| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Graduate School Neurosciences Amsterdam (F.P.M.K., J.-N.Z., C.W.P., M.A.H., D.F.S.), Netherlands Institute for Brain Research, 1105 AZ Amsterdam ZO, The Netherlands; Department of Endocrinology (L.J.G.G.), Free University Hospital, 1007 MB Amsterdam, The Netherlands; and Anhui Geriatric Institute (J.-N.Z.), The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230032 China
Address all correspondence and requests for reprints to: Frank P. M. Kruijver, M.D., or Prof. Dick F. Swaab, M.D., Ph.D., Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands. E-mail: F.Kruijver{at}nih.knaw.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Gender identity (i.e. the feeling to be male or to be female) is an important trait of a subject. Transsexuals experience themselves as being of the opposite sex, despite having the biological characteristics of one sex (19, 20, 21). In line with the hypothesis that in transsexuals sexual differentiation of the brain contrasts with that of the genetic and physical characteristics of sex, our group has recently found that the size of the central subdivision of the BST (BSTc) was within the female range in genetically male-to-female transsexuals (22). In that study the, BSTc was defined on the basis of its vasoactive intestinal polypeptide innervation, which is probably mainly derived from the amygdala (23). A crucial question resulting from that study was, therefore, whether the difference according to gender in the BSTc is based on a neuronal difference in the BSTc itself or rather a reflection of a difference in innervation from the amygdala. To see whether the BSTc itself has a neuronal organization that is opposite to that of the genetic and genitalial characteristics of transsexuals, we determined the number of somatostatin (SOM)-expressing neurons in the BSTc, which is the major neuronal population in this structure (23).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, 42 brains of patients were analyzed (for
an overview see Table 1
). The brains of
34 reference subjects (9 presumed heterosexual males, 9 homosexual
males, 10 presumed heterosexual females, and 6 male-to-female
transsexuals) ranging from 20–53 yr of age, together with six brains
(three males and three females) of patients with sex hormone disorders
were obtained at autopsy, after the required permissions had been
obtained. Twenty-six of the reference subjects were the same as used in
the earlier study of Zhou et al. (22), whereas eight new
patients (five females, two males, and one homosexual man) were
included because not enough sections were left for the present study. A
Turner syndrome patient (S6) and a castrated (orchiectomized) male
patient (S5) were included in the sex hormone disorder group [n =
6; see the legend to Fig. 1; S1, S2, S3, and M2 were
also used in the study of Zhou et al. (22)]. A nontreated
individual with strong cross-gender identity feelings (S7), which were
already present since his earliest childhood, was also analyzed. In
addition, we had the exceptional opportunity to be able to study the
first collected brain ever of a female-to-male transsexual (FMT). The
brains were matched for age, postmortem time, and duration of formalin
fixation. Neuropathology of all subjects was systematically performed
by Dr. W. Kamphorst (Free University, Amsterdam, The Netherlands), Dr.
D. Troost (Academic Medical Centre of the University of Amsterdam,
Amsterdam, The Netherlands), or Prof. F. C. Stam (Netherlands
Brain Bank, Amsterdam, The Netherlands). Subjects had no primary
neurological or psychiatric diseases, unless stated otherwise.
|
|
Brains were weighed, generally followed by 37 days of fixation in 4% formaldehyde at room temperature. The hypothalamic area was subsequently dissected, dehydrated, and embedded in paraffin. Serial 6-µm frontal sections were cut on a Leitz microtome, mounted on SuperFrost/Plus (Menzel-Gläser, Braunschweig, Germany; Art. No. 041300) slides, and subsequently dried overnight on a hot plate at 58 C.
