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The Journal of Neuroscience, October 15, 2002, 22(20):9005-9014
A Model System for Study of Sex Chromosome Effects on Sexually
Dimorphic Neural and Behavioral Traits
Geert J.
De Vries1,
Emilie F.
Rissman2,
Richard
B.
Simerly3,
Liang-Yo
Yang4,
Elka M.
Scordalakes2,
Catherine J.
Auger1,
Amanda
Swain5,
Robin
Lovell-Badge6,
Paul S.
Burgoyne6, and
Arthur P.
Arnold4
1 Center for Neuroendocrine Studies, University of
Massachusetts, Amherst, Massachusetts 01003-9333, 2 Department of Biology, University of Virginia,
Charlottesville, Virginia 22904, 3 Division of
Neuroscience, Oregon Regional Primate Research Center, Beaverton,
Oregon 97006, 4 Department of Physiological Science,
University of California, Los Angeles, California 90095-1606, 5 Chester Beatty Labs, Institute of Cancer Research, London
SW7 3RP, United Kingdom, and 6 Division of Developmental
Genetics, Medical Research Council National Institute for Medical
Research, Mill Hill, London NW7 1AA, United Kingdom
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ABSTRACT |
We tested the hypothesis that genes encoded on the sex chromosomes
play a direct role in sexual differentiation of brain and behavior. We
used mice in which the testis-determining gene (Sry) was
moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an
Sry transgene onto an autosome), so that the
determination of testis development occurred independently of the
complement of X or Y chromosomes. We compared XX and XY mice with
ovaries (females) and XX and XY mice with testes (males). These
comparisons allowed us to assess the effect of sex chromosome
complement (XX vs XY) independent of gonadal status (testes vs ovaries)
on sexually dimorphic neural and behavioral phenotypes. The phenotypes
included measures of male copulatory behavior, social exploration
behavior, and sexually dimorphic neuroanatomical structures in the
septum, hypothalamus, and lumbar spinal cord. Most of the sexually
dimorphic phenotypes correlated with the presence of ovaries or testes
and therefore reflect the hormonal output of the gonads. We found,
however, that both male and female mice with XY sex chromosomes were
more masculine than XX mice in the density of
vasopressin-immunoreactive fibers in the lateral septum. Moreover, two
male groups differing only in the form of their Sry gene
showed differences in behavior. The results show that sex chromosome
genes contribute directly to the development of a sex difference in the brain.
Key words:
Y chromosome; X chromosome; sexual differentiation; lateral septum; androgens; sex chromosome; Sry; sex
determination
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INTRODUCTION |
All sex differences in mammalian and
avian development originate ultimately from the action of genes located
on the sex chromosomes. For example, the Y-linked gene Sry
directs the sexual differentiation of the mammalian gonad by committing
that tissue to a testicular rather than ovarian pattern of development
(Goodfellow and Lovell-Badge, 1993 ). Once testes or ovaries develop,
however, differences in their secretions induce sexual differentiation
of non-gonadal tissues (Jost, 1958), including the brain. Testosterone,
or its metabolites such as estradiol, act on the brain during critical periods of development to induce masculine patterns of neural differentiation that lead to sex differences in brain and behavior (Phoenix et al., 1959; Goy and McEwen, 1980; Arnold and Gorski, 1984).
In some cases, however, sexual dimorphisms in non-gonadal tissues are
difficult or impossible to explain as the result of gonadal steroid
action. (1) In several mammalian species, male embryos develop faster
than female embryos (Erickson, 1997) before differentiation of the
gonads. X- and Y-linked genes contribute to this sex difference
(Burgoyne et al., 1995). (2) In the tammar wallaby, several sexually
dimorphic structures, such as pouch and scrotum, differentiate before
the gonads have differentiated (Renfree and Short, 1988). (3) Neurons
dissociated from embryonic male or female rat brain cultured under
identical conditions nevertheless grow in a sexually dimorphic manner,
with female cultures developing more tyrosine hydroxylase (TH) or
prolactin-immunoreactive neurons, although the cells are harvested
before the onset of sexually dimorphic gonadal secretions (Beyer et
al., 1991, 1992a,b). (4) Numerous X-linked genes are expressed
differently in males and females and can lead to sex differences in
traits. In New World primates, for example, females express more
X-linked photopigment alleles than males, which generates a sex
difference in retinal photosensitivity (Jacobs, 1998; Dulai et al.,
1999). (5) In zebra finches, genetic females that possess testes
develop a feminine neural circuit for song (Wade and Arnold, 1996), and
it has so far proved difficult to prevent masculine neural
differentiation by manipulating gonadal steroid action in genetic males
(Arnold, 1997).
Comparisons of behavioral phenotypes across mouse strains have revealed
that the Y chromosome encodes genes that influence specific sexually
dimorphic behaviors or neural phenotypes (Maxson, 1992, 1996a,b;
Guillot et al., 1995; Hensbroek et al., 1995; Sluyter et al., 1995,
1996). Y chromosome dosage also appears to have significant behavioral
effects in humans (Ratcliffe et al., 1990). These studies support a
role for the Y chromosome in the development of male neural and
behavioral phenotypes.
In the present paper we introduce a powerful mouse model system for
testing the idea that X- or Y-linked genes contribute directly to
sexual differentiation of the brain via nonhormonal mechanisms. Our
test involves comparisons of the brain and behavior of mice that have
the same gonads but possess different complements of sex chromosomes.
We identify one sexual dimorphism in brain, the density of vasopressin
(VP)-immunoreactive (ir) fibers in the lateral septum, which is more
masculine in mice with XY sex chromosomes than in mice with XX
chromosomes. The difference is likely a direct effect of genes encoded
on the sex chromosomes.
