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Brain Research, 537 (1990) 141-148 141
Elsevier
BRES 16178
An enlarged suprachiasmatic nucleus in homosexual men
D.E Swaab and M.A. Hofman
Netherlands Institute for Brain Research, Amsterdam (The Netherlands)
(Accepted 17 July 1990)
Key words: Suprachiasmatic nucleus; Sexually dimorphic nucleus; Human hypothalamus; Vasopressin neuron; Homosexuality; Acquired
immune deficiency syndrome
Morphometric analysis of the human hypothalamus revealed that the volume of the suprachiasmatic nucleus (SCN) in homosexual men is
1.7 times as large as that of a reference group of male subjects and contains 2.1 times as many cells. In another hypothalamic nucleus which
is located in the immediate vicinity of the SCN, the sexually dimorphic nucleus (SDN), no such differences in either volume or cell number
were found. The SDN data indicate the selectivity of the enlarged SCN in homosexual men, but do not support the hypothesis that homosexual
men have a 'female hypothalamus'.
INTRODUCTION MATERIALS AND METHODS
The suprachiasmatic nucleus (SCN) of the hypothala-
mus is a cell group located in the basal part of the
mammalian brain (Fig. 1). It is considered to be the
principal component of the biological clock generating
and coordinating hormonal, physiological and behavioral
circadian rhythms 24'25'29. In addition it is thought to be
involved in reproduction 3°'33. Because of the differences
in circadian rhythms found in relation to sex 11'42 and the
attenuation of circadian rhythmicity with aging as well as
in Alzheimer's disease 5'2a'41 the human SCN has been
studied with particular reference to these conditions 2~'
32,33. We found, for example, a marked cell loss in the
human SCN in late onset Alzheimer's disease 32'33 drop-
ping to values which were only about 30% of the cell
number found in normal adults.
In order to investigate whether cell loss in the SCN
also occurs in other types of dementias, we subsequently
examined the SCN in postmortem brains of subjects with
early onset Alzheimer's disease as well as of patients who
died with an AIDS-dementia complex 27. Our studies
revealed that the SCN indeed was as strongly affected in
early onset as in late onset Alzheimer's disease 19.
However, we found that the SCN was not smaller in
subjects with AIDS-dementia complex than in the
reference group, but rather appeared to be considerably
enlarged. Subsequent research, as reported in the present
paper, suggested that the enlarged SCN is related neither
to the AIDS-dementia complex nor to AIDS per se but
rather to homosexuality.
Subjects
For the present study the brains of 34 subjects were investigated
(Table I). The required, separate permission for brain autopsy was
obtained either from the patients themselves or, in case of
dementia, from partners or relatives. The reference group consisted
of brains of 18 male subjects from 22 to 74 years of age (39.9 + 3.6
years; mean + S.E.M.). General pathology and neuropathology
were performed either at the Free University of Amsterdam (Dr. W.
Kamphorst) or at the Academic Medical Center of the University
of Amsterdam (Dr. D. Troost). Sexual preference of the subjects of
the reference group was generally not known. The homosexual male
group consisted of 10 non-demented AIDS subjects (aged 25-43;
36.7 + 2.1 years). AIDS patients were diagnosed according to the
Centers for Disease Control 6. As a control group the same
parameters were measured in 6 non-demented heterosexuals (4
males, 2 females; aged 21-73 years; 36.7 + 7.5 years) who also died
from AIDS. Two contracted AIDS by blood transfusion, two by
sexual contact and two were drug addicts. Excluding the two female
subjects from the group of heterosexuals with AIDS, in order to
confine the analysis exclusively to male subjects, did not in any way
affect the .outcomes. Sexual preference of the Subjects of
the
homosexual and heterosexual AIDS groups was registered in the
clinical records. Only after the measurements were performed, was
it established from the records whether or not the AIDS patients
belonged either to the non-demented or demented homosexual
groups or to the heterosexual group.
