 |
Figure 1: Schematic frontal section through two
subdivisions of the bed nucleus of the stria terminalis (BST) that are hatched. III: third
ventricle; AC: anterior commissure; BSTc and BSTv: central and ventral subdivisions of the
BST; FX: fornix; IC: internal capsule; LV: lateral ventricle; NBM: nucleus basalis of
Meynert; OT: optic tract; PVN: paraventricular nucleus; SDN: sexually dimorphic nucleus;
SON: supraoptic nucleus. We have studied the hypothalamus of six
male-to-female transsexuals (T1-T6); this material that was collected over the last eleven
years. We searched for a brain structure that was sexually dimorphic, but not influenced
by sexual orientation, as male-to-female transsexuals may be "oriented" to
either sex with respect to sexual behaviour. Our earlier observations showed that the
paraventricular nucleus (PVN), sexually dimorphic nucleus (SDN) and suprachiasmatic
nucleus (SCN) did not meet these criteria ([6] and unpublished data). Although there is no
accepted animal model for gender identity alterations, the bed nucleus of the stria
terminalis (BST) turned out to be an appropriate candidate to study for the following
reasons. First, it is known that the BST plays an essential part in rodent sexual
behaviour [3,4]. Not only have oestrogen and androgen receptors been found in the BST
[8,9], it is also a major aromatization centre in the developing rat brain [10]. The BST
in the rat receives projections mainly from the amygdala and provides a strong input in
the preoptic-hypothalamic region [11,12]. Reciprocal connections between hypothalamus, BST
and amygdala are also well documented in experimental animals [13-15]. In addition, sex
differences in the size and cell number of the BST have been described in rodents which
are influenced by gonadal steroids in development [16-18]. Also in humans a particular
caudal part of the BST (BNST-dspm) has been reported to be 2.5 times larger in men than in
women [19].
The localization of the BST is shown in figure 1. The central part of the BST (BSTc) is
characterized by its somatostatin cells and vasoactive intestinal polypeptide (VIP)
innervation [20]. We measured the volume of the BSTc on the basis of its VIP innervation
(Fig. 2).
 
 
Figure 2: Representative sections of the BSTc
innervated by vasoactive intestinal polypeptide (VIP). A: heterosexual man; B:
heterosexual woman; C: homosexual man; D: male-to-female transsexual. Bar=0.5 mm. LV:
lateral ventricle. Note there are two parts of the BST in A and B: small sized medial
subdivision (BSTm), and large oval-sized central subdivision (BSTc).
The BSTc volume in heterosexual men (2.49±0.16 mm3) was 44% larger than in
heterosexual women (1.73±0.13 mm3) (P<0.005) (Fig. 3). The volume of the
BSTc of heterosexual and homosexual men was found not to differ in any statistically
significant way (2.81±0.20 mm3) (P=0.26). The BSTc was 62% larger in
homosexual men than in heterosexual women (P<0.005). AIDS did not seem to influence the
size of the BSTc: the BSTc size of two heterosexual AIDS-infected women and three
heterosexual AIDS-infected men remained well within the range of the corresponding
reference group (Fig. 3). The AIDS-infected heterosexuals were therefore included in the
corresponding reference group for statistical purposes. A small volume of the BSTc
(1.30±0.23 mm3) was found in the male-to-female transsexuals (Fig. 3). Its
size was only 52% of that found in the reference males (P<0.005) and 46% of the BSTc of
homosexual males (P<0.005). Although the mean BSTc volume in the transsexuals was even
smaller than that in the female group, the difference did not reach statistical
significance (P=0.13). The volume of the BSTc was not related to age in any of the
reference groups studied (P>0.15), indicating that the observed small size of the BSTc
in transsexuals was not due to the fact that they were, on average, 10 to 13 years older
than the hetero- and homosexual men.
The BST plays an essential role in masculine sexual behaviour and in the regulation of
gonadotrophin release, as shown by studies in the rat [3,4,21]. There has been no direct
evidence that the BST has such a role in human sexual behaviour but our demonstration of a
sexually dimorphic pattern in the size of the human BSTc, which is in agreement with the
previously described sex difference in a more caudal part of the BST (BNST-dspm) [19],
indicates that this nucleus may also be involved in human sexual or reproductive
functions. It has been proposed that neurochemical sex differences in the rat BST may be
due to effects of sex hormones on the brain during development and in adulthood [22,23].
Our data from humans however, indicate that BSTc volume is not affected by varying sex
hormone levels in adulthood. The BSTc volume of a 46-year-old woman who had suffered for
at least 1 year from a tumour of the adrenal cortex that produced very high blood levels
of androstenedione and testosterone, was within the range of that of other women (Fig. 3:
S1). Furthermore, two postmenopausal women (aged over 70 years) showed a completely normal
female-sized BSTc (Fig. 3: M1, M2). As all the transsexuals had been treated with
oestrogens, the reduced size of the BSTc could possibly have been due to the presence of
high levels of oestrogen in the blood. Evidence against this comes from the fact that
transsexual T2 and T3 both showed a small, female-like BSTc (Fig. 3), although T2 stopped
taking oestrogen about 15 months before death, since her prolactin levels were too high
and T3 stopped hormone treatment since a sarcoma was found about three months before
death; also a 31-year-old man who suffered from a feminizing adrenal tumour which induced
high blood levels of oestrogen, nevertheless had a very large BSTc (Fig. 3: S2).
Figure 3: Volume of the BSTc innervated by VIP
fibres in presumed heterosexual males (M), homosexual males (HM), presumed heterosexual
females (F) and male-to-female transsexuals (TM). The six transsexuals are numbered T1-T6.