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MATERIALS AND METHODS |
Mouse stocks
All mice were random-bred MF1 except for the Y chromosome, which
derived from strain 129 (129/SvEv-Gpi1c Y)
(Simpson et al., 1999). Some mice possessed a variant of
Y129 deleted for the testis-determining
gene Sry [Tdym1 mutation of
Lovell-Badge and Robertson (1990); Gubbay et al. (1992)], here
designated Y . Thus,
XY mice are female (defined by the
presence of ovaries). In some mice an Sry transgene was
inserted into an autosome, creating XYSry mice that possess
testes and are fully fertile males (Mahadevaiah et al., 1998).
XYSry males were bred with
normal MF1 XX females to produce four genotypes: XX females,
XY females,
XYSry males, and
XXSry males. Comparison of these four groups (using two-way
ANOVAs; see below) allowed assessment of the independent effects of sex
(male vs female) and sex chromosome complement (XY vs XX) and their interaction. All
four genotypes occur in the same litters so that prenatal and postnatal
environment and litter effects are distributed across groups. In
addition, XY129 males (MF1 males of the
same strain as the other mice except that the
Y129 chromosome was not deleted for
Sry) were compared with
XYSry males to test for any
differences attributable to the different Sry genes
(transgenic vs endogenous).
Experiment 1
Mice were bred at the Medical Research Council National
Institute for Medical Research. Genotype was determined by PCR analysis of the presence of the Y long-arm gene family Ssty and of
the Sry transgene at the time of weaning and again after
behavioral analysis. As adults (7-8 weeks of age) the mice were
shipped to the University of Virginia (Charlottesville, VA), where they
were tested for aggression, copulatory behavior, and social
exploration. The results on aggressive behavior will be presented in a
separate publication. The mice were anesthetized and perfused with
fixative (see below). Fixed brains and spinal cords were shipped to
other laboratories for histological analysis of several sexually
dimorphic neural systems, including the vasopressin (VP) innervation of the lateral septum, tyrosine hydroxylase (TH)-immunoreactive (ir) neurons of the anteroventral periventricular nucleus of the preoptic region (AVPV), and the spinal nucleus of the bulbocavernosus (SNB), as
described below. All handling of mice and analyses were conducted by
investigators who were unaware of the genotypes of the mice. At the
University of Virginia, males were housed individually. Females were
housed in groups of two to three for 2 months until they were treated
as described below and housed individually. Mice were housed on a 12 hr
light/dark cycle (lights off at 1:00 P.M. EDT) and received ad
libitum access to food (Purina mouse chow 5001) and water.
Behavioral tests: surgery, hormone treatment, and social
exposure. Mice used as experimental subjects were gonadectomized bilaterally. Testes were collected and weighed for later indirect check
of genotype (XXSry males have smaller testes than
XYSry males). Surgery was
conducted under ketamine/xylazine anesthesia (xylazine, 100 mg/kg;
ketamine, 10 mg/kg). At the time of surgery each mouse received a
subcutaneous SILASTIC capsule [1.02 mm inner diameter (id) × 2.16 mm outer diameter (od)] in the midscapular region filled with 7 mm of testosterone. For the behavior tests, the group sizes were
XY = 10, XYSry = 17, XXSry = 15,
XY = 19, and XX = 17.
Stimulus animals. One week after surgery each animal was
given exposure to other mice 3-5 hr before the dark cycle (Wersinger et al., 1997). Gonad-intact C57BL/6J males served as stimulus animals
for both social exposure and social exploration tests. For the social
exposure and sex behavior tests, female C57BL/6J adults were
ovariectomized and received a subcutaneous implant of estradiol
benzoate dissolved in sesame oil (50 µg in 0.025 ml) infused into a
SILASTIC implant (1.98 mm id × 3.17 mm od) sealed with SILASTIC
adhesive. The females received a subcutaneous injection of progesterone
(P) [100 µg in 0.025 ml sesame oil; method of Rissman et al.
(1997)] 2-5 hr before each sex test. These same females were also
used for social exploration tests, in which no P injections were given.
Masculine mating tests. Starting 3 weeks after gonadectomy,
each mouse was tested for masculine sexual behavior beginning 2-3 hr
after the start of the dark cycle according to the procedure of
Wersinger and Rissman (2000a). Briefly, tests were conducted in the
dark in clear Plexiglas cages (18 × 38 cm) placed on a mirror
stand to allow ventral viewing and permit the observer to distinguish
between mounts with or without intromission. The tests lasted for 30 min, or until the test animal ejaculated. If the pair was engaged in
mounting at the end of 30 min, the test was continued until either the
test animal ejaculated or the pair stopped interacting for 5 min.
During the tests, the latencies and numbers of each attempted mount,
mounts with thrusts, mounts with intromission, and ejaculation were
recorded as were the numbers of thrusts and intromissions per mount.
For data analysis of behavioral latencies and frequencies, only tests
that included the behavior of interest were scored.
Social exploration tests. Social exploration was tested 1 week after the sexual behavior test 2-3 hr after the start of the dark
cycle, using a procedure described in Wersinger and Rissman (2000b). A
Plexiglas test box was divided into three areas. Anesthetized stimulus
mice were placed in each of the two end chambers, an intact male at one
end and an ovariectomized estrogen-implanted female at the other end.
The number of visits to each stimulus animal, the total amount of time
spent with stimulus animals, and the amount of time each mouse spent
sniffing each stimulus animal were recorded (Wersinger and Rissman,
2000b). The social exploration test is sensitive to central processing
of social stimuli, but also to individual differences in basic sensory
processing, for example in olfactory sensitivity.
