Cent. Eur. J. Biol. • 3(1) • 2008 • 39-47
DOI: 10.2478/s11535-008-0003-z
Central European Journal of Biology
Prenatal testosterone improves the spatial
learning and memory by protein synthesis
in different lobes of the brain in the male
and female rat
Research Article
Cristian Gurzu*, Vlad Artenie, Lucian Hritcu, Alin Ciobica
Department of Molecular and Experimental Biology,
Biology Faculty, University „Alexandru Ioan Cuza”,
700505, Iasi, Romania
Received 16 July 2007; Accepted 3 January 2008
Abstract: T he high density of the steroid hormone receptors in the structures of temporal lobe involved in learning and memory, such as the
hippocampus, perirhinal cortex, entorhinal cortex and amigdaloid complex, shows that there must be a direct relationship between
gonadal hormones and organizational effects of steroid hormones in those structures during development of the nervous system.
The present study was undertaken in order to investigate the effect of testosterone administration during the third week of gestation
on the spatial memory formation of the offspring rats and the level of soluble proteins in the temporal lobe and frontal lobe of brain,
as evidence of important organizational effects of androgens during prenatal development in brain sexual dimorphism. Animals have
received testosterone undecanoate on days 14, 15, 16 and 19, 20, 21 of gestation. Learning and memory tests were started 100 days
after the testosterone treatment. At the end of the experiments, the temporal and frontal lobes of brain were removed for assessing
the level of soluble proteins. Testosterone treatment significantly improved spontaneous alternations percentage of male offspring in
Y-maze task comparative with female offspring and reference memory in radial 8 arm-maze task (decreasing in number of reference
memory errors in both male and female offspring groups), suggesting effects of both short and long-term memory. Also, testosterone
significantly increased the brain soluble protein level of treated female rats in 14-16 prenatal days compared with the control group as
well as the brain soluble protein level of treated male rats. These results suggest that steroid hormones play an important role in the
spatial learning and memory formation by means of protein synthesis in different lobes of the brain.
Keywords: Testosterone • Spatial memory • Frontal lobe • Temporal lobe • Protein synthesis
© Versita Warsaw and Springer-Verlag Berlin Heidelberg. 1. Introduction
In most mammals, the hippocampus has a welldocumented role in spatial memory acquisition [1-3].
Previous studies, which have shown sex-dependent
differences in hippocampal morphology and physiology,
suggest a modulatory role for sex steroids in hippocampal
function [4-9].
In the medial temporal lobe, there is a group of
interconnected structures of limbic system which
appears to be of great importance for declarative
memory consolidation [10,11]. The key structures are
the hippocampus, the nearby cortical areas, and the
pathways that connect these structures with other parts
of the brain [12-14].
In these structures were found a high density of
steroid receptors, especially androgen receptors [15,16]
suggesting there must be some relationship between
androgen receptors and development of these structures
during pregnancy.
The study of sexual dimorphism in humans and
other animals has focused on the role of hormones in
organization and differentiation during development
[9,17-19]. The literature of androgen effects on spatial
memory in animals and humans is complex and
contradictory [5,7,20].
* E-mail: [email protected]
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Prenatal testosterone improves the spatial
learning and memory by protein synthesis in different
lobes of the brain in the male and female rat
Some evidence suggested a positive correlation
between testosterone and spatial ability [21-24].
Support for the hypothesis that gonadal steroids
influence memory is found from studies in which
castration of male rats inhibits performance, and
treatment of intact and ovariectomized female rats with
testosterone improved spatial performance [19,25].
Furthermore, neonatal treatment of intact male rats with
cyproterone acetate, an androgen antagonist, feminized
their performance in a spatial learning task, as adults
[26].
Studies on organizational effects of androgens and
estrogen, on spatial ability have almost exclusively
targeted the first 10 postnatal days to be critical for
the establishment of sex differences [19]. However,
functional gonadal steroids can be detected in the blood
stream of the rat in as early as the last week of gestation
[27,28]
In the learning process, there are two components:
short-term memory for recent events - working memory
[29] and long-term acquired memory for past events reference memory [30].
The current conceptual framework for the
consolidation of memory proposes the involvement
of de novo protein synthesis and modulation of gene
expression [31-33].
The possible role of new protein synthesis in memory
has been investigated extensively since the introduction
of drugs in the 1969s that selectively inhibit the assembly
of protein from messenger RNA [13].