The patients with abnormal sex hormone levels are numbered S1-S4. M1 and M2:
postmenopausal women. Bars indicate mean±SEM. Open symbols: individuals who died of AIDS.
METHODS. Brains of 42 subjects matched for age, postmortem time and duration of formalin
fixation were investigated. The autopsy was performed following the required permission.
For immunocytochemical staining of VIP, the paraffin sections were hydrated and rinsed in
TBS (Tris-buffered-saline: 0.05 M tris, 0.9% NaCl, pH 7.6). The sections were incubated
with 200 µl anti-VIP (Viper, 18/9/86) 1:1000 in 0.5% triton in TBS overnight at 4° C.
The immunocytochemical and morphometric procedures were performed as described extensively
elsewhere [25-27]. In brief, serial 6 m m sections of the BSTc were studied by means of a
digitizer (Calcomp 2000) connected to a HP-UX 9.0, using a Zeiss microscope equipped with
a 2.5x objective and with 10x (PLAN) oculars. Staining was performed on every 50th section
with anti-VIP. The rostral and caudal borders of the BSTc were assessed by staining every
10th section in the area. The volume of the BSTc was determined by integrating all the
area measurements of the BSTc sections that were innervated by VIP fibres. In a pilot
study, the size of the BSTc was measured on both sides in eight subjects (five females and
three males) and no left-right asymmetries were observed: the left BSTc (1.71±0.16 mm3)
was comparable in size to that of the right BSTc (1.83±0.30 mm3) (P=0.79). No
asymmetry was observed in the BNST-dspm either [19]. The rest of our study was therefore
performed on one side of the brain only. Brain weight of the male transsexuals (1385±75
g) was not different from that of the reference males (1453±25 g) (P=0.61) or that of the
females (1256±35 g) (P=0.23). The cause of death of the six transsexuals was suicide
(T1), cardiovascular disease (T2,T6), sarcoma (T3), AIDS, pneumonia, pericarditis (T4) and
hepatitic failure (T5). Sexual orientation of the subjects of the reference group (12 men
and 11 women) was generally not known, but presumably most of them were heterosexual.
Sexual orientation of nine homosexuals was registered in the clinical records [28].
Differences among the groups were tested two-tailed using the Mann-Whitney U test. A 5%
level of significance was used in all statistical tests.
Our results might also be explained if the female-sized BSTc in the transsexual group
was due to the lack of androgens, because they had all been orchidectomized except for T4.
We therefore studied two other men who had been orchidectomized because of cancer of the
prostate (one and three months before death: S4 and S3, respectively), and found that
their BSTc sizes were at the high end of the normal male range. The BSTc size of the
single transsexual who had not been orchidectomized (T4) ranged in the middle of the
transsexual scores (Fig. 3). Not only were five of the transsexuals orchidectomized, they
all used the antiandrogen cyproterone acetate (CPA). A CPA effect on the BSTc does not
seem likely, because T6 had not taken CPA for the past 10 years, and T3 took no CPA during
the two years before death and still had a female-sized BSTc.
In summary, our observations suggest that the small size of the BSTc in male-to-female
transsexuals cannot be explained by differences in adult sex hormone levels, but is
established during development by an organizing action of sex hormones, an idea supported
by the fact that neonatal gonadectomy of male rats and androgenization of the female rats
indeed induced significant changes in the number of neurons of the BST and suppressed its
sexual dimorphism [17,18].
Considered together with information from animals, then our study supports the
hypothesis that gender identity alterations may develop as a result of an altered
interaction between the development of the brain and sex hormones [5,6]. The direct action
of genetic factors should also be considered on the basis of animal experiments [24].
We found no relationship between BSTc size and the sexual orientation of transsexuals,
that is, whether they were male-oriented (T1,T6), female-oriented (T3,T2,T5), or both
(T4). Furthermore, the size of the BSTc of heterosexual men and homosexual men did not
differ, which reinforced the idea that the reduced BSTc size is independent of sexual
orientation. In addition, there was no difference in BSTc size between early-onset
(T2,T5,T6) and late-onset transsexuals (T1, T3), indicating that the decreased size is
related to the gender identity alteration per se rather than to the age at which it
becomes apparent. Interestingly, the very small BSTc in transsexuals appears to be a very
local brain difference. We failed to observe similar changes in three other hypothalamic
nuclei, namely, PVN, SDN or SCN in the same individuals (unpublished data). This might be
due to the fact that these nuclei do not all develop at the same time, or to a difference
between these nuclei and the BST with respect to the presence of sex hormone receptors or
aromatase. We are now studying the distribution of sex hormone receptors and the aromatase
activity in various hypothalamic nuclei in relation to sexual orientation and gender.
Acknowledgements
We thank Mr. B. Fisser, Mr. H. Stoffels, Mr. G. van der Meulen, and Ms. T. Eikelboom
and Ms. W.T.P. Verweij for their help, and Drs. R.M. Buijs, M.A. Corner, E. Fliers, A.
Walter and F.W. van Leeuwen for their comments. Brain material was provided by the
Netherlands Brain Bank (coordinator Dr. R. Ravid). This study was supported by NWO.
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Correspondence and requests for materials to:
J.-N. Zhou, M.A. Hofman and D.F. Swaab
Graduate School Neurosciences Amsterdam
Netherlands Institute for Brain Research
Meibergdreef 33
1105 AZ Amsterdam ZO
The Netherlands
L.J.G. Gooren
Department of Endocrinology
Free University Hospital
1007 MB Amsterdam
The Netherlands
Email: lgooren@inter.nl.net |