Immunocytochemistry
Sections were hydrated and rinsed in aquadest 2x 5 min and Tris-buffered saline [TBS; 0.05 M Tris, and 0.9% NaCL (pH 7.6)] for 30 min. To enhance antigen retrieval [for a review see Shi et al. (24)], sections were put in a plastic jar [filled with a Citrate 0.05 M (pH 4.0) buffer solution] and heated to boiling (120 C) for 10 min at 700 W in a microwave oven (Miele Electronic M696, Darmstadt, Germany). After cooling down for about 10 min, the sections were washed in TBS for 3x 10 min and preincubated in TBS (pH 7.6) containing 5% nonfat dry milk (Elk, Campina bv., Eindhoven, The Netherlands) to reduce background staining. Subsequently, a circle was drawn around the sections with a Dakopen (Glostrup, Denmark; Code No. S 2002) to prevent the antibody from diffusing. The sections were: 1) incubated with 300-µL rabbit antisomatostatin [SOMAAR, 8/2/89; dilution 1:500; for details and specificity see Van de Nes et al. (25)] in 0.5% Triton X-100 (Sigma, Steinheim, Germany), 0.25% gelatin, and 5% nonfat dry milk TBS solution [supermix-milk (pH 7.6)] overnight at 4 C; 2) washed in TBS-milk 3x 10 min, followed by a second incubation with goat antirabbit IgG antiserum (Betsie, NIBR, Amsterdam, The Netherlands; dilution 1:100) in supermix for 60 min; 3) washed in TBS-milk 3x 10 min; 4) incubated with rabbit peroxidase-antiperoxidase (dilution 1:1000 in supermix) for 30 min; 5) rinsed 3x 10 min in 0.05 M Tris-HCL (Merck, Darmstadt, Germany; pH 7.6); 6) incubated in 0.05 mg/mL 3,3'-diaminobenzidine (Sigma), 0.25% nickel ammonium sulphate (BDH, Poole, UK) in 0.05 M Tris-HCL (pH 7.6) containing 0.01% H2O2 (Merck) for 15 min; 7) washed in aquadest for 10 min; 8) dehydrated in ethanol; and 9) mounted in Entallan.
Morphometry
Every 50th section stained for SOM along the rostro-caudal
axis of the BSTc on one side of the brain (22) was used for analysis
with the help of a specially developed program on an IBAS
(Kontron Electronik, Munich, Germany) image analysis
system. The image analysis system was connected to a scanning stage
control box (MCU, Carl Zeiss, Oberkochem, Germany) and had
a Sony B/W CCD-camera for image acquisition. Both the scanning stage
and the camera were mounted on a microscope (Carl Zeiss)
equipped with planapo objectives. To provide optimal contrast and
homogenous illumination of the section the voltage of the light source
was set maximally. The light was reduced by neutral gray filters
(0.03/0.12/0.5/Schott; Mainz, Germany) to improve light contrast. For
each section, the analysis consisted of the following steps: By
using the plan x2.5 objective of the microscope, a low magnification
image covering the BSTc area was obtained and loaded into the IBAS
image memory. In this image the BSTc was outlined manually on the
basis of the distribution of the SOM immunoreactivity in neurons
and fibers (see Fig. 3).
Subsequently, the image analyzer covered the outlined area with a grid
of rectangular fields, each with the size of the area displayed by the
camera when the x40 objective was installed. By a random
systematic sampling procedure, 50% of the fields (which were for at
least 80% covered by the outlined area) were selected for analysis.
Taking into account the aberration of the optical axis between the
x2.5 and the x40 objective, the pixel positions of the selected
rectangular fields in the 2.5 image were converted into scanning stage
coordinates to position the corresponding areas of the BSTc in front of
the camera when using the x40 objective. After the x40 objective
was installed, the image analyzer moved the scanning stage
automatically to the coordinates of the selected fields. In each field,
SOM-positive neurons containing a nucleolus were counted manually,
taking into account the exclusion lines according to Gundersen (26).
Neurons with double nucleoli were never seen. The spectrum of neuronal
sizes was equally distributed among the different groups.
|
Statistics
Differences among the groups were statistically evaluated by the nonparametric Kruskal-Wallis multiple comparison test. Differences between the groups were analyzed two-tailed using the Mann-Whitney U test with a 5% experiment wise error rate (sequential Bonferroni method). Throughout this study values are expressed as mean ± SEM. A significance level of 5% was used in all statistical tests.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The number of SOM neurons in the BSTc of heterosexual men (32.9 ±
3.0 x 103) was 71% higher than that in
heterosexual women (19.2 ± 2.5 x 103)
(P < 0.006), whereas the number of neurons in
heterosexual and homosexual men (34.6 ± 3.4 x
103) was similar (P = 0.83). The
BSTc number of neurons was 81% higher in homosexual men than in
heterosexual women (P < 0.004). The number of neurons
in the BSTc of male-to-female transsexuals was similar to that of
females (19.6 ± 3.3 x 103)
(P = 0.83) (see also Figs. 1
and 2). In addition, the
neuron number of the FMT was clearly in the male range (see Fig. 1).
