1Department of Physiology, Kaohsiung Medical College, Kaohsiung 807; and 2School of Nursing, Mei-Ho Institute of Technology, Pingtung 900, Taiwan
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ABSTRACT |
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Hsu, Hseng-Kuang, Rei-Cheng Yang, Huei-Chuan Shih, Ya-Lun Hsieh, U-Yang Chen, and Chin Hsu. Prenatal Exposure of Testosterone Prevents SDN-POA Neurons of Postnatal Male Rats From Apoptosis Through NMDA Receptor. J. Neurophysiol. 86: 2374-2380, 2001. The role of N-methyl-D-aspartate (NMDA) receptor in mediating the effect of testosterone exposure prenatally on neuronal apoptosis in the sexual dimorphic nucleus of the preoptic area (SDN-POA) of rats was studied. The endogenous testosterone was diminished by prenatal stress (PNS) or simulated by testosterone exposure (TE) to understand the effect of testosterone on NR1 (a functional subunit protein of NMDA receptor) expression and neuronal apoptosis. To further study whether the testosterone, after being converted into estradiol, modulates NR1 expression, 4-androstein-4-ol-3,17-dione (ATD; an aromatase inhibitor) was used to block the conversion of estradiol from testosterone. The expressions of the NR1 mRNA and NR1 subunit protein were quantified by RT-PCR and western blotting analysis, respectively. In addition, a noncompetitive antagonist of NMDA receptor, MK-801, was used to find out whether blockage of NMDA receptor affects the naturally occurring apoptosis in SDN-POA. The results showed the following. 1) Expression of perinatal NR1 subunit protein in the central part of the medial preoptic area of male rats was significantly higher than that of females, especially on postnatal days 1 and 3. 2) The testosterone level of male fetuses on embryonic day 18 was significantly higher than that of females, while the testosterone level of TE females or PNS males was similar to that of intact males or intact females, respectively. 3) The apoptotic incidence of intact male rats was significantly less than that of females, and the apoptosis was stimulated by PNS in male or inhibited by TE in female. 4) The expression of NR1 subunit protein could be inhibited by PNS or ATD-treatment in male, while stimulated by TE in female. 5) NR1 mRNA showed no significant difference among intact male, PNS male, ATD-treated male, TE female and intact female rats. 6) The low apoptotic incidence of male rats was significantly increased when NMDA receptor was blocked by MK-801. These results suggest that testosterone, after being converted to estradiol, may prevent the SDN-POA neurons of male rats from apoptosis through enhancing the expression of NR1 at the posttranscriptional level.
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INTRODUCTION |
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Morphological sexual dimorphism
has been found in the medial preoptic area (MPOA), which is the major
site of glutamate receptor (Brann 1995). Glutamate
regulates GnRH release (Brann and Mahesh 1997
) and
controls neuronal survival (Ikonomidou et al. 1999
) via
N-methyl-D-aspartate receptor (NMDAR). The
sexual dimorphic nucleus of POA (SDN-POA), a circumscribed region
within the medial part of the MPOA, of male rats exhibits about
seven-fold greater in nuclear volume than females (Gorski et al.
1980
). This difference in the nuclear volume has been
attributed to the prevention of neurons from apoptosis developed by the
influences of circulatory androgen after being converted to estrogen
during the perinatal period (Davis et al. 1996
;
Dohler et al. 1986
). A previous report indicated that
estradiol treatment increased the number of NMDA receptor binding sites
in the rat hippocampus (Woolley et al. 1997
) and
modulated NMDAR function via regulation of the
NR1 subunit protein (Gazzaley et al.
1996
), which is mandatory for channel activity
(Moriyoshi et al. 1991
; Nakanishi 1992
).
Our previous results showed that the NR1
expression in POA of neonatal male rats was higher than that of females
(Hsu et al. 1999
). Since only male fetuses showed a
testosterone peak, and moderate NMDA receptor activation is involved in
the survival signal of the neuron (Ikonomidou et al.
1999
; Takadera et al. 1999
), it is reasonable to
speculate that the high testosterone level evoked during
embryonic days 17-19 in male rats may contribute to this
sexual dimorphism of perinatal NR1 expression and
subsequent postnatal neuronal survival. Thus the aims of the study are
1) to investigate the time correlation between perinatal
testosterone peak and NR1 expression; 2) to determine the effect of testosterone administration
prenatally, after converting into estradiol, on the neonatal
NR1 expression and neuronal apoptosis; and
3) to evaluate the neonatal NMDA receptor blockage on
enhancing the apoptosis in the SDN-POA neurons at postnatal day
8.
