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Original Studies |
and ß Immunoreactivity in the Human Supraoptic Nucleus in Relation to Sex and Aging1
Netherlands Institute for Brain Research (T.A.I., F.P.M.K., R.B., D.F.S.), Amsterdam 1105 AZ, The Netherlands; and Department of Histology and Embryology, Kursk State Medical University (T.A.I.), Kursk 305033, Russia
Address all correspondence and requests for reprints to: D. F. Swaab, M.D., Ph.D., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.
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and ß immunoreactivity in the dl-SON. Hypothalami of 34
controls were subdivided into 4 groups within a 50-yr boundary (young
men, young women, elderly men, and elderly women). The AVP part of the
dl-SON of young women contained 50 times more neurons with ERß
nuclear staining than that in young men and 250 times more than that in
elderly women. In addition, young women also showed more ERß
cytoplasmic staining than young men and elderly women. In contrast to
the ERß immunoreactivity, no differences were found in the number of
ER-positive neurons in the 4 groups, but the age and sex pattern of
ER staining was basically opposite that of ERß. Significant
correlations between the percentage of ERß- and ER-positive and
-negative AVP neurons and age were found in women, but not in men. Our
data demonstrate for the first time a strong decrease of ERß and an
increase of ER immunoreactivity in AVP neurons of the dl-SON of
postmenopausal women. Both receptor changes are proposed to participate
in the activation of the AVP neurons in postmenopausal women. |
Introduction |
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50 yr of age), whereas
these neurons appeared to become activated in postmenopausal women (6, 7). Our data suggest an inhibitory role of estrogens in the activity of
AVP neurons in the human SON, and we presumed that these changes were
estrogen receptor (ER) mediated. ERs were shown to be abundantly
expressed in the SON in the rat (8, 9), the ewe (10), and the monkey
(11, 12). To date two genomic subtypes of ER have been cloned in humans
and rodents: ER (13) and ERß (14). They play different roles in
gene regulation; ER activates and ERß inhibits transcription in
HeLa cells (15). Interestingly, the primate hypothalamus contains more
ERß messenger ribonucleic acid (mRNA) than ER (16). In the rat
SON, ERß mRNA, but not ER mRNA, was found (9), and AVP cells were
demonstrated to colocalize ERß and ERß mRNA (17, 18). To date, no
information is available on the presence of ER and ERß in the
human hypothalamus. The aim of the present study was to analyze ERß
and ER expression in the AVP neurons of the human SON in relation to
age and sex. We studied groups of subjects subdivided within a 50-yr
boundary, which is the mean age of the menopause (19), to find out
whether there was a sex difference in ER immunoreactivity in AVP
neurons and a menopause-associated ER decrease in women. Such changes
are of particular interest, because prominent sex and age differences
in the incidence of hypertension and cardiovascular diseases have been
reported. Premenopausal women have a 3–4 times lower prevalence of
hypertension as age-matched men, whereas in women after the menopause
this sex difference is reversed (20, 21). All measurements were
performed in the dorsolateral part of the SON (dl-SON), in which
90–95% cells are vasopressinergic (1, 22). In the present study the
small number of oxytocin (OT) neurons that are localized preferentially
in the cap of the dl-SON (1) was identified on the basis of OT-stained
adjacent sections, and only a semiquantitative analysis of ERs
immunoreactivity was performed in this small group of neurons. |
Materials and Methods |
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Brains from 34 control subjects (16 males and 18 females)
without a primary neurological or psychiatric disease and ranging in
age from 20–94 yr (mean ± SEM, 54.9 ± 3.5)
were obtained at autopsy (see Table 1
for clinicopathological
information). The hypothalami were dissected and fixed in 4%
formaldehyde in phosphate-buffered saline (pH 7.4) at room temperature,
for about 2 months. The fixed hypothalami were dehydrated in graded
ethanols, embedded in paraffin, and cut serially in 6-µm coronal
sections. For anatomical orientation, every 50th section was mounted on
chrome-aluminum sulfate-coated glass slides, deparaffinized, hydrated,
and stained with thionine (0.5%). Two adjacent sections per patient in
the middle of the dl-SON were mounted for immunocytochemistry.