The number of neurons in transsexuals was 40% lower than that found in
the heterosexual reference males (P < 0.04; see the
legend to Fig. 1) and 44% lower than that found in the homosexual
males (P < 0.02). Including patients S2, S3, and S5 in
the male group and S1, S6, and M2 in the female group or S7 in the
transsexual group to increase the number of their respective gender
groups enhanced the level of significance among the groups
(P < 0.001 for SOM neuron number). There seemed to be
no clear difference in the BSTc number of neurons between early onset
(T2, T5, T6) and late-onset transsexuals (T1, T3), indicating that
their smaller number of neurons is related to the gender identity
per se rather than to the age at which it became apparent.
No indication was found for a relationship between cause of death and
BSTc neuron numbers. Analysis of the BSTc volumes showed a similar
pattern of differences among the groups with heterosexual men having a
BSTc volume of 4.60 ± 0.28 mm3, similar to
that in homosexual men (5.00 ± 0.39 mm3)
(P = 0.76). The BSTc volume of females (3.38 ±
0.41 mm3) and that of transsexuals (3.58 ±
0.19 mm3) did not differ either
(P = 0.50). The volumes of all males, regardless of
sexual orientation, vs. all females or vs. all
genetic male transsexuals were statistically highly significant
(P 
0.01). The FMT had a BSTc volume in the male
range (4.80 mm3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Analysis of the total number of SOM neurons of the human BSTc in
individual patients with highly different hormone levels does not give
any indication that changes in sex hormone levels in adulthood change
the neuron numbers. Because the transsexuals had all been treated with
estrogens, at least for some time (see Table 2), the reduced neuron numbers of the
BSTc could theoretically be due to the presence of high levels of
circulating estrogens. Arguments against this possibility come from the
finding that transsexuals T2 and T3 both showed a small BSTc (Fig. 1),
despite the fact that T2 stopped taking estrogens about 15 months
before her death because of hyperprolactinemia, and T3 no longer
received hormone treatment when a sarcoma was found about 3 months
before she died. T5 continued to take estrogens until 3 months before
death and had even more SOM neurons than T3, whereas T1 and T6
continued to take estrogens until death and even had higher SOM neuron
numbers than T2 and T3 (Fig. 1). Furthermore, a 31-yr-old man (S2), who
suffered for at least 1 yr from a feminizing adrenal tumor that
produced high blood levels of estrogens, still had a BSTc neuron number
in the normal male range (the latest highest serum estradiol levels
before death varied between 577–779 pmol/L; the normal range is
50–200 pmol/L).
|
), indicating that orchiectomy did not cause any decrease in SOM
neuron numbers. Not only were five of the transsexuals orchiectomized,
they all used the antiandrogen cyproterone acetate (CPA). However, an
effect of CPA reducing the number of SOM neurons of the BSTc is highly
unlikely because S5 had taken CPA during the last 2 yr of his life and
his BSTc neuron number was at the upper end of the male range, whereas
T6 had not taken CPA for the past 10 yr, and T3 took no CPA during the
last 2 yr before her death, and they still had relatively low numbers
of SOM neurons.
The BSTc SOM neuron numbers of two postmenopausal women [73- (M2) and
53-yr-old (P)] and of a 25-yr-old woman with Turner syndrome (S6:
complete 45,X0, with ovarian hypoplasia) were completely within the
normal female range (Fig. 1). If high estrogen levels would have a
reducing effect on BSTc neuron numbers, the opposite effect (high
neuron numbers) would be expected in the postmenopausal women and the
Turner syndrome patient due to their low endogenous sex hormone level
status. However, this was not the case. Noteworthy is that according to
the available clinical data the two postmenopausal women did not
receive any estrogen replacement therapy either. Although the Turner
syndrome patient had been receiving hormone replacement therapy since
she was 16 yr of age, her neuron numbers were even higher than P,
whereas she had almost the same BSTc neuron number as M2 who did not
receive such a therapy. Again, this argues against the probability of
an estrogen-induced reduction effect on the number of SOM neurons.
