at all because
the INAH3 measurements of pre- and post-menopausal
women did not differ. Earlier, a female-sized INAH3
volume in homosexual men had been reported (LeVay,
1991; Byne et al., 2001). However, the number of neurons
in the INAH3 of homosexual and heterosexual men did not
differ as demonstrated in previous work (Byne et al., 2001).
In the present study we found both female volume and
neuron number in the INAH3 of male-to-female transsexual persons. As previously shown (LeVay, 1991; Byne et al.,
2001) INAH3 volume may thus be related to sexual
orientation, whereas our data show that INAH3 neuron
number may be related to gender identity. This finding is
in contrast to the earlier observations in the BSTc where
3142
Fig. 8 Representative photomicrographs of the uncinate nucleus in three different groups in consecutive sections (A, B and C: NBB #
00131 male 25 years old; D, E and F: NBB # 01011 female 46 years old; G, H and I: NBB # 84037 transsexual male-to-female 44 years old).
A, D and G are low-power immunocytochemical stainings of NPY, INAH3 and 4: interstitial nucleus of the anterior hypothalamus 3 and 4,
3V: third ventricle. Scale bar = 500 mm. B, E and H: details of the innervation by NPY fibers and C, F and I: details of the thionin staining.
Scale bar = 63 mm. Note that the male group shows a larger INAH3 and, in C, also a larger number of cells than the transsexual and female
groups. The neuronal size is larger in INAH3 subdivision than in INAH4 (C, F and I). It is also noticeable how INAH3 contains more
neurons and extension in the male group (C) than in the other two groups (F and I).
3143
3144
Innervation
A remarkably dense and continuous band of NPY fibers
was found between the infundibular nucleus, paraventricular area including the uncinate nucleus and the medial and
the medial and lateral subdivisions of the bed nucleus of
the stria terminalis (BSTm and BSTl), while an extra dense
NPY fiber network was present in both subnuclei of the
uncinate (Fig. 3). Cell bodies containing NPY were seen in
the infundibular nucleus and in the BSTm and BSTl
subdivisions, whereas the BSTc showed clearly fewer NPY
cells and fibers than the other BST subnuclei. Rostrally, a
NPY connection between the lateral BST and the uncinate
nucleus was observed (Fig. 3), while more caudally the BST
and the infundibular nucleus were connected by NPY fibers
(not shown). The NPY fibers between the infundibular
nucleus, the uncinate nucleus and the lateral and medial
subdivisions of the BST are of special interest since the
Conclusions
Our data reveal a sex-atypical INAH3 volume and neuron
number in transsexual male-to-female people to be in the
female range, while the values of a female-to-male subject
were in the male range. Differences in adult testosterone
levels can only partly explain the observed differences in the
INAH3 subdivision of transsexual people while estrogen
levels do not seem to have an influence. In male-to-female
subjects the number of neurons in the INAH3 does not
seem to be related to sexual orientation, nor to the onset
time of transsexuality, but rather to atypical early femalebiased gender. The differences observed between the INAH3
structure, its innervation in relation to sexual orientation
and gender identity and its putative connection to the BSTc
suggest that these two nuclei, together with the SDN-POA
(= intermediate nucleus, = INAH1 and 2) and the SCN
(Swaab et al., 1985) are part of a complex network involved
in various aspects of sexual behaviour. For the INAH4
subdivision of the uncinate nucleus, the only difference
found among the groups was in relation to its shape, which
was similar in all genetically male groups studied.
3145
Acknowledgements
We thank Bart Fisser, Unga Unmehopa, Rawien Balesar,
Arja A. Sluiter, Joop Van Heerikhuize and Ton Puts for
their technical help, Wilma Verweij for secretarial help,
Jenneke Kruisbrink for her literature resource help and Mrs
Terry Reed, Dr Michel Hofman and Dr Ronald W.H.
Verwer for their critical comments. Brain material was
provided by the Netherlands Brain Bank (coordinator Dr
Inge Huitinga).
Funding
Spanish government (MEC); postdoctoral fellowships
(MEC/Fulbright, EX2006-0930); from Cajamadrid bank;
from GIRES and Mermaids.
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