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Current dogma holds that differences in brain development and behavior between males and females depend
primarily on gonadal steroid hormones, especially testosterone and its metabolites that induce the masculine
pattern and inhibit the female pattern of brain development. However, there is also evidence that genetic
factors may act directly on the developing brain contributing to these differences. Until recently, this
alternative view has been difficult to document, but Dewing et al provide new and convincing evidence for
non-hormonal genetic effects.
Their work was done in a mouse embryo 10.5 days after conception. This is just before the first sign of
sexual differentiation of the genital ridges occurs, thus the influence of gonadal hormones could be
excluded. Their strategy was to harvest whole heads from the embryos, isolate RNA into separate pools
for males and females and then analyze for differential gene expression in the male and female brains.
For screening analysis, they used gene (microarray) chip (Affymetrix) technology which allowed the
relative expression of nearly 10,000 characterized mouse genes and over 3,000 less well defined expressed
sequences (Expressed Sequence Tags – ESTs) to be determined. The normalized gene chip results reported as
fold change or difference between male and female brain RNA revealed 36 genes or ESTs with enhanced
expression in females and 18 genes or ESTs with enhanced expression in males. These genes exhibited a
significant fold difference of greater than 1.1 and 7 genes or ESTs for each sex displayed a fold
difference of 2.0 or more. The gene showing highest differential expression in females was Xist,
which was 18.5 fold higher in females, while genes showing the highest differential expression in
males included DEAD box peptide (Dby) and eukaryotic translation initiation factor 2,Y (Eif2s3Y) with
fold differences of 10.0 and 8.8, respectively. Xist maps to the X chromosome, while the latter two genes
reside on the Y chromosome.
Real-time quantitative analysis (RT-PCR) of littermate-matched male and female embryonic brain RNA confirmed
and validated the results of the gene chip screening for a small number of genes based on their potential
roles in brain development. The authors concluded that developmental differences in male and female brains
in mice are due in part to the differential expression of genes before gonadal secretion starts.
First Editor's Comment: This is an important paper that documents the differential
expression of genes in the male and female brain prior to any influence from gonadal hormones. If confirmed,
it will have a substantial impact on understanding how genetic factors influence brain development. The design
of the study allows for the identification of non-hormonal factors that act before the gonads are formed.
However, there is no reason to think that genes act through mechanisms that do not involve gonadal hormones
after gonadal hormone secretion begins, although other investigational approaches will be needed to demonstrate
this. Dewing and colleagues provide no insight into the nature of the non-hormonal mechanisms through which genes
may act before the appearance of gonadal hormones, although they could presumably be multiple and diverse.
One should note that the most dramatic differences were found for genes whose expression is expected to be limited
to one sex or the other. For example, one would expect genes located on the Y chromosome to be expressed only in
the male brain and Xist mRNA, which is expressed only by the inactive X chromosome in XX females, to be detected
only in the female brain. That they were detected at all, seemingly reflects how the assays distinguish negative
results from background signals. When these results are excluded the differences were diminished. Microarray gene
chip and related approaches for studying gene expression are relatively new and evolving rapidly as is bioinformatics,
the discipline that deals with analysis of the vast amounts of data this technology generates. Its novelty combined
with the complexity of its data has led to a certain amount of caution in the biomedical field with regard to the
biological significance of microarray results. Initially, a 2-fold difference in expression was considered an
informal threshold for biological significance. Many of the results in this study fall below this level and
therefore would not be considered significant by this criteria even though they are statistically significant.
However, as the analytical methods advance, the threshold is being progressively lowered such that a cut-off,
such as the 1.1-fold difference used in this paper, is becoming acceptable. It is still probably wise, however,
to view small differences in gene expression with caution until they are confirmed by others and placed in a
biological context.
William A. Horton, MD
Second Editor's Comment: The findings of this study are important and exciting, and will
likely contribute to a transformation of the dominant conceptual model regarding sexual differentiation of somatic
phenotype, brain, and behavior. There is a risk that the findings may be misinterpreted in a manner potentially
harmful to the clinical decision-making process in cases involving intersexuality. The findings force us to rethink
the classic view of brain sexual differentiation and behavior which posits that the role of genes in the development
of sex differences is restricted to the process of sex determination, i.e., the development of a bipotential and
undifferentiated gonad into either an ovary or a testis. Evidence of a direct role of genes (not mediated by sex
hormones) may lead clinicians to question the flexibility in decision-making they may currently exercise when sex
assignment is in question. But should they?
The basic finding of the study is that over 50 candidate genes are differentially expressed in the brains of male
and female mice, ostensibly prior to gonadal production of sex hormones. Although a remarkable observation, these
findings are not necessarily relevant for one psychological outcome variable of great importance in intersex cases,
that is the stability of gender identity across the lifespan. (Gender identity refers to the individual's self
identification as either girl/woman or boy/man.) Readers of media reports of this article will likely draw different
conclusions. The headline of one well-publicized report of this study states "Sexual Identity Hard-Wired by Genetics."
1 Quotes within the article imply that gender identity springs directly from our genome.
If so, then how do we account for the consistent finding in the literature that 46,XY individuals with complete
androgen insensitivity syndrome develop an unambiguous gender identity as girls, and later women?2
The conflict between research findings and their interpretation is likely more apparent than real and is promoted by
an oversimplification of the process of psychosexual differentiation in humans. An individual's gender identity need
not be congruent with their gender-role (which refers to behaviors that differ in frequency or level between males
and females in this culture and time such as toy play or maternal interest), and sexual orientation (the pattern
of sexual arousal). At the present time, the clinical research literature suggests that gender identity generally
conforms with the gender of rearing, even when gender assignment is discordant with genetic sex. The picture is
quite different, however, with respect to the variables of gender-role behavior and sexual orientation. It is clear
that many new findings will stem from the line of research described in this report. However, it would be unfortunate
if these data were to be interpreted as suggesting that gender assignment must conform with genotype to foster a stable
gender identity.
David E. Sandberg, PhD
References - (linked to  )
- Reuters Health. Sexual identity hard-wired by genetics.
http://www.nlm.nih.gov/medlineplus/news/fullstory_14351.html
- Hines M, et al. Arch Sex Behav 2003;32:93-101.
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Current GGH - Vol. 24 No. 1
Dewing P, et al. Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation. Mol Brain Res
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