Finally, the BSTc neuron number of a 46-yr-old woman who had suffered
for at least 1 yr from a virilizing tumor of the adrenal cortex (that
produced very high blood levels of androstendione and testosterone) was
also clearly within the lower spectrum of that of other women (Fig. 1;
S1: latest androstendione serum level before death was 48.0 ng/mL; the
normal range for women is 0.4–3.5 ng/mL; the latest serum testosterone
level before death was 26.82 nm/L; the normal range for women is
1.04–3.30 nm/L). Thus, an increasing effect of testosterone on the
BSTc neurons does not seem likely to be the case either. Furthermore,
it should be noted that the FMT stopped taking testosterone 3 yr before
death while having a BSTc neuron number clearly within the male
range.
In conclusion, estrogen treatment, orchiectomy, CPA treatment, or
hormonal changes in adulthood did not show any clear relationship with
the BSTc SOM neuron number. In addition, we had the unique opportunity
to study the brain of an 84-yr-old man (S7) who also had very strong
cross-gender identity feelings but was never orchiectomized, sex
re-assigned, or treated with CPA or estrogens. Interestingly, this man
had also a low BSTc SOM neuron number that was fully in the female
range (see Fig. 1, S7). This case provides an additional argument
against the view that orchiectomy, CPA, or adult estrogen treatment of
the transsexuals would be responsible for the reduced somatostatinergic
neuron numbers. Moreover, studies that investigated the effects of
estrogen treatment on hypothalamic SOM neurons in (castrated) rats are
also not in support of such an effect. Estrogen treatment does not
reduce the amount of SOM messenger RNA (mRNA) in neurons but even
enhances its neuronal expression (28). Moreover, another animal study
indicates that, although changes occur in the hypothalamic neuronal
expression of SOM mRNA due to castration or testosterone treatment of
male rats, no differences in hypothalamic SOM neuron numbers are
induced at all by either of such treatments (29). This observation is
also in agreement with the control SOM neuron numbers of the castrated
male patients (S3, S5) and testosterone-exposed (S1) female patient.
Together, all these data clearly indicate that sex hormone-mediated
reduction (or enhancement) effects on transsexual BSTc neurons in
adulthood are extremely unlikely to be the underlying mechanism of the
observed somatostatinergic BSTc differences.
In short, our findings seem to support the hypothesis that the somatostatinergic sex differences, the female number of SOM neurons in the BSTc of the male-to-female transsexual brain and the male number of SOM neurons in the BSTc of the FMT are not the result of changes of sex hormone levels in adulthood. Instead, the neuronal differences are likely to have been established earlier during development [see also Zhou et al. (22), and for functional differences see Cohen-Kettenis et al. (30)]. In line with this reasoning are the developmental data on the rat BST showing that adult volumes and neuron numbers of BST subdivisions are orchestrated by androgen exposure during early brain development (31, 32). Such a mechanism is also in agreement with data of Breedlove (33, 34) showing that perinatal androgens but not adult variations in androgen exposure induce differences in the total neuron number of the rat spinal nucleus bulbocavernosus. Apart from such well known irreversible "organizing" effects of sex hormones on the developing brain, the possibility of a direct action of genetic factors on sexual differentiation of the brain should not be ruled out (35).
We are aware of the fact that our data are based on postmortem brain
material derived from a heterogeneous patient population of which each
individual’s clinical status might have had an impact on the brain.
However, despite that we were still able to find striking sexual
dimorphic differences (that become even more significant if patients
S1, S2, S3, S5, S6, S7, and M2 are included in their respective gender
groups; see statistics and the legend to Fig. 1). An exciting
additional new finding came from the FMT who revealed a "masculine"
BSTc, which is completely in line with the sexual brain paradigm (7, 22, 30, 36, 37, 38, 39, 40).