It is well established in a variety of species and
training paradigms, that protein synthesis is required for
the formation of long-term memory [34-36].
The present study employed the influence of prenatal
testosterone in cognitive performances on offspring rats
by means of behavioural tests for spatial working memory
and reference memory and a possible correlation with
soluble protein level in the frontal lobe and the temporal
lobe of brain as evidence for important organizational
effects of androgens during prenatal comparative with
postnatal development in brain sexual dimorphism. The
rationale for administration of testosterone during 14, 15,
16 and 19, 20, 21 gestational days was to investigate
when the brain is most receptive to androgens and if
the brain receptivity could have important effects in later
behaviour during adulthood.
2. Experimental Procedures
2.1. Subjects
Fifteen female albino Wistar rats (200-250 g) obtained
from the Faculty of Biology of Iasi were housed five per
cage, in large cages, with a male rat. After copulation
(0 day) the male was removed and the pregnant
females were maintained at room temperature (22ºC)
under standard 12:12 h light – dark cycle. Food and
water were available ad libitum. Rats were treated in
accordance with the guidelines of animal bioethics from
the Act on Animal Experimentation and Animal Health
and Welfare Act from Romania and all procedures were
in compliance with the European Council Directive of 24
November 1986 (86/609/EEC).
2.2. Prenatal treatment
Steroid hormones were administrated intraperitoneally
(i.p.), during pregnancy period: five pregnant female
received testosterone undecanoate (2 mg/kg b.w. i.p.,
SIGMA) in peanut oil (FLUKA) during prenatal days 14,
15, 16, five pregnant females received testosterone
undecanoate (2 mg/kg b.w. i.p., SIGMA) in peanut
oil (FLUKA) during prenatal days 19, 20, 21 and five
pregnant females (control) received injections with
peanut oil.
Thirty days following birth, the animals were weaned
and separated by type of treatment into colony cages
(3 animals per cage). At 40 days, animals were singly
caged. Learning and memory tests were started 100
days after the steroid hormones treatment.
2.3. Y-maze task
Short-term memory was assessed by spontaneous
alternation behaviour in the Y-maze task. The Y-maze
used in the present study consisted of three arms (35
cm long, 25 cm high and 10 cm wide) and an equilateral
triangular central area. The rat was placed at the end of
one arm and allowed to move freely through the maze
for 8 min. An arm entry was counted when the hind paws
of the rat were completely within the arm. Spontaneous
alternation behaviour was defined as entry into all three
arms on consecutive choices.
The number of maximum spontaneous alternation
behaviours was than the total number of entered minus
2 and percent spontaneous alternation was calculated
as (actual alternations/maxim alternations) X 100 [37].
Spontaneous alternation behaviour is considered to
reflect spatial working memory, which is a form of shortterm memory.
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C. Gurzu et al.
The radial-arm maze with 8 arms was used to asses
the reference memory. The apparatus extending away
from a central platform at equal angles. A stand placed
under the central platform raised the maze 40 cm above
the floor of the room. Each arm of the maze was 48
cm long, 12 wide and 13 cm high. Five arms (1, 2, 4, 5
and 7) were baited with a food pellet. At the beginning
of each session (one session per day for 12 days), the
rat was placed on the central platform and allowed to
choose arms in the maze until all the food pellets were
obtained.
The order of arms chosen was recorded, and a
response was defined when the central point of its body
was more than 10 cm in the arm. Measures consisted
of the number of correct choices (defined as successful
entry into a baited arm to eat the food pellet), the number
of working memory errors (defined as entry to an arm
that previously contained food but had already been
consumed) and the number of reference memory errors
(defined as entry to an unabated arm). The time taken to
consume all five rewards was recorded.
2.5. Tissue collection
After the behavioural tests, all rats were anesthetised,
rapidly decapitated and whole brain was removed. The
frontal and temporal lobes were collected. Each of the
brain tissue samples was weighed and homogenized
with a glass homogenizer in bidistilled water (1 g
tissue/10 ml bidistilled water).
Samples were centrifuged 15 min at 3000 x g.
Following centrifugation, the supernatant was separated
and pipetted into tubes.
2.6. Protein assay
The amount of proteins in the supernatant obtained after
brain tissue homogenate centrifugation was measured
using Bradford dye-binding method, with bovine serum
albumin as standard [38].
2.7. Statistical analysis
The results of radial 8 arm-maze were analyzed using
two-way ANOVA (ANOVA: two-factor without replication).