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METHODS |
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Animals
In conducting the research described in this study, the authors
adhered to the guidelines of the National Institutes of Health for the
use of experimental animals, and all experiments were approved by the
Animal Committee of the Kaohsiung Medical University. Long-Evans rats
were mated. The presence of plug under the cage on the next day was
marked embryonic day 1 (ED1). Pregnant rats were
housed individually under a controlled temperature (22 ± 0.5°C)
and on a 14 h:10 h light:dark cycle. Food and water were available
ad lib. The sex of the rat pups was confirmed by the presence of
intra-abdominal testes in male rats after sampling. Under light ether
anesthesia, the brain tissues were isolated from rat pups individually
at ED18, ED19, ED21, postnatal day 1 (PND1), PND3, PND5, and PND7 for the
analysis of NR1 subunit protein expression. To
dissect the brain tissue containing the SDN-POA, perpendicular cuts to
the ventral floor of the brain were made at the center of the optic
chiasm and the rostal margin of the third ventricle. Bilateral cuts
were placed at about 0.5 mm from the ventricle. The tissue was served
from the rest of the brain by a horizontal cut at a depth of 1 mm
(Takagi and Kawashima 1993).
Testosterone-exposed (TE) female and prenatal-stressed (PNS) male rats
Subcutaneous injections of testosterone propionate (TP; 2 mg/0.1
ml/d) in sesame oil were given to mother rats from ED16
through ED18 (Dodson and Gorski 1993). Female
pups were chosen as TE female. The other group of pregnant rats was
stressed from gestational day 16 to day 18. The
stress treatment utilized followed the method by Ward and Weisz
(1984)
with some modification. In brief, it consisted of
placing females individually into a 11 × 5 × 5-cm Plexiglas
holder for 2 h daily. The male pups were chosen as PNS males.
Increased urination and defecation was observed as an indicator of the
stress, which effectively induced low circulatory levels of
testosterone in fetal males at ED18 (Anderson et al.
1985
; Ward and Weisz 1980
). Serum levels of
testosterone were measured at the afternoon of ED18 by
radioimmunoassay (RIA).
RIA of testosterone
The serum samples were extracted with diethyl ether and allowed
to freeze in a dry ice-ethanol mixture. The ether was decanted into
another tube and dried under ventilation at 38°C. The dried residue
was dissolved in 0.01 M PBS (pH 7.4) containing 0.1% gelatin. Aliquots
of the PBS-gelatin-dissolved steroids were used for RIA as previously
described without further chromatographic separation of testosterone
(Yu et al. 1988). The sensitivity of the testosterone assay was 1.25 pg/assay tube, and the intra-assay and inter-assay coefficients of variation were 2.95 and 10.3%, respectively.
In situ apoptosis analysis: TUNEL staining
Blocks of brain tissue containing the SDN-POA were embedded in
paraffin. Serial sections in the frontal plain were cut at 10 µm
thickness. The SDN-POA was included about 18-20 tissue sections in
total. One slide was selected every two or three sections. Therefore
about eight sections were chosen and counted in each rat brain sample.
The tissue sections were first de-paraffinized by washing the sections
in two changes of xylene and then in two changes of ethanol. They were
then washed sequentially in 90% ethanol, 80% ethanol, 70% ethanol,
and TBS, and the glass slide around the specimen was carefully dried
(Lucassen et al. 1995; Shi et al. 1991
).
Then, the specimens were subjected to proteinase K (20 µg/ml)
digestion (25°C, 20 min). Three percent hydrogen peroxide (incubated
at room temperature for 5 min or more) was used to quench endogenous
peroxidase activity. DNA strand breaks were identified by TUNEL assay
using the KLENOW FragEL DNA fragmentation detection kit (Calbiochem,
Cat No. QIA21) following the manufacturer's protocol. The cells were
counter stained with methyl green. Negative controls were processed
identically except that terminal deoxynucleotidyl transferase
(TdT) was not added. The incidence of apoptosis was derived
from the quotient of apoptotic nucleus number divided by the sum of
total neuron numbers in each section. All measurements were performed
by a single person.