Although our collection of male-to-female transsexual brains is small, it offers new opportunities to explore neurobiological correlates of transsexualism, as has previously been done in relation to sexual orientation (4, 5, 6). The development of high resolution imaging techniques may allow in vivo volume measurements of particular brain areas in much larger groups of transsexuals, which could extend our findings in the distant future. Although brain imaging proved to be useful in visualizing [e.g. septo-hypothalamic brain injuries leading to hypersexuality or altered sexual preference (9, 10)], precise neuroanatomical delineation of small brain structures such as the BSTc or neuronal counts are, at present, not possible using such techniques.
Taking into account the aforementioned limitations of our studies, the present study of SOM neurons in the human BSTc provides unequivocal new data supporting the view that transsexualism may reflect a form of brain hermaphroditism such that this limbic nucleus itself is structurally sexually differentiated opposite to the transsexual’s genetic and genital sex. It is conceivable that this dichotomy is just the tip of the iceberg and holds also true for many other sexually dimorphic brain areas.
Because the sexually differentiated brain in general (41) may be the basis of sex differences in the prevalence of many neurobiological diseases and disorders (7), more studies are needed to further unravel the potential determinants of the sexual dimorphic brain and its related clinical disorders.
|
Acknowledgments |
|---|
; and
Gerben van der Meulen for making the photographs. Tini Eikelboom, Wilma
Verweij, and Olga Pach are thanked for their secretarial help. Mariann
Fodor is thanked for critically reading the manuscript. Brain material
was provided by the Netherlands Brain Bank (coordinator Dr. Rivka
Ravid). Received October 13, 1999.
Accepted January 11, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. N. Sharpe Gender Recognition in the UK: a Great Leap Forward Social Legal Studies, June 1, 2009; 18(2): 241 - 245. [PDF]
|
|
|
|
 |
|
 A. Garcia-Falgueras and D. F. Swaab A sex difference in the hypothalamic uncinate nucleus: relationship to gender identity Brain, December 1, 2008; 131(12): 3132 - 3146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Berglund, P. Lindstrom, C. Dhejne-Helmy, and I. Savic Male-to-Female Transsexuals Show Sex-Atypical Hypothalamus Activation When Smelling Odorous Steroids Cereb Cortex, August 1, 2008; 18(8): 1900 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Tugnet, J. C. Goddard, R. M Vickery, D. Khoosal, and T. R Terry Current management of male-to-female gender identity disorder in the UK Postgrad. Med. J., October 1, 2007; 83(984): 638 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Genazzani, N. Pluchino, S. Luisi, and M. Luisi Estrogen, cognition and female ageing Hum. Reprod. Update, March 1, 2007; 13(2): 175 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J Giltay, K. H Kho, B. A Blansjaar, M. M Verbeek, P B. H Geurtz, J. M Geleijnse, and L. J G Gooren The sex difference of plasma homovanillic acid is unaffected by cross-sex hormone administration in transsexual subjects J. Endocrinol., October 1, 2005; 187(1): 109 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Wylie Gender related disorders BMJ, September 11, 2004; 329(7466): 615 - 617. [Full Text] [PDF]
|
|
|
|
 |
|
 H. F. L. Meyer-Bahlburg Gender Identity Disorder in Young Boys: A Parent- and Peer-Based Treatment Protocol Clinical Child Psychology and Psychiatry, July 1, 2002; 7(3): 360 - 376. [Abstract] [PDF]
|
|
|
|
 |
|
 W. C. J. Chung, G. J. De Vries, and D. F. Swaab Sexual Differentiation of the Bed Nucleus of the Stria Terminalis in Humans May Extend into Adulthood J. Neurosci., February 1, 2002; 22(3): 1027 - 1033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. P. M. Kruijver, A. Fernández-Guasti, M. Fodor, E. M. Kraan, and D. F. Swaab Sex Differences in Androgen Receptors of the Human Mamillary Bodies Are Related to Endocrine Status Rather Than to Sexual Orientation or Transsexuality J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 818 - 827. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
46 yr of age): adrenal cortex tumor for more than 1 yr,
causing high cortisol, androstendione, and testosterone levels. S2 (
31 yr of age): feminizing adrenal tumor that induced high blood levels
of oestrogens. S3 (