Histology
Brains were generally weighed after removal followed by fixation
in formaldehyde at room temperature. No sic, nificant differences in
postmortem delay were found among the 3 groups considered (P >
0.2). In contrast, the fixation times in the reference group were on
average longer than in the homosexual male group (40 + 3 days and
29 + 4 days, respectively, P < 0.05). Since the length of the fixation
period and the SCN volume and cell number were not found to be
correlated (P > 0.3) the difference in fixation time between these
groups will not affect the outcome. After fixation, the hypothalamic
area was dissected, dehydrated and embedded in paraffin. Serial 6
Correspondence: D.F. Swaab, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.
0006-8993/901503.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
TABLE I
Brain material
All patients were male subjects, except for two female patients in the group of heterosexuals who died from AIDS (patient nos. 33 and 34).
143
Patient Age Brain Suprachiasmatic nucleus Postmortem Fixation Clinical diagnosis
number (years) weight delay (days)
(g)* Volume Total cell AVP cell (h)
(ram 3) number number
(xlO 3) (xlo 3)
Reference group
1 22 1570 0.380 46.82 12.93
2 23 1310 0.379 58.98 9.92
3 27 1330 0.129 17.75 3.45
4 27 1560 0.196 22.87 3.97
5 28 1510 0.303 40.69 5.45
6 28 1450 0.233 43.27 9.89
7 29 1400 0.279 60.51 5.24
8 31 1330 0.339 74.99 9.25
9 37 1370 0.133 18.76 1.87
10 37 1510 0.169 24.79 2.95
11 41 1440 0.464 84.55 13.73
12 42 1510 0.214 54.31 9.60
13 43 1260 0.195 58.93 10.75
14 47 1620 0.138 24.02 2.24
15 59 1350 0.260 33.77 6.35
16 61 1400 0.285 56.97 8.10
17 63 1420 0.305 53.38 8.93
18 74 1410 0.246 37.00 7.34
Homosexuals (AIDS)
19 25 1530 0.534 106.75 18.40
20 30 1480 0.669 101.29 21.80
21 30 1640 0.413 59.11 15.57
22 32 1440 0.473 99.94 12.26
23 39 >1320 0.432 117.59 10.83
24 41 >1240 0.634 147.73 20.61
25 42 1340 0.109 24.66 2.70
26 42 1340 0.481 90.91 12.15
27 43 >1260 0.279 56.47 16.94
28 43 >1340 0.375 64.20 10.29
15 35
13 11
40 28
24 40
23 32
24
13
29
39
48
120
22
23
24
4
22
32
13
46
41
30
35
46
44
42
53
39
53
51
35
48
47 28
4 31
24 26
49 11
24 28
12 34
4 35
19 30
2 96
24 17
Heterosexuals (AIDS)
29 21 1500 0.154 36.96 3.35 17 26
30 30 1430 0.092 23.82 2.56 8 35
31 30 1340 0.244 40.77 6.97 8 26
32 32 1340 0.128 43.56 1.48 11 131
33 34 1400 0.181 40.38 5.14 12 24
34 73 >1090 0.258 55.88 4.38 48 38
Lymphatic leukemia
Encephalitis in brainstem
Coma
Drug addiction; sepsis (Staphylococcus aureus)
Medial cerebral artery aneurysm; vena cave superior
syndrome; lung emboli
Guillain-Barr6 syndrome
Congenital heart disease; cardiac failure
Multiple trauma; small subarachnoidal hemorrhage
Bronchopneumonia
Alcohol intoxication, combined with benzodiazepines
Cerebral contusion, lung emboli
Metastatic bronchogenic carcinoma; pneumothorax
Non-Hodgkin lymphoma; sepsis
Amyotrophic lateral sclerosis/spinal muscular
atrophy
Pulmonary emphysema; pneumothorax
Myocardial infarction; cardiac failure
Myocardial infarction; cardiac failure
Cardiac failure; bronehopneumonia
AIDS, pneumonia
AIDS, cytomegalic infections
AIDS, Pneumocystis carinii pneumonia
AIDS, Pneumocystis carinii pneumonia
AIDS, progressive multifocal leukoencephalopathy
AIDS, bronchopneumonia, cytomegalic infections
and toxoplasmosis
AIDS, disseminated Karposi sarcoma and
generalized mycobacterium avium infection
AIDS, cytomegalic meningoencephalitis
AIDS, disseminated Karposi sarcoma and pneumonia
AIDS, Pneumocystis carinii pneumonia, Karposi
sarcomas, cytomegalic infections
AIDS, mycobacterial infections pneumonia,
cerebrovascular accident
AIDS, Pneumocystis carinii pneumonia, lung
tuberculosis, toxoplasmosis, heroin addiction
AIDS, disseminated non-Hodgkin lymphoma
infections, drug use
AIDS, cytomegalic infections
AIDS, disseminated histoplasmosis
AIDS, pneumonia, epilepsia
* Inequality signs indicate that parts of the brain were already removed directly after autopsy before weighing the brain.