4-Androstein-4-ol-3,17-dione (ATD; an aromatase inhibitor) treatment
Pregnant Long-Evans female rats received daily subcutaneous
injections of ATD (5 mg in 0.1 ml propylene glycol; n = 4) or propylene glycol (0.1 ml; n = 4) from days
10-22 of pregnancy (day of impregnation = day 1)
(Houtsmuller et al. 1994). Within 9 h of birth,
male pups from an ATD or propylene glycol-treated mother were killed.
Brain tissue containing SDN-POA was sampled for estimation of the
expression of NR1.
Dizocilpine hydrogen maleate (MK-801) treatment
Neonatal rat pups were injected with vehicle or an NMDA receptor
blocker, MK-801 (0.4 mg/kg body wt sc), on PND1
(Ikonomidou et al. 1999; Speiser et al.
1991
). Then, brain was sampled on PND8 for tissue
section and TUNEL stain.
Western blot analysis of NR1 subunit protein
The brain tissues containing SDN-POA were isolated from rat pups
for the analysis of NR1 expression. The cell
membrane was prepared as described previously (Hsu et al.
1999). Nine volumes of dissecting buffer (50 mM Tris-acetate,
pH 7.4, 10% sucrose, 5 mM EDTA) were added to each sample and
homogenized in glass homogenizer for 12-15 strokes. The suspension was
subsequently centrifuged for 30 min at 16,000 g, and the resulting
pellets were resuspended, rehomogenized, and stored at
70°C. The
protein concentration was estimated using the Bio-Rad protein
microassay procedure (Bradford 1976
). Equal amounts of
protein (15 µg) were heated in boiling water for 5 min and were
separated by 7.5% SDS-polyacrylamide gels. The gel was transferred
onto polyvinylidene difluoride (PVDF) transfer membrane by
electroblotting for 1 h (100 V), and the membrane was blocked
overnight at 4°C by the Tween-Tris buffer saline solution (t-TBS)
containing 5% nonfat dry milk and 0.1% Tween 20. Blot was incubated
with NR1 antibody (monoclonal; PharMingen, San
Diego, CA) at a 1:1,000 dilution in t-TBS containing 5% nonfat milk
and 0.1% Tween 20 for 1 h. Then it was washed in t-TBS (20 mM
Tris base, 0.44 mM NaCl, 0.1% Tween-20, pH 7.6), and incubated for
1 h with goat anti-mouse IgG (HRP conjugated, Santa Cruz) diluted
to 1:2,000 in t-TBS containing 5% nonfat dry milk and 0.1%
Tween 20. The blot was subsequently washed for 1 h in the t-TBS.
Immunoreactive protein was visualized by enhanced chemiluminescence (ECL, Amersham) according to the manufacturer's specifications (Siegel et al. 1994
).
RT-PCR of the NR1 mRNA
The mRNA level of NR1 was determined by
RT-PCR, which is widely used in neuroendocrine studies concerned with
small tissue sample (Zamorano et al. 1996). Total RNA
was isolated from tissue block containing SDN-POA. First strand cDNA
was reverse transcribed from 5 µg total RNA. The PCR primers used
were 5' primer: 5'-AACCTGCAGAACCGCAAG-3', and 3' primer:
5'-GCTTGATGAGCAGGTCTATGG-3' for NR1. The cDNA of
-actin was also amplified as an internal control. The primers used
were 5' primer: 5'-CTACAATGAGCTGCGTGTGG-3'. 3' primer:
5'-TAGCTCTTCTCCAGGGAGGA-3'. The amplification profile involved
denaturation at 94°C for 1 min, primer annealing at 45°C for 1 min,
and extension at 72°C for 1 min. This cycle was repeated 30 times
(Younkin et al. 1993
). The amplified 333 bp cDNA of
NR1, which spanned a portion of the reported
sequence for the rat NR1 cDNA (Moriyoshi
et al. 1991
) and 450 bp DNA of
-actin, was
ethanol-precipitated, dried, and dissolved in Tris/EDTA buffer and
observed after 1.8% agarose gel electrophoresis under ultraviolet light.
Statistical analysis
All data were analyzed with one-way ANOVA followed by Scheffé method. A 95% confidence limit was accepted as statistically significant.
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RESULTS |
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NR1 subunit protein expression in the SDN-POA of male and female rats during perinatal period
A 116 KD protein was identified by NR1 monoclonal antibody in the membrane preparation of tissue from SDN-POA of neonatal rats. The protein expression of NR1 in intact male rat was higher than that of intact females from ED18 to PND7, which is more significant (P < 0.01) at neonatal days 1 and 3 (Fig. 1).