The results for protein assay were analyzed using
Student’s t-Test. All results are expressed as means
± SEM; F values for which P<0.05 were regarded as
significant.
3. Results
3.1. Effects of testosterone treatment on shortterm memory in Y-maze task
Prenatal treatment with testosterone undecanoate (2
mg/kg/day) in different days (14,15,16 and 19,20,21
gestational days) of pregnancy significantly improved
spontaneous alternation percentage in Y-maze task in
both male and female offspring rats (n=8 per group),
suggesting effects on short-tem memory (Figure 1).
Testosterone treatment significantly improved the
spontaneous alternation percent in both male and
female treated groups in 14,15,16 days of gestation
(female 30.24%, male 32.29% P<0.001) compared with
both male and female treated groups in 19,20,21 days
of gestation (female 29.27%, male 31.46% P<0.001)
and compared with control groups (female 17.50%,
male 18.50%), respectively.
35,00
Spontaneous alternation ( %)
2.4. Radial-arm maze task
*
*
30,00
25,00
20,00
15,00
10,00
5,00
0,00
Female Male Female Male Control Control
14-16
14-16 19-21
19-21 female male
Figure 1.
The spontaneous alternation in Y maze test in male and
female offspring rats. The data are means ± SEM. (n=
8 per group). *P < 0,001 vs. control group.
3.2. Effects of testosterone treatment on longterm memory in radial 8 arm-maze task
Regarding spatial ability in the radial 8 arm-maze task,
the results showed that steroid hormones administered
during 14, 15, 16 and 19, 20, 21 gestational days had
important effects in development of the structures
involved in spatial memory. In both treated-groups,
testosterone undecanoate significantly decreased
numbers of reference memory errors during 12 days
of training in radial 8 arm-maze, suggesting effects on
long-term memory comparative with control groups
(Figures 2, 3). The number of reference memory errors
were significantly impaired in male (F(2,35) = 7.82,
p<0.002) and female (F(2,35) = 6.45, p<0.006) treated
group in 14-16 prenatal days comparative with in 1921 prenatal days and control group, suggesting that
testosterone treatment improved the male and female
spatial cognitive performance, particularly in the 14-16
days of treatment.
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The mean of errors in reference memory during 12 days of
test in 8 radial arm maze for male treated groups during
14-16 and 19-21 prenatal days. The data are means ±
SEM F(2,35)=7.82, p<0.002.
These results suggest that the improvement of
cognitive performance is lead by the organizational
effects of androgens in the brain, which could be more
important during 14-16 gestational days rather than 1921 gestational days.
3.3. Bradford protein assay in brain rat
homogenate tissue
The results obtained by the Bradford protein assay of the
brain tissue homogenate showed significant differences
of soluble protein levels between female and male rats
treated with testosterone comparative with the control
groups.
In the testosterone female treated-rats (during 1416 prenatal days), there was a higher concentration
of soluble proteins in frontal lobe (124.774 mg/g brain
tissue, P<0.05) (Figure 4) and in temporal lobe (100.415
mg/g brain tissue, P<0.05) (Figure 5) comparative with
treated group during 19-21 prenatal days (71.41 mg/g
brain tissue of frontal lobe and 56.30 mg/g brain tissue
of temporal lobe) (Figures 4, 5) or control group (60.50
mg/g brain tissue of frontal lobe and 44,80 mg/g brain
tissue of temporal lobe) (Figures 4, 5).
Female control
140
Female testosterone
*
120
100
80
60
40
20
0
days of treatments
14-16
Figure 4.
mg solubile proteins/ g brain tissue
Figure 3.
The mean of errors in reference memory during 12 days of
test in 8 radial arm maze for female treated groups during
14-16 and 19-21 prenatal days. The data are means ±
SEM. F(2,35)=6.45, p< 0.006.
19-21
The amount of soluble proteins in brain tissue from the
frontal lobe in female rats. The data are means ± SEM.
(P*< 0.05).
Female control
120
Female testosterone
*
100
80
60
40
20
0
14-16
Figure 5.
days of treatments
19-21
The amount of soluble proteins in brain tissue from the
temporal lobe in female rats. The data are means ±
SEM. (P*< 0.05).
In the testosterone male treated groups (during
14-16 prenatal days) there was a higher concentration
of soluble proteins in temporal lobe (86.03 mg/g brain
tissue, P<0.05) (Figure 6) comparative with frontal lobe
(73.89 mg/g brain tissue, P<0.05) (Figure 7) and with 1921 prenatal days treated group (78.47 mg/g brain tissue
of temporal lobe, 72.81 mg/g tissue of frontal lobe) or
control group (67.21 mg/g brain tissue of temporal lobe,
63.93 mg/g brain tissue of frontal lobe, P<0.05).