Tissue collection. At the end of behavioral testing the mice
were anesthetized with sodium pentobarbital. Blood was collected for
testosterone radioimmunoassay. The mice were perfused briefly with
0.9% saline followed by 5% acrolein in 0.1 M
sodium phosphate buffer (PB), pH 7.6. The bodies of the mice were sent
to the University of California Los Angeles for histological analysis
of the SNB. Fixed brains were placed in a solution of 30% sucrose in
PB and shipped to the University of Massachusetts (Amherst, MA).
Brains were blocked into 2- to 4-mm-thick transverse slices. The slices that included the septum and the AVPV were split into a dorsal and
ventral half by a horizontal cut at the level of the crossing of the
anterior commissure. The portion containing the AVPV was frozen and
shipped to the Oregon Regional Primate Research Center (Beaverton, OR)
for analysis of TH immunoreactivity in the AVPV. The other samples were
stored in PB-buffered sucrose solution at 4°C until they were
sectioned transversely at 35 µm with a freezing microtome for
analysis of VP immunoreactivity in the lateral septum.
Testosterone radioimmunoassay. Testosterone levels were
determined by radioimmunoassay conducted by the University of Virginia Core Ligand and Assay Laboratory (Charlottesville, VA). Samples were
run in duplicate in a single assay. The range of the assay was from 0.1 to 25.0 ng/ml. The average intra-assay coefficient of variability was
10.8%. Two animals were excluded from further analysis because they
possessed exceptionally high levels of testosterone at the time they
were killed.
Vasopressin immunocytochemistry. Sections were processed for
VP immunoreactivity at room temperature unless stated otherwise. VP
immunoreactivity was located with rabbit anti-VP serum (ICN Laboratories, Costa Mesa, CA) in a 1:4000 dilution for 90 min at
37°C, followed by detection of the primary antibody by biotinylated goat anti-rabbit serum (Vector Laboratories, Burlington, CA) and the
avidin-biotin complex ABC detection system (Vector Elite Kit, Vector
Laboratories) followed by visualization of the antibody complex using
nickel-intensified DAB as the chromogen as described in Villalba et al.
(1999). Microscopic images were captured under bright-field
illumination using a CCD camera linked to a computer. The density of
VP-ir fibers in the lateral septum was examined in the section that
contained the highest fiber density in these areas [corresponding to
Fig. 30 in the atlas of Paxinos and Franklin (1998)]. Fiber
density was analyzed by computerized gray-level thresholding using the
NIH Image software. The light intensity and camera setting were kept
constant across the sections to standardize measurements. Fiber density
was expressed as the number of pixels covered by VP-ir fibers in an
image of a 0.25-mm-square sampling area immediately bordering the
ventricular wall. Group sizes were XY = 10, XYSry = 17, XXSry = 13, XY = 15, and
XX = 13.
Tyrosine hydroxylase histochemistry of the AVPV. To
determine the number of dopaminergic neurons in the AVPV of each
animal, 20-µm-thick frozen sections through the preoptic region were
cut on a sliding microtome and collected in chilled potassium PBS. Dopaminergic neurons were labeled by incubating tissue sections at
4°C for 72 hr in a 1:1000 dilution of an antiserum directed against
TH (EugeneTech, Allendale, NJ), which was localized with an
affinity-purified goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (BioSource International, Camarillo, CA) as described in
detail previously (Sawchenko and Swanson, 1981). The sections were
mounted, counterstained with ethidium bromide for cytoarchitectonic orientation (Schmued et al., 1982), and coverslipped with buffered glycerol. The number of TH-ir neurons within the AVPV was counted in
every third section using fluorescence microscopy and then corrected
with Abercrombie's method (Abercrombie, 1946). The counted objects did
not differ in size, and the section thickness did not vary between
experimental groups; thus the results provide estimates of the relative
number of TH-ir neurons in different groups, not absolute cell numbers.
The group sizes were XY = 8, XYSry = 11, XXSry = 13, XY = 16, and
XX = 14.
Spinal nucleus of the bulbocavernosus. Motoneurons were
discriminated from non-motoneurons in the region of the SNB using immunostaining for Islet-1, a motoneuronal marker (Ericson et al.,
1992, 1996). The lumbosacral region of spinal cords was immersed in
30% sucrose in 0.1 M PB and then
frozen-sectioned horizontally on a sliding microtome at 40 µm and
processed for Islet-1-like immunoreactivity at room temperature. After
three rinses in 0.05 M Tris-buffered saline
(TBS), sections were incubated in 1% sodium borohydride (in 0.1 M PB). Sections were then incubated in TX100 solution (0.05 M TBS with 0.3% Triton X-100)
with 4% normal horse serum (NHS) and 1% hydrogen peroxide for 30 min
and then reacted with mouse anti-Islet-1 antibody (39.4D5,
Developmental Studies Hybridoma Bank, Department of Biological
Sciences, University of Iowa, Iowa City, IA) at a concentration of 1:80
in TX100 solution with 2% NHS for 60 min. Sections were then incubated
with the secondary antibody (horse anti-mouse IgG; Vector Elite Kit,
Vector Laboratories) in TX100 solution with 2% NHS for 60 min and
reacted with ABC reagent, and then 0.05 M TBS
with 0.05% DAB and 0.003% H2O2. Some spinal sections
were processed without the primary or secondary antibody, in which case
no Islet-1-like staining was observed.