Fig. 1.
Thionin staining (A,C) and vasopressin staining
(B,D)
of the 6/zm hypothalamus adjacent sections of patient no. 28. In the overview
the SDN (A) and SCN (B) are indicated by arrowheads. The asterisk marks the site of a blood vessel and ventricular wall that corresponds
with the asterisk in the higher magnification (C and D, respectively). The bars indicate 1 mm (A and B) or 0.1 mm (C and D). OC, optic
chiasm; PVN, paraventricular nucleus; SCN, suprachias.matic nucleus; SDN, sexually dimorphic nucleus of the preoptic area; SON, supraoptic
nucleus; III, third ventricle.
144
0.6
0.5
OA
E
E
e 0.3
E
0.2
o.1
SUPRACHIASMATtC NUCLEUS SEXUALLY DIMORPHIC NUCLEUS
Reference Homo- Hereto-
group sexuals sexuals
(AIDS) (AIDS)
Reference Homo - Heteeo-
group sexuals sexuals
(AIDS) (AIDS)
125
A 100
%
L 75
E
E
5O
u
~- 25
SUPRACHIASMATIC NUCLEUS
B
r~1 r~
SEXUALLY DIMORPHIC NUCLEUS SUPRACHIASMATIC NUCLEUS
il
/ml
0
x
v
n
E
E
C
>
20
C
15
10
5
Reference Homo- Hetero- Reference Homo- Hetero- Reference Homo- Hetero-
group sexuals sexuals group sexuals sexuals group sexuals sexuals
(AIDS) (AIDS) (AIDS) (AIDS) (AIDS) (AIDS)
Fig. 2. A: volume of the human SCN and SDN as measured in 3 groups of adult subjects: (1) a male reference group (n = 18); (2) male
homosexuals who died from AIDS (n = 10); and (3) heterosexuals who died from AIDS (n = 6; 4 males and 2 females). The values indicate
medians and the standard deviation of the median 23. The differences in the volume of the SCN between homosexuals and the subjects from
both other groups, are statistically significant. (lr.~uskal-Wallis multiple comparison-test, *P < 0.05; **P < 0.01; ***P < 0.001). Note that
none of the parameters measured in the SDN (Fig. 2A,B) showed significant differences among the 3 groups (P always > 0;4). B: total number
of cells in the human SCN and SDN. The SCN in homosexual men contains 2.1 times as many cells as in the reference group of male subjects
and 2.4 times as many cells as the SCN in heterosexual AIDS patients. C: the number of vasopressin neurons in the human SCN (the human
SDN does not contain vasopressin-producing cells ='aL~. The SCN in homosexual men contains, on average, 1.9 times as many vasoprcssin-
(VP) producing neurons as the reference group of male subjects and 3.6 times as many VP neurons as the SCN in heterosexual AIDS patients.
Notice that the SCN of heterosexual individuals who died from AIDS, contains less vasopressin cells than the subjects from the reference group.