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Immunocytochemical detection of ERß was performed as follows: deparaffinization in xylene and graded ethanols, rinsing in distilled water twice for 5 min each time, rinsing in Tris-containing buffered saline (TBS)-high salt (pH 7.6) twice for 5 min each time, microwave pretreatment in 0.1 mol/L citrate buffer (pH 6.0) twice for 5 min each time at 700 watts, washing in TBS-high salt twice for 5 min each time, incubation in milk-TBS for 1 h at room temperature, washing in TBS-high salt twice for 10 min each time, and incubation with a primary polyclonal goat anti-ERß antibody corresponding to an amino acid sequence mapping at the amino-terminus of ERß of human origin (catalogue no. sc-6820, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:50 in Supermix (0.25% gelatin and 0.5% Triton X-100 in TBS, pH 7.6) for 1 h at room temperature and at 4 C overnight. The next day the sections were washed in milk-TBS twice for 10 min each time; washed in TBS-high salt twice for 5 min each time; incubated with secondary biotinylated antigoat IgG (Vector Laboratories, Inc., Burlingame, CA) 1:200 in Supermix for 1 h at room temperature; washed in milk-TBS twice for 10 min each time; washed in TBS-high salt for 10 min; incubated with avidin-biotin complex (ABC, Elite kit, Vector Laboratories, Inc.) 1:800 in Supermix for 1 h at room temperature; washed in TBS-high salt twice for 10 min each time; incubated with biotinylated tyramine diluted 1:500 in TBS plus 0.01% hydrogen peroxide (H2O2) for 15 min at room temperature; washed in TBS-high salt twice for 10 min each time; incubated with ABC complex as described above; rinsed in 0.05 mol/L Tris-HCl (pH 7.6); incubated in Tris-HCl containing 0.05 mg/ml 3,3'-diaminobenzidine (Sigma, St. Louis, MO), 0.01% H2O2, and 0.2% nickel ammonium sulfate; washed in Tris-HCl twice for 10 min each time; dehydrated in graded ethanols; cleared in xylene; and coverslipped with Entellan mounting medium (Merck & Co., Darmstadt, Germany).
ER staining was performed as follows: deparaffinization in xylene
and graded ethanols, rinsing in distilled water twice for 5 min each
time, rinsing in TBS (pH 7.6) twice for 5 min each time, water bath
pretreatment in 0.05 mol/L Tris-HCl buffer (pH 7.6) for 30 min at 90 C,
washing in TBS twice for 5 min each time, incubation in milk-TBS for
1 h at room temperature, washing in TBS for 5 min, and incubation
with a primary polyclonal rabbit anti-ER antibody recognizing the
carboxyl-terminus epitope of the ER (Santa Cruz Biotechnology, Inc., catalogue no. sc-542) diluted 1:100 in Supermix-milk
(0.25% gelatin, 0.5% Triton X-100, and 5% milk powder in TBS, pH
7.6) for 1 h at room temperature and at 4 C overnight. The next
day the sections were washed in milk-TBS three times for 10 min each
time; washed in TBS for 5 min; incubated with secondary biotinylated
antirabbit IgG (Vector Laboratories, Inc.) 1:200 in
Supermix-milk for 1 h at room temperature; washed in TBS three
times for 10 min each time; incubated with ABC (Elite kit, Vector Laboratories, Inc.) 1:800 in Supermix for 1 h at room
temperature; rinsed in 0.05 mol/L Tris-HCl (pH 7.6); incubated in
Tris-HCl containing 0.05 mg/ml 3,3'-diaminobenzidine
(Sigma), 0.01%
H2O2, and 0.2% nickel
ammonium sulfate; washed in Tris-HCl twice for 10 min each time;
dehydrated in graded ethanols; cleared in xylene; and coverslipped with
Entellan mounting medium (Merck & Co.).
To localize the area containing OT neurons at the dorsal side of the SON (1), adjacent sections were stained with a monoclonal mouse antibody (A1–28). Briefly, after rehydration sections were incubated with A1–28 (1:200), biotinylated horse antimouse IgG (1:200; Vector Laboratories, Inc.), ABC complex (1:800), and 0.05 mol/L Tris-HCl containing 0.05 mg/ml 3,3'-diaminobenzidine, 0.01% H2O2, and 0.