SNB motoneurons were counted within eight consecutive sections
comprising a 320-µm-thick horizontal slab with as most dorsal limit
the section that contained the most dorsal Islet-1-ir cells ventral to
the central canal. Cells were counted only if they expressed
Islet-1-immunoreactivity and were located within 175 µm of the
midline and were between the lumbar enlargement and the level of the
sacral region at which the spinal cord width was >1.2 mm. In addition,
nuclear size and diameter of 20 Islet-1-positive neurons was measured
by using NIH Image morphometric software. Abercrombie's correction was
used to correct neuron number (Abercrombie, 1946). The group sizes were
XY = 9, XYSry = 9, XXSry = 8, XY = 9, and
XX = 9.
Statistical tests. All data were analyzed by ANOVAs followed
by planned comparisons (Tukey-Kramer tests) to test for differences between pairs of groups. Some of the behavioral data were analyzed by
ANOVA on ranks if they failed to meet criteria for a normal distribution. For all analyses except that of VP-ir fibers in the
lateral septum (see experiment 2), two basic ANOVAs were run. The first
was a two-way ANOVA on the four groups of mice that were progeny of
XYSry fathers and XX mothers,
with two factors of sex chromosome complement
(XY vs XX) and sex (male vs female or
Sry vs no Sry). This analysis allowed us to
determine whether there was a main effect of sex that would indicate a
phenotypic difference correlating with the presence of testes or
ovaries, or a main effect of sex chromosome complement
(XY vs XX), or an interaction. The
predictions of the present paper were that (1) there would be a main
effect of sex chromosomes or an interaction of sex chromosomes and sex,
or both, and (2) planned comparisons would show a difference between
XYSry males and
XXSry males, or between XY
females and XX females, or both. The second analysis was a one-way ANOVA comparing two male groups (XY vs
XYSry), which tests whether
the Sry transgene has a different effect than the endogenous
Sry on the dependent variables measured. Our hypothesis was
that there would be no difference in that analysis. When we found a
significant difference between male groups, we conducted a further
nested ANOVA with litter as the nested variable. This analysis
determined whether within-group differences among litters could be
eliminated as a significant contributor to the between-group difference
and whether the difference was robust enough to survive the loss of
power that is inherent in the nested analysis. The  level for all
tests was 0.05.
Experiment 2
A second experiment was run to replicate the finding in the
first experiment that there was a significant effect of sex chromosome complement on VP-ir fiber density in the lateral septum. Mice of the
same genotypes as in experiment 1 were bred at National Institute for
Medical Research (NIMR; Mill Hill, London, UK) and then shipped to the
University of Massachusetts. There they were gonadectomized and
implanted with SILASTIC capsules filled with T as in experiment 1. Three weeks later, the mice were killed and processed for VP
immunoreactivity as in experiment 1. The tissue of one XXSry
male was excluded from analysis because of excessively high background
staining. The statistical analysis of VP-ir paralleled that for other
dependent variables, except that experiment number was included as a
third factor and data were standardized per experiment as
z-scores (z = (x  m)/sec where m = group mean and
s = standard deviation). Thus, we conducted a three-way
ANOVA on the XY versus XX
(±Sry) genotypes [three factors were sex chromosomes (XX
vs XY), sex (male vs female), and
experiment number]. A second, two-way ANOVA was used to compare XY and
XYSry groups in the two
experiments [factors were Sry (endogenous Sry in
XY vs Sry transgene in
XYSry) and experiment
number]. Group sizes were XY = 7, XYSry = 7, XXSry = 8, XY = 10, and
XX = 10.
Experiment 3
The first two experiments showed opposite differences between
XY females and XX females in the density
of VP-ir fibers in the lateral septum. To test the hypothesis that this
inconsistency was the result of variability induced by uncontrolled
exposure of fetal females to androgens originating from adjacent males in utero (vom Saal and Bronson, 1978), litters of mice were
produced containing only females.
XYYq-delRIII
males (Mahadevaiah et al., 2000) were mated to MF1 females. The Yq-del chromosome is of RIII strain origin and carries a deletion of Yq
(Conway et al., 1994). Although initially sterile, older males become
fertile because of random loss of one or the other Y in a proportion of
spermatogonia. Most of these males produce only female offspring (XX
and XY genetically identical to those in
experiments 1-3), apparently because of selection against Yq-del sperm
in the female tract (P. S. Burgoyne, unpublished results). Mice were
bred at NIMR. In experiment 3A they were shipped to the University of
Virginia where they underwent surgery, hormone implantation, and
behavioral testing similar to that described for experiment 1 (data not
shown), after which fixed brains were shipped to the University of
Massachusetts for analysis of septal VP-ir fiber density. In experiment
3B the mice were shipped directly to the University of Massachusetts, where they were treated as in experiment 2 before measuring septal VP-ir fiber density. A two-way ANOVA was used to compare XX and XY groups in the two experiments
(factors were sex chromosome complement, XX vs
XY, and experiment number). The group
sizes were XX = 7 and 16 and XY = 7 and 17 in experiments 3A and 3B, respectively.
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RESULTS |
Experiment 1
Testosterone levels
At the time the mice were killed, plasma testosterone levels were
not statistically different among the groups because they all had
received identical treatment with testosterone during testing: XY males
(2.17 ± 0.27 ng/ml; mean ± SEM),
XYSry males (2.21 ± 0.13), XXSry males (1.81 ± 0.13),
XY females (3.01 ± 0.61), and XX
females (3.68 ± 1.27). The slightly higher but not significantly
different levels in females, if anything, would tend to diminish any
sex differences in dependent variables that are sensitive to both adult
and perinatal circulating levels of testosterone, such as the density
of VP-ir fibers in the lateral septum. It should not influence,
however, phenotypes that are insensitive to the level of testosterone
in adulthood, for example, the numbers of neurons in the SNB or AVPV.