#m frontal sections were cut on a Leitz microtome, mounted on
chrome-alum-coated slides, hydrated, brought to phosphate-buff-
ered saline (PBS) and each 50th section was stained with thionin for
orientation. Volume and cell numbers were determined in two
hypothalamic nuclei, the suprachiasmatic nucleus (SCN) and the
sexually dimorphic nucleus (SDN) zz'32'34. The SDN (Fig. 1), which
is identical with the intermediate nucleus 3 and twice as large in
males as~in females, was included as a reference nucleus in order to
test the hypothesis of 'female differentiation of the hypothalamus'
13 15 ,
as a biological explanation for male homosexuality ' . The SDN
was visualized in thionin-stalned sections 31 (Fig. 1) whereas for the
SCN vasopressin -- one of the main neurotransmitters or neuro-
modulators in this structure -- was used as a marker 32 (Fig. 1).
lmmunocytochemistry
For immunocytochemistry the hydrated sections were rinsed in
PBS, pH 7.4 for 10 rain, after which they were: (1) incubated with
anti-AVP (Truus, 18/9/85) 1:800 in 0.5% Triton in PBS overnight at
4 °C; (2) washed in PBS (2 x 10 rain); (3) incubated with goat
anti-rabbit serum (Betsie) 1:50 in PBS for 30 rain; (4) washed inPBS
(2 x 10 min); (5) incubated with pcroxidase-antipcroxidas¢ (PAP)
1:500 to 1:1000 for 30 min; (6) washed in PBS (2 x 10 rain); (7)
rinsed in 0.05 M Tris-HCI (Merck), pH 7.6; (8) incubated in 0.05
mg/ml 3,3-diaminobenzidine (Sigma) in 0.05 M Tris-HCl, pH 7.6,
0.01%
H202
(Merck) for 10 rain; (9) washed in aqua dest; (10)
dehydrated in ethanol and mounted in Entellan. In the sections
stained with anti-vasopressin, the borders of the SCN can be
delineated reliably 32 (Fig. 1).
Morphometry
Area measurements of the vasopressinergic SCN and its cell
nuclei were performed unilaterally by means of a digitizer (Calcomp
2000) connected to a VAX 11/780, using a Zeiss microscope
equipped with 10x and 40x (PLAN) objectives, respectively, and
with 12.5x (PLAN) oculars. In order to describe the shape of this
nucleus 32 the rostrocaudal axis, the maximal cross-sectional area
covered by vasopressin cells and fibers and the SCN volume were
determined. The rostrocaudal axis was determined by staining every
25th section with anti-vasopressin starting from the lamina termi-
nalis and ending at the eandal end of the optic chiasm. The rostral
and caudal borders of the SCN were assessed by staining every 10th
section in the area, and by determining the sections in which,
respectively, the first and the last vasopressin cells were present.
The maximal cross-sectional SCN area covered with vasopressin
cells in the rostrocandal series was determined as a separate
parameter. The volume of the SCN was determined by integrating
all the area measurements 37 of the SCN sections that contained
immunocytochemically stained cells.
In addition, the vasopressin and total cell number were deter-
mined in the SCN of each subject. The number of vasopressin cells
per unit SCN volume was estimated using a discrete 'unfolding'
procedure 39, which included the modification proposed by Cruz-
Orive 9
and a correction for section thickness (6/~m). The total SCN
cell number was estimated by counting the profile density per unit
area in thionin-counterstained material by means of the same
procedure. For this purpose the section with the maximal SCN area
was selected and nuclear profiles were determined per subject. The
computer programs for these procedures were developed by Dr.
R.W.H. Verwer at our Institute (for details, see ref. 32).
The volume and total cell number of the SDN were measured in
the same way at the same side of the brain in thionin-stained
sections (for details, see ref. 31).
Differences among the groups were tested two-tailed using the
Kruskal-Wallis multiple comparisons test statistic 7. Throughout this
study values are expressed as medians + the standard deviation of
the median. The critical level for statistical significance was taken to
be 5%.