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Serum levels of testosterone in the intact male, TE female, PNS male and intact female rat on ED18
The testosterone level of male fetus at ED18 was significantly higher than that of females. As expected, the serum levels of TE female and PNS male were similar to intact male and intact female, respectively (Fig. 2).
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Effect of prenatal exposure of testosterone on the apoptotic incidence in the SDN-POA of rat at PND8
The incidence of neuronal apoptosis per 1,000 cells in the SDN-POA of male rat (1.2 ± 0.73%, mean ± SD) was significantly lower than that in females (4.17 ± 2.16%; P < 0.01). The apoptotic incidence per 1,000 cells in PNS males was significantly increased to 4.14 ± 1.02% (P < 0.01), while apoptotic incidence in TE female was significantly decreased to 0.69 ± 0.19% (Fig. 3).
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Effect of prenatal exposure of testosterone on the expression of NR1 subunit protein and RT-PCR product of NR1 mRNA during neonatal stage
The neonatal protein expression of NR1 in the SDN-POA of male rats was decreased when the endogenous critical testosterone peak at ED18 was diminished by prenatal stress or ATD treatment. In contrast, when the high testosterone level was simulated by testosterone exposure in female fetus, the neonatal protein expression of NR1 was higher in comparison with that of intact females (Fig. 4). However, there was no quantitative difference of the RT-PCR products of NR1 mRNA among the intact male, PNS male, and TE female and intact female rats (Fig. 5A) or among control male, ATD-treated male and control female rats (Fig. 5B).
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Effect of neonatal MK-801 treatment on the incidence of neuronal apoptosis in the SDN-POA of male and female pups at PND8
The incidence of neuronal apoptosis per 1,000 cells in the SDN-POA of male rat (1.5 ± 0.63%) was significantly lower than that in females (4.86 ± 0.63%; P < 0.01). The apoptotic incidence per 1,000 cells in MK-801-treated males was significantly increased to 6.10 ± 0.40% (P < 0.01), while no significant difference between MK-801-treated (3.22 ± 1.20%) and intact female rats (Fig. 6) was observed.
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DISCUSSION |
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The hypothalamic MPOA plays an important role in the initiation
and coordination of all endocrine systems, and some functions, i.e.,
gonadotropin secretion and prolactin secretion, have been shown to be
sexually dimorphic. The hormone milieu of male and female fetuses is
strictly different. The male fetus, but not female, exhibits a
testosterone peak during embryonic days 18-20 (Ward
and Weisz 1980). It has been well known that the
characteristic sex differences in physiological functions depends not
on the genetic sex of the animal, but on the exposure of the brain to androgen during a critical period of development. Although it appears
that the perinatal development of sexual phenotype in the rodent brain
is determined by exposure to estradiol generated locally via
aromatization of androgen (Arai et al. 1996
;
Dohler et al. 1986
), the mechanisms underlying these
processes are not fully understood. A previous report indicated that,
in the SDN-POA, conversion of testosterone to estradiol by neuronal
aromatase during development appears to be a prerequisite for sexual
differentiation (McCarthy et al. 1993
). Aromatase
activity appears to be sexually dimorphic in POA, enzyme activity being
higher in the male (McLusky et al. 1985
). Besides,
aromatase activity rises rapidly to peak levels before birth in the rat
hypothalamus (Lephart et al. 1992
). In the present
results, the enhancement of the NR1 subunit
protein expression, but not mRNA expression of
NR1, by the prenatal testosterone exposure was
blocked by ATD, an aromatase inhibitor. Although ATD may act both as an
androgen receptor blocker and as an aromatization inhibitor
(Kaplan and McGinnis 1989
), the recent report indicated that ATD decreased the immunostaining of brain aromatase-immunoreactive cells (Foidart et al. 1995
) and decreased the aromatase
mRNA concentration in the POA (Balthazart 1997
).
Therefore we were inclined to believe that testosterone is converted
into estradiol to affect NR1 expression at the
posttranscriptional level.
Few studies have directly addressed the mechanisms underlying estradiol
regulation of NMDARs. Posttranscriptional control of
NR1 protein expression has been demonstrated
previously in PC12 cells (Sucher et al.