Masculine sexual behavior
In analyses of specific behaviors we included only data from mice
that displayed the behavior. Female mice had shorter latencies to mount
than did males and also shorter latencies to mount with thrusts (Fig.
1). There was a main effect of sex on
latency to mount (F(1,59) = 19.42;
p < 0.00005) but no significant effect of sex
chromosomes or a significant interaction. The same pattern was found
for latency to thrust: a main effect of sex
(F(1,45) = 6.02; p < 0.02) (Fig. 1), but no significant effect of sex chromosomes or a
significant interaction. Mount latencies were not different between XY
and XYSry males, but
XYSry males began thrusting
sooner than XY males (F(1,21) = 6.05; p < 0.025). The latter difference was also significant
when a nested ANOVA was performed
(F(1,21) = 6.11; p < 0.04). Although XY males began thrusting later than
XYSry males, they tended to
ejaculate sooner (F(1,16) = 4.31;
p = 0.057).
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Figure 1.
Latencies to mount, thrust, and ejaculate during
sex behavior tests (means ± SEM). Within the four genotypes
generated in the core cross, males
(XYSry and XXSry)
had longer latencies to mount and to thrust than did females
(XY and XX) (p < 0.00005). XY males, which derived from a different cross, had longer
latencies to mount than XYSry
males. Group sizes left to right were as
follows: 8, 15, 16, 15, and 13 for latency to mount; 7, 14, 14, 10, 7 for latency to thrust; 5, 11, and 10 for latency to ejaculate.
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Males displayed more total mounts than females
(F(1,67) = 12.13; p < 0.001) (Fig. 2), but there was no
significant effect of sex chromosomes or a significant interaction.
None of the other behavioral measures (frequencies of mounts with
thrusts or numbers of mounts in the first 30 min after mating) common
to both males and females were significantly different. Total numbers
of mounts tended to be greater for
XYSry males than for XY males
(F(1,23) = 4.07; p = 0.057) (Fig. 2), but none of the other variables showed any
statistically significant differences. There was a trend for the
interval between the onset of intromissions and ejaculation to be
shorter in XY males (32.2 ± 8.4 min) than in
XYSry males (66.6 ± 6.95 min; F(1,17) = 4.28;
p = 0.056).
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Figure 2.
Total mounts (means ± SEM) during sexual
behavior tests with receptive female partners. Within the core cross,
males (XYSry and
XXSry) mounted more than did females
(XY and XX) (p < 0.001). Groups sizes for groups left to
right were 8, 15, 16, 15, and 13.
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To determine whether animals of different genotypes were more or less
likely to engage in certain types of masculine behavior, we conducted
 2 and Fisher exact tests. When a 2 × 2 analysis was done to examine the effect of sex chromosomes or an
interaction of sex chromosomes and sex, we found that the numbers of
mounters and nonmounters did not differ among the four groups. However,
when we examined the frequency of animals that did and did not display
mounts with thrusts, we found that males were more likely to thrust
than females (2(3) = 13.3;
p < 0.01). No differences were found in the
frequencies of XY and XYSry
males that mounted, mounted with thrusts, mounted with intromissions, or ejaculated.
Social exploration
Males spent significantly more total time sniffing, particularly
sniffing stimulus females, than did females (main effect of sex,
F(1,62) = 13.60, 18.37 for all
sniffing and sniffing a female, respectively; p < 0.0005) (Fig. 3), but there were no significant effects of sex chromosomes nor was there an interaction. There was a trend for a sex difference in the total number of visits
[males visited more than females
(F(1,68) = 3.94; p = 0.051)] (Fig. 4), and males visited
stimulus males more often than did females (main effect of sex,
F(1,68) = 4.27; p < 0.05) (Fig. 4). XY males did more visiting and visited the anesthetized
males more frequently than did the
XYSry males
(F(1,25) = 5.37, 4.68, respectively;
p < 0.042) (Fig. 4). When the male groups were
compared using a nested ANOVA, the difference was also significant for
visits to the male (F(1,25) = 5.32;
p < 0.044). A trend in this same direction was noted
for visits to the female (F(1,25) = 3.62; p = 0.07; data not shown).
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Figure 3.
Time spent sniffing an anesthetized female in a 10 min social exploration test (means ± SEM). Within the core cross,
males (XYSry and
XXSry) spent more time sniffing the stimulus female than
did females (XY and XX)
(p < 0.0005).
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Figure 4.
Numbers of visits (means ± SEM) to the
chambers in the three-chambered box, regardless of whether it contained
a male or female stimulus animal (top), and visits to
the chamber that contained the anesthetized male in the social
exploration tests. Males (XYSry and
XXSry) paid more visits to the stimulus male than did
females (XY and XX) (p < 0.05). XY males paid more visits to the chambers with the stimulus
male or female and also visited the stimulus male more often than did
XYSry males
(p < 0.05).
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Spinal nucleus of the bulbocavernosus
The number of SNB motoneurons was greater in males than in females
(Fig. 5), replicating the sex difference
found in rats and mice (Breedlove and Arnold, 1980; Wee and Clemens,
1987; Hauser and Toran-Allerand, 1989; Wagner and Clemens, 1989). In
the two-way ANOVA, there was a significant effect of sex
(F(1,31) = 33.9; p < 0.0001), but no significant effect of sex chromosomes and no
significant interaction. The male groups did not differ significantly from each other.
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Figure 5.
Number of SNB motoneurons (means ± SEM).
Males of the core cross (XYSry and
XXSry) had more SNB neurons than females
(XY and XX) did (p < 0.0001) but did not differ from XY males derived from a different
cross.