RESULTS
The SCN volume in homosexual males was 1.73 times
larger than in the male subjects of the reference group
(0.463 + 0.066 mm 3 and 0.267 + 0.030 mm 3, respec-
tively; P < 0.01) (Fig. 2A) and contained 2.09 times as
many cells (97.5 + 14.6 x 103 and 46.7 + 7.6 x 103,
respectively; P < 0.001) (Fig. 2B). Similar differences
between these two groups were found for the number of
vasopressin neurons (15.0 + 2.1 x 103 and 7.9 + 1.3 x
103, respectively; P < 0.01) (Fig. 2C). In addition, the
rostrocaudal axis of the SCN was longer in homosexual
males than in the male subjects of the reference group
(2.37 + 0.35 mm and 1.47 + 0.42 mm, respectively; P <
0.02), whereas no such differences were found in the
maximal cross-sectional area of the nucleus. In other
words, the enlarged volume of the SCN in male homo-
sexuals is mainly due to an extension of this nucleus in
145
rostrocaudal direction, as a result of which homosexuals
have a more elongated SCN than heterosexuals. Because
the SCN in heterosexual patients who died from AIDS,
was not significantly different in volume or total cell
number from that of the reference group, an enlarged
SCN seems not to be related to the terminal course of
illness or to AIDS (Table I) but rather to homosexuality.
Since the number of vasopressin neurons was smaller in
the heterosexual AIDS group than in the reference group
(P < 0.05) (Fig. 2C), while the total cell number was
unaltered (Fig. 2B), AIDS seems to be accompanied by
a reduction in the number of neurons expressing vaso-
pressin. This might imply that the number of vasopressin
neurons in homosexual men without AIDS may even be
higher than observed in Fig. 2. Cell numbers in the SDN
of the reference group, the male homosexuals and the
heterosexual subjects did not differ significantly implying
some degree of selectivity for the SCN enlargement in the
hypothalamus of homosexual men.
DISCUSSION
The prominent theory on sexual orientation, i.e. that
it develops as a result of an interaction between the
developing brain and sex hormones 13'15 does not seem to
be supported by our data on the SDN. Maternal stress 14
or chemicals 34 are thought to influence the process of
sexual differentiation of the brain. According to D6rner's
hypothesis, male homosexuals would have a female
differentiation of the hypothalamus. This hypothesis was,
however, solar based solely on indirect evidence, i.e. the
existence of a positive feedback on luteinizing hormone
secretion in some homosexual men following injection of
estrogens 13'15. However, according to Gooren 16A7, this
phenomenon is probably related to changes in testicular
function rather than to sexual orientation and in his
studies could be demonstrated as often in homosexual as
in heterosexual men. D6rner's hypothesis became di-
rectly testable when we found that the SDN of the
preoptic area of the human hypothalamus contains twice
as many cells in men as in women22'31'34: The SDN was
first described in the rat ~8 where it appears to be involved
in male sexual behavior 2A2'36. Neither the SDN volume
nor the cell number in the hypothalamus of homosexual
men who died from AIDS, however, differed from that
of the male reference group in the same age range 34. The
present data confirm and extend this observation with a
heterosexual control group of subjects also suffering from
AIDS. The fact that no difference in SDN cell number
was observed between homo- and heterosexual men who
died from AIDS (P = 0.50) refutes the most global
formulation of D6rner's hypothesis that male homosex-
uals have 'a female brain'.
146
The present data revealed that the volume of the SCN
in homosexual men is 1.7 times as large as that of a
reference group. Since the SCN in the former group also
contains 2.1 times as many cells, this difference cannot be
atu:ibuted to differences in shrinkage. The difference in
SCN cell number in relation to sexual orientation can,
however, not be directly related to sexual differentiation
of the brain since no differences in SCN volume or cell
number were found between males and females 21'32. The
possibility cannot be excluded, yet, that sex hormone
levels during brain development do play some part in this
phenomenon (see below).