Male control
mg solubile proteins / g brain tissue
Figure 2.
mg solubile proteins/ g brain tissue
Prenatal testosterone improves the spatial
learning and memory by protein synthesis in different
lobes of the brain in the male and female rat
100
Male testosterone
*
90
80
70
60
50
40
30
20
10
0
14-16
Figure 6.
days of treatment
19-21
The amount of soluble proteins in brain tissue from the
temporal lobe in male rats. The data are means ± SEM.
(P*< 0.05).
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mg solubile proteins / g brain tissue
C. Gurzu et al.
90
Male control
Male testosterone
80
70
60
50
40
30
20
10
0
14-16
Figure 7.
days of treatment
19-21
The amount of soluble proteins in brain tissue from the
frontal lobe in male rats. The data are means ± SEM.
4. Discussion
Previous studies have demonstrated that administration
of testosterone during the first week of life improved the
performance of female rats but impaired the performance
of males in radial arm-maze and water-maze, possibly
due to its organizational effects on the development of
the dentate gyrus [7]. Similar organizational effects were
described earlier in rhesus monkeys. When females
were treated with testosterone during development,
they showed object discrimination equivalent to that of
untreated males [39].
It was hypothesized that testosterone and/or its active
metabolites dihydro-testosterone and estrogen may be
regulating developmental processes (e.g., neurogenesis,
neuronal cell death, neuron migration, neuronal soma
growth, neuronal differentiation) prenatally, which may
significantly influence masculinization and feminization
of hippocampal target sites, and ultimately of adult
spatial ability. It has been shown that androgens appear
to be responsible in regulating masculinizing effects in
the CA1-CA3 volume and pyramidal neuron soma size
in hippocampus [19].
These studies indicated a relationship between early
androgen exposure and later spatial ability. The ability
of androgen, possibly following conversion to estrogen
[40,41], to affect the development of cognitive abilities
wanes after the first 10 days of life [9]. Furthermore,
in gonadally intact adult male rodents, exposure to
testosterone or other anabolic steroids for 1-3 months
did not affect acquisition or retention in the standard
reference memory version of the water maze in male
rats or voles [39,42].
The results of the experiment showed that prenatal
administration of testosterone undecanoate during third
week (days 14, 15, 16 and 19, 20, 21) of pregnancy
improved the cognitive performances in spatial working
memory and reference memory of male and female
offspring rats versus control. The cognitive performances
were superior for treated male than treated female rats
with testosterone during prenatal development.
Also, the effects of testosterone could be more
important in the development of brain during 14, 15, 16
than 19, 20, 21 prenatal days.
These cognitive performances could be correlated
with an increase of the amount of brain soluble proteins
in frontal and temporal lobes, in a different manner in
male and female rats.
Thus, in female rats treated with testosterone during
14-16 prenatal days the amount of brain soluble protein
was significantly increased in frontal and temporal lobes
compared to females treated during 19-21 prenatal
days and the amount of protein was higher in the frontal
lobe than the temporal lobe in all female groups. These
results could be explained by an augmentative role of
testosterone which improves the organizational effects
of estrogens in the brain during prenatal development of
nervous system. However, in the male rat, the amount of
brain soluble protein is higher in temporal lobe than the
frontal lobe in all treated male groups These results could
be correlated with high density of androgen receptors
in structures of temporal lobe, which are involved in
learning and memory.
These results are supported by studies in humans,
using in vivo magnetic resonance imaging, where
specific regions of cerebral cortex have sex differences
in volume relative to cerebrum size [43]. Thus, women
had larger cortical volumes, relative to cerebrum size,
than men in the majority of the frontal and medial
paralimbic brain regions. Significantly larger volumes
in women than men were seen in the precentral gyrus,
frontoorbital cortex, superior frontal and lingual gyri.
Men had larger volumes, relative to cerebrum size, in
frontomedial cortex, the amygdala and hypothalamus
[43].
Previous work has demonstrated that protein
synthesis is critical for consolidation of long–term
memory in a spatial memory task [44]. It has been
demonstrated that a lack of protein synthesis can block
or impair memory function. However, it has not been
shown that retention is directly related to the amount of
protein synthesis that accure after training [34].