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TH-ir neurons in the AVPV
Among the four genotypes that were progeny of
XYSry fathers, females (XX
and XY) had more TH neurons in the AVPV
than did their brothers
(XYSry and XXSry)
(F(1,50) = 27.8; p = 0.000003) (Fig. 6). There was no
significant effect of sex chromosomes and no significant interaction.
XY males were significantly different from
XYSry males in both the basic
and nested ANOVAs (F(1,19) = 9.23 and
13.2; p < 0.008). Related to this is the result that
XY males did not differ from XX in the number of TH-ir neurons, in
distinct contrast to C57BL/6 mice, which show a difference between XX
and XY (Simerly et al., 1997).
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Figure 6.
Number of TH-ir neurons in the AVPV (means ± SEM). Within the core cross, males
(XYSry and XXSry)
had fewer neurons than females (XY and XX) did
(p < 0.000003). Paradoxically, XY males had
a feminine number of TH-ir neurons and therefore significantly more
TH-ir neurons than did XYSry males
(p = 0.08).
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Experiments 1-3
Vasopressin fiber density in the lateral septum
In Experiment 1, the tissue for males and females was fixed and
immunostained at different times, so that the most appropriate comparisons in that experiment are within sex (Fig.
7). In experiment 1, XYSry males were more
masculine (greater fiber density) than XXSry males, but
XY females were less masculine than XX
females. Males had higher density of VP-ir fibers than females, which
replicates the sex difference found consistently in rats and mice
(Mayes et al., 1988; De Vries and Al-Shamma, 1990; Wang et al., 1993).
The magnitude of this sex difference found in experiment 1, however,
could have been partly an artifact of staining differences caused by
separate processing of male and female tissues. In experiment 2, all
tissue was stained and analyzed at the same time. The difference
between XYSry and
XXSry males was replicated in experiment 2, but not the difference between XY and XX females.
The three-way ANOVA showed a main effect of sex chromosomes
(F(1,85) = 4.9; p = 0.0296), and a main effect of sex [males greater than females
(F(1,85) = 90.6; p < 0.000001)]. Planned comparisons indicated that there was an effect of
sex chromosomes in males but not females (Tukey-Kramer;
p < 0.05). There was a significant interaction of
experiment number and sex chromosomes
(F(1,85) = 4.63; p < 0.034) because the difference between XY
and XX, collapsing across sex, emerged in the second but not the first
experiment (Tukey-Kramer; p < 0.05). There was a
significant interaction of experiment number and sex
(F(1,85) = 16.4; p < 0.00012), which reflects the artificially larger difference between sexes in the first experiment than in the second experiment. There was
no main effect of experiment number. There was no significant difference between XY and
XYSry males
(F(1,37) = 2.62; p > 0.05).
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Figure 7.
Density of vasopressin immunoreactive fibers in
the lateral septum (means ± SEM). Black and
hatched bars represent the results from the first and
second experiments, respectively. Within the core cross, males
(XYSry and XXSry)
had a higher density than did females (p < 0.000001). XYSry males had a higher
density than XXSry males (p < 0.05), showing an effect of sex chromosome complement on this
trait.
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Because XY females had higher density of
VP-ir fibers than XX females in experiment 2 but not experiment 1, we
examined this dependent variable in females derived from all-female
litters in experiment 3. In this experiment, two batches of animals
were stained and analyzed at different times and then compared in a single two-way ANOVA. XY females had a
significantly higher density of VP-ir fibers than XX females
(F(1,43) = 6.37; p < 0.02) (Fig. 8). There was no interaction between order of the experiment and genotype.
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Figure 8.
Density of vasopressin immunoreactive fibers in
the lateral septum in mice from all-female litters (means ± SEM).
XY females had a higher density than XX females
did (p < 0.02).
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DISCUSSION |
By measuring sexually dimorphic neural and behavioral traits in
mice of one sex that possess different sex chromosomes (XY vs XX), we
hoped to determine whether the sex chromosomes contribute to normal
sexual differentiation of the brain and behavior. Our hypothesis was
that a masculine complement of sex chromosomes (XY) causes the brain to
develop in a more masculine and less feminine manner, compared with a
feminine complement (XX). The panel of sexually dimorphic brain and
behavioral phenotypes chosen for analysis included masculine copulatory
and social behaviors known to be expressed more in males than females,
and neural dimorphisms that are either male biased (SNB, septum) or
female biased (AVPV) and occur at various levels of the neuraxis.
Because development of these sexual dimorphisms is known in each case
to be potently influenced by gonadal secretions (Wagner and Clemens,
1989; Wang et al., 1993; Rissman et al., 1997; Wersinger and Rissman,
2000a), we suspected that any effects of sex chromosomes might be subtle.
We detected an effect of the sex chromosomes on one measure, the
density of VP-ir fibers in the lateral septum.
XYSry males were more
masculine on this trait than XXSry males, and when females
from all-female litters were examined,
XY females were more masculine than XX
females. In contrast, there was no effect of sex chromosomes on the
other phenotypes in the XX females, XY
females, XYSry males, and
XXSry males, although these phenotypes varied by sex. The
results suggest that sex chromosome genes contribute to some
sex-specific patterns of brain differentiation. However, this
contribution appears to be much smaller for the traits analyzed than
the organizing effects of gonadal hormones, because the differences between male and female mice of the same chromosomal sex were much
larger than the differences between XY
and XX mice of the same sex. Because the relative variability in
exposure to androgens from adjacent male fetuses is a plausible source
of variability in the phenotype (vom Saal and Bronson, 1978), modest
effects of intrauterine androgen may have masked the relatively smaller
effects of sex chromosomes on VP-ir fiber density in females in the
first experiment. The consistently higher VP-ir fiber density found in
XY mice in the all-female litters in the third experiment supports this
possibility. Because a masculine complement of sex chromosomes
(XY) led to greater masculinization of
this trait than did a feminine complement (XX), the difference between
XY and XX mice is in the same direction
as the sex difference in this trait. Although we have emphasized direct
sex chromosome gene influences on neural development, the difference
between groups in the present experiments could be the result of sex
chromosome gene actions on any tissue that influences neural development.