The association between a large SCN (and, in partic-
ular, an increase in the number of neurons) and male
homosexuality raises a number of questions about the
way it might have arisen. It appears very unlikely that
homosexual behavior would increase the neuronal num-
ber in any brain structure. The nerve cells of the SCN are
postmitotic from a few years of age onwards, if not
earlier 35. An increase in stainability of vasopressin
neurons due to homosexual behavior is also unlikely,
since the vasopressin cell densities do not differ among
the 3 groups (P > 0.2). Although such a functional
interpretation of the data cannot be totally excluded, the
development of SCN cell numbers suggests that the
explanation for the large SCN in homosexual men most
likely may be found in early brain development. At birth,
the SCN contains only 13-20% of the adult number of
vasopressin and total cells, but in the postnatal period
development is rapid. Cell counts reach a peak around
13-16 months after birth 35 and are then of a similar
magnitude as in adult homosexual men. In the reference
group, the vasopressin and total cell numbers subse-
quently decline to the adult value of about 35% of the
peak values. In homosexual men, therefore, this pro-
grammed postnatal cell death in the SCN seems to be
limited. The observation that a similarly enlarged SCN
was present in a woman with Prader-Willi syndrome 33, a
congenital luteinizing hormone-releasing hormone defi-
ciency in which sex hormone levels are very
low 4,
suggests that the interaction with sex hormones in some
stage of development might be essential for the pro-
grammed SCN cell death. The possibility of sex hor-
mones playing some role in SCN development is rein-
forced by an observation of S6dersten et al. 3°. They
showed that the amplitude of the daily rhythm in sexual
behavior, for which the SCN is responsible, is enhanced
by anti-estrogen treatment of the neonatal animal. This
observation and the large SCN in Prader-Willi syn-
drome 33 suggests that a larger SCN, as reported here for
homosexual men, may relate to a difference in the
interaction with sex hormones during development. This
possibility should be tested in animal experiments.
Dementia as a result of
AIDS 26
does not seem to affect
the size and cell number of the SCN in a significant
manner, as can be concluded from comparing a group of
4 demented homosexual patients who died from AIDS,
with the group of 10 non-demented homosexual AIDS
patients from the present study. However, because of the
dementia, these 4 cases were not included in the present
study.
One might argue that the present finding of an
enlarged SCN in male homosexuals who died from
AIDS, holds only for a particular subset of homosexual
men, i.e. those with a high number of frequently
changing sexual partners with whom anal receptive sexual
techniques were performed 1°'38. This possibility, i.e. that
an enlarged SCN may be related to, e.g. the level of
sexual activity rather than to homosexuality certainly
warrants further study. Experiments in rats, however,
have shown a close correlation between sexual activity
and SDN size 2. Our observation that the size of the SDN
in homosexual men did not differ from that of the male
reference group nor from that of the heterosexual men
that died from AIDS, does not support this possibility.
An alternative explanation for the enlarged SCN
found in male homosexuals is that it might be related to
hypogonadism in adulthood, as has been found in AIDS
patients 8. The observation that the SCN in heterosexual
male AIDS patients is not enlarged seems to exclude this
possible explanation, but homosexual men who did not
die of AIDS should certainly be studied in the future. In
this respect, it is interesting that we observed an enlarged
SCN in two male-to-female transsexuals who did not
suffer from AIDS 33.
The conclusion of the present paper is that the SDN
data do not support the global hypothesis that homosex-
ual men have a 'female brain '13'15. An association was
found, however, between sexual orientation in men and
SCN size, from which the functional implications are
momentarily not clear. However, various observations in
animals suggest that the SCN, apart from being the
biological clock, may be involved in reproductive proces-
ses
30'33.
The SCN is also activated around puberty a. In
addition, lesions of the SCN area in the female rat
attenuated positive feedback response of gonadotropic
hormones to estrogens 2°'4°. The relationship between a
large SCN and homosexuality is, of course, not neces-
sarily a causal one. Animal experimental research has to
reveal whether the SCN is causally implicated in sexual
orientation, or whether SCN size and sexual preference
are influenced by a common factor during develop-
ment.
Acknowledgements. The
authors wish to thank Mr. B. Fisser and
Ms. T. Wesseling for
their technical assistance,
Mr. B. Schillings for
147
his secretarial help, Mr. H. Stoffels for drawing the figures and Mr.
G. van der Meulen for his assistance with the photography, Dr. S.A.
Danner for his scientific assistance, the NIBR scientists and Drs.
J.J.M. Bedaux, E. Fliers, L.J.G. Gooren, J. Joosse, S.A.L.M.
Kooijman and E Portegics for their critical remarks. Brain material
was obtained from the Netherlands Brain Bank (coordinator Dr. R.
Ravid).
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