1993). Moreover, Gazzaley et al. also indicated that estradiol
enhances the function of NMDA receptor via posttranscriptional
regulation of NR1 expression (Gazzaley et
al. 1996
). It is reasonable to predict that the
posttranscriptional regulation may be due to an increase in rate of
protein translation and/or an alteration in the rate of protein
degradation. Although the classical cellular mechanism of steroid
hormone regulation of gene transcription seems unable to explain the
posttranscriptional modification by estradiol, we could not exclude the
possibility that estradiol modulates the expression of
NR1 subunit protein indirectly via estrogen receptors.
The NMDA receptor is a heteromeric complex of several subunits, most
likely consisting of an NR1 subunit and
NR2 subunits that affect function and
ligand-binding characteristics during development (Adams et al.
1999; Buller et al. 1994
; Monyer et al.
1992
; Sheng et al. 1994
; Zhong et al.
1995
). In the developing CNS of rat, the
NR1 gene is expressed in virtually all neurons, whereas the four NR2 transcripts display distinct
expression patterns (Monyer et al. 1994
). Most
attractively, the hypothalamus shows a predominance of both the mRNA
and protein of NR2D receptor subunit (Dunah et al. 1996
; Goebel and Poosch
1999
; Monyer et al. 1994
), and the
NR2D mRNA is co-localized with estrogen receptor
(ER) mRNA in the rat hypothalamus (Watanabe et al.
1999
). Since one genomic DNA fragment, corresponding to the
putative 3'-untranslated region (UTR) of NR2D
gene, contained at least four half palindromic estrogen-responsive
elements, estrogen possibly regulates NR2D via
its 3'-UTR regulatory region in rat hypothalamus (Watanabe et
al. 1999
). It suggested that NR2D subunit
may play an important role in brain development and estradiol may
regulate NMDA receptor activity by modulating
NR2D mRNA level in an ER-dependent manner. Recent
evidence suggests that the functional NMDA receptor is probably a
pentamer (Hawkins et al. 1999
; Premkumar and
Auerbach 1997
). However, it is not known whether
NR1 expression is modulated by
NR2 subunit protein and the mechanisms regulating
the assembly of the NMDA receptor are not well characterized. A more
detailed characterization of estradiol-modulated NMDA receptor subunit protein expression remains to be elucidated.
Our recent report indicated that the marked expression of NMDA receptor
in male fetuses might protect the SDN-POA neurons from a naturally
occurring neuronal death partly by modulating prenatal testosterone
levels (Hsu et al. 2000). The present results indicate
that prenatal exposure of testosterone enhances the neonatal NR1 subunit protein expression. Furthermore,
blockage of neonatal NMDA receptor diminished the protective effect of
testosterone exposure on neuronal apoptosis in SDN-POA of male rats. It
has been reported that activation of NMDA receptor results in an
increase in intracellular Ca2+ loading
(Dayanithi et al. 1995
), NFkB nuclear translocation
(Guerrini et al. 1995
), and some gene expressions, such
as brain-derived neurotrophic factor (BDNF) that has been
reported as possibly mediating the anti-apoptotic effect of NMDA in
cerebelar granular neurons (Aliaga et al. 1998
;
Bhave et al. 1999
; Tabuchi et al. 2000
).
Calcium may promote cell survival through CaM-K kinase activation of
PKB, which in turn phosphorylates and dissociated BAD (a
distant member of the Bcl-2 family that promotes cell death) from
Bcl-xL-BAD complex (Korsmeyer 1999
; Porter
1999
; Yano et al. 1998
). Considerable evidence
also indicates that NFkB induces the expression of anti-apoptotic gene
products, such as MnSOD, inhibitor-of-apoptosis proteins
(IAPs) (Wang et al. 1998
), calbindin, and Bcl-2
(Mattson et al. 2000
). It suggests that NMDA receptor may play an important role in neuronal apoptosis during sexual development.
In conclusion, the present study provides the first evidence that the prenatal exposure of testosterone, after being converted into estradiol, protects neonatal SDN-POA neurons from naturally occurring neuronal apoptosis by enhancing the expression of neonatal NMDA receptor. The molecular mechanism of NMDA receptor activation in mediating the prevention of neurons from apoptosis needs further investigation.
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ACKNOWLEDGMENTS |
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This work was supported by Grant NSC-89-2320-B037-018 from the National Science Council, Republic of China.
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FOOTNOTES |
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Address for reprint requests: C. Hsu, Dept. of Physiology, Kaohsiung Medical College, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan (E-mail: m685003{at}cc.kmu.edu.tw).
Received 7 March 2001; accepted in final form 3 August 2001.
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REFERENCES |
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