The sex chromosome effect could originate via a number of different
genetic mechanisms. (1) Y genes (encoded on the nonrecombining region
of the Y) might masculinize the trait or inhibit feminine development.
Sry and six other Y-linked genes are reported to be
expressed in mouse brain (Kay et al., 1991; Agulnik et al., 1994;
Zambrowicz et al., 1994; Lahr et al., 1995; Greenfield et al., 1996;
Ehrmann et al., 1998; Mayer et al., 1998; Mazeyrat et al., 1998; Xu et
al., 2002). The sex chromosome effect cannot be attributed to the
action of Sry, however, because Sry was present as a transgene in the genome of both
XYSry and XXSry
males, and was absent in both female groups. (2) A double dose of one
or more X genes might inhibit masculine development or promote feminine
development of the trait in XX mice. Although most X genes are thought
to be expressed in a single dose in each XX somatic cell because of
inactivation of one of the X chromosomes, some X genes escape
inactivation (Carrel et al., 1999) and could therefore be present in
the brain in a higher dose in XX than in XY mice, as has been
documented for several X genes (Xu et al., 2002). The X chromosome
appears to have an unusually high number of genes implicated in
brain-specific functions, which could contribute to the observed sex
chromosome effect (Zechner et al., 2001). (3) Parent of origin effects
(maternal or paternal imprinting) could activate or inactivate X
chromosome genes differentially on maternally or paternally derived X
chromosomes (Leighton et al., 1996; Skuse et al., 1997). Because
XY mice have only a maternally derived X
chromosome, whereas XX mice have X chromosomes derived from both
parents, these imprinting effects could give rise to different X gene
dosages in XY versus XX brain.
The present data support the idea that a masculine genome has a
masculinizing effect on the density of septal VP-ir fibers and that
this sex chromosome effect is not found in other sexually dimorphic CNS
systems (SNB and AVPV) or in several measures of reproductive and
social behaviors mediated by diverse brain circuits. Might
XY and XX mice have experienced
different levels of gonadal or adrenal steroids, so that this effect of
the sex chromosomes represents merely another example of the well known
masculinizing effects of testosterone? Indeed, perinatal testosterone
injections masculinize septal VP fiber density in rats (Wang et al.,
1993). The sex chromosome effect reported here, however, is unlikely to
be mediated by group differences in androgen secretion. Had the
XY genotype caused a greater secretion
of testosterone perinatally relative to the XX genotype, the difference
in these groups should have been detected in more than one phenotype
because most of the phenotypes measured are masculinized by
testosterone or its metabolites, probably during different but
overlapping critical periods (Vale et al., 1973; Wagner and Clemens,
1989; Wang et al., 1993; Simerly et al., 1997). Thus, each system can
be considered a sensitive barometer of levels of gonadal steroids
during perinatal development, and the lack of sex chromosome effects in
most of the systems is evidence against a sex chromosome effect on the levels of circulating gonadal steroids. We conclude that the sex chromosome effect is specific to only one of the hormone-sensitive phenotypes that we measured and does not reflect a broad increase in
steroid secretion or action. More interesting would be a sex chromosome
effect on the cellular and molecular systems that respond to gonadal
steroids (e.g., receptors, receptor cofactors, etc.), or on the level
of sex steroid synthesis in the brain itself (Schlinger et al., 2001).
Whatever the molecular mechanism, the sex chromosome effect does not
require a masculine endocrine environment because it was detected in
both males and females.
Previous results could be interpreted to suggest that factors other
than gonadal hormones contribute to sex differences in VP-ir fiber
density. In rats, neonatal gonadectomy of male rats reduced the VP-ir
fiber density to the level of control females, whereas neonatal
testosterone treatment prevented these changes. Neonatal testosterone
treatment of females, however, failed to increase VP-ir fiber
innervation to that of control males (Wang et al., 1993). Although this
discrepancy in testosterone effect is consistent with a sex chromosomal
effect on the sexual differentiation of VP-ir fiber density, prenatal
gonadal hormone levels may have contributed to sex differences in
testosterone sensitivity. Because it is difficult to mimic throughout
development a female endocrine environment in XY males or a male
endocrine environment in XX females, it has previously not been easy to
eliminate sex differences in endocrine effects to focus on a role for
the sex chromosomes in brain development. The genetic approach outlined
in this paper therefore offers significant advantages for analyzing the
relative contributions of sex chromosomal genes versus gonadal hormones in sexual differentiation.