These data could be correlated with recent studies
that has demonstrated the effect of anisomycin (protein
synthesis inhibitor) which impaired spatial learning and
memory in Morris water maze task by administration
in the hippocampus of the adult male rats or in the
basolateral nucleus of the amygdala [45].
Early studies have provided evidence of an increase
in expression of specific structural proteins and their
mRNA in neuronal plasticity processes such as neuronal
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Prenatal testosterone improves the spatial
learning and memory by protein synthesis in different
lobes of the brain in the male and female rat
growth and synapse formation which could underlie the
molecular mechanisms of learning and memory [46,47].
Changes in the expression of mRNA enconding certain
structural components may underlie the mechanism
of plastic response of neurons. Some evidence is
accumulating that sex steroids play a significant role in
regulating the expression of certain structural proteins
and their mRNA in sex steroid-sensitive tissues [48-52].
Tubulin, the main component of microtubules, is one of
the major cytoskeletal proteins in neurons of the central
nervous system [46]. Growth-associated protein 43
KD (GAP-43) is concentrated in axonal growth cones
and has been implicated in axonal elongation and
synaptogenesis [46].
It has been reported that the amount of mRNA coding
for tubulin isolated from neonatal rat POA-hypothalamus
is higher in males than that in females and that amount in
females is increased by neonatal exposure to androgen
or estrogen [48,53]. In addition, estrogen and androgen
induce neuritic growth in cultured rat pheochromocytoma
PC 12 cells in which estrogen receptor and androgen
receptor genes are transfected, respectively [54]. It is also
reported that in the developing hypothalamus, androgen
modifies the monoclonal antibody immunoreactivity
in radial glia which may be responsible for neuronal
organization [49]. These organizational actions of sex
steroids seem to induce permanent sexual dimorphism
in synaptic connections. Although it is not known how
sex steroids act on developing brain tissue, resulting in
sexual difference in neuronal circuitry, it is possible to
speculate that axons of sex steroid-sensitive neurons
exposed to sex steroids during developmental period
would grow and branch more rapidly than sex steroidinsensitive neurons [55].
It has been demonstrated that in the CA1 field of
hippocampus, the Schaffer collateral axons from CA3
field use glutamate as their transmitter. Glutamate
produces LTP by acting postsynaptically on at least
two types of receptors: NMDA receptors and AMPA
receptors. Also, three kinds of protein kinases have
been activated during LTP in the formation of long term
memory: calcium-calmodulin protein kinase II [56],
protein kinase C [57] and the tyrosine kinase [58]. Once CaM-KII is activated by calcium, it acts
on itself, autophosphorylating the 286th amino acid.
Once autophosphorylation occurs, CaM-KII travels
to the postsynaptic density of dendritic spines where
it phosphorylates AMPA receptors and makes them
more sensitive to glutamate. Long-term potentation
causes AMPA receptors to move into dendritic spines
and appears also to cause the formation of additional
synapses [59].
Also, it was reported that the anti-androgen flutamide
inhibited the rapid effects of testosterone on Ca2+ pointing
[60].
Thus, testosterone could increase the calcium
concentration in nervous cells which activate the CaMKII. It is possible that testosterone is involved in synthesis
of calcium-calmodulin protein kinase, an enzyme, which
has been shown to be, implicated in the formation of
new synapses and the glutamate receptors.
This hypothesis could explain the molecular
mechanism which underlies the role of testosterone in
learning and memory and synaptic plasticity of neurons
in hippocampus and other structures of the limbic
system, which has been demonstrated by treatments
with dehydroepiandrosterone and flutamide on
hippocampal CA1 spine synapse density in male and
female rats [61].
One conclusion of this experiment is that prenatal
testosterone has different effects in the brain of female
and male rats. Thus, testosterone could have a more
important organizational effect in the frontal lobe of
female rats than male rats during 14-16 prenatal days
and important organizational effects in the temporal lobe
of male rats in some prenatal period. Previous studies
shows that these structures are involved in learning and
memory, the frontal and temporal cortices in short term
memory and the hippocampus is implicated in spatial
function [62]. The hypothesis about the role of prenatal
androgens in spatial learning and memory could be
supported by increase of cognitive performances in
working memory and reference memory for all treated
animals and also by an increase in the level of proteins
in the frontal and temporal lobe of treated rats.
These results explain that a lack of testosterone during
the prenatal period could affect the development of the
frontal lobe and structures of the limbic system.
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