The failure to find a sex chromosome effect on the number of TH-ir
neurons in the AVPV in the present study might appear to conflict with
the finding of Beyer et al. (1991) that female cultures of rat
embryonic diencephalic neurons express higher levels of dopamine (see
also Sibug et al., 1996). Because Beyer et al. (1991) attributed
this sex difference to nonhormonal factors such as cell-autonomous
actions of genes encoded on the sex chromosomes, one might have
expected a sex chromosome effect on the number of TH-ir neurons in the
AVPV. The lack of an effect could indicate, for example, that sex
chromosome effects are exerted on diencephalic dopamine neurons in
areas other than the AVPV, or that the effects found in
vitro are not manifested in vivo (Reisert et al., 1990; Lieb et al., 1996). Interestingly, mesencephalic neurons from the same
four sibling genotypes of the present experiment
(XY, XX,
XYSry, XXSry) were
recently grown in vitro using conditions similar to that of
Beyer et al. (1991), and a strong sex chromosome effect could be
detected (Carruth et al., 2002). That is, cultures consisting of
XY or
XYSry cells developed more
TH-ir neurons than those derived from XX or XXSry cells, so
that sex chromosome complement, not gonadal status of the embryos, was
the major determinant of group differences in TH neuron number. It is
not yet known whether this sex chromosome effect is found in dopamine
neuron populations in vivo.
XY males differed from XYSry
by several measures: latency to thrust, the number of total visits and
visits to males in the social exploration tests, and the number of
TH-ir AVPV neurons. These differences are potentially attributable
either to the fact that XY and
XYSry come from different
crosses (thus the groups may have experienced different environments or
had uncontrolled differences in genetic background) or to a difference
in the effect of the Sry transgene versus that of endogenous
Sry. The latter seems more likely, given that the nested
litter analysis showed that the variation among litters within groups,
which is the result of environment and chance genetic variation, is
exceeded by the variation between litters across groups, where the
difference in Sry comes into play. These behavioral
differences could reflect Sry effects on the brain or other
tissues. The Sry transgene mRNA may be expressed at higher
levels in embryonic gonadal ridge than that encoded by the endogenous
Sry (A. Swain, unpublished observations). Because the
differences between XY and
XYSry males were found in
several but not all phenotypes, it is not clear whether the effect of
Sry is mediated by an increase in androgen secretion or via
a nonhormonal mechanism. Alternatively, the effects could be on
nongonadal tissue outside of the brain. For example, the number of
total visits and visits to males in the social exploration tests may be
caused by differences in olfactory responsiveness (Paredes et al.,
1998; Dominguez-Salazar et al., 2002). However, given that hypothalamic
dopamine has been implicated in male sexual behavior (Hull et al.,
1999), Sry effects on TH-ir neurons (Beyer et al., 1991) and
male sexual behavior may be related.
Previous studies in mice have proved or suggested that the Y chromosome
contains genes that influence various neural and behavioral traits,
including aggressive behavior (Maxson et al., 1979; Maxson, 1992, 1999;
Roubertoux et al., 1994; Guillot et al., 1995; Sluyter et al., 1996),
the distribution of hippocampal mossy fibers (Hensbroek et al., 1995),
dopamine systems (Sluyter et al., 1995), and brain serotonin (Tordjman
et al., 1995). Moreover, Morris water maze learning performance has
been reported to be more masculine in C57BL/6J
XYPOS female mice than in XX females
(Stavnezer et al., 2000), although the fetal gonads of such females
might contain some testicular tissue (Taketo et al., 1991), which could
cause masculinization via a hormonal mechanism. These studies, together
with the present findings, are consistent with a role for sex
chromosome genes in neural and behavioral sexual differentiation.
Interestingly, the same Y chromosomal factors that influence aggressive
behavior (Sluyter et al., 1996) influence VP-ir fiber density in the
lateral septum of mice selected for short or long attack latencies
(Compaan et al., 1993) and may therefore have contributed to the
differences found in the present study. These differences in VP-ir
fiber density and aggressive behavior may also be causally related
because VP has been implicated in aggressive behavior in rats and voles
(Koolhaas et al., 1990, 1991; Winslow et al., 1993).
We have introduced a powerful model system for examining the separate
and interactive effects of sex chromosomes and gonadal secretions on
sexually dimorphic phenotypes. The dissociation of chromosomal and
gonadal sex in these mice allows, for the first time, a strong test of
the direct role of sex chromosomes in sexual differentiation of the
brain and other somatic tissues. Importantly, these mice offer the
ability to test the role of a masculine (XY) versus feminine (XX)
complement of sex chromosomes under both masculine and feminine
hormonal conditions. Group differences are sensitive to the effects of
both X- and Y-linked genes. These mice will prove useful in further
studies to investigate the role of sex chromosomes in differentiation
of other sexually dimorphic phenotypes and the molecular basis for sex
chromosome-induced somatic sexual differentiation.
Although the present results indicate that at least one sexual
dimorphism in mouse brain is influenced by the complement of sex
chromosome genes, the results are also compatible with the strong web
of evidence that has already proven the dominant role for gonadal
steroids in the induction of sex differences in brain and behavior
(Arnold, 2002; De Vries and Simerly, 2002).
| |
FOOTNOTES |
Received Jan. 31, 2002; revised July 16, 2002; accepted July 22, 2002.
This work was supported by National Institutes of Health (NIH) Grants
R01 MH57759 and K02 MH01349, and by National Institute of Child Health
and Human Development/NIH through cooperative agreement U54 HD28934 as
part of the Specialized Cooperative Centers Program in Reproduction
Research. Islet-1 antibodies were developed by J. Ericson, S. Thor, T. Edlund, T. M. Jessell, and T. Yamada and obtained from the
Developmental Studies Hybridoma Bank maintained by The University of
Iowa, Department of Biological Sciences (Iowa City, IA), under contract
NO1-HD-7-3263. We thank Nancy Forger, Xia Li, and Melissa Kirigiti for assistance.
Correspondence should be addressed to Geert J. De Vries, Center for
Neuroendocrine Studies, Department of Psychology, University of
Massachusetts, Amherst MA 01003-9333. E-mail:
devries{at}cns.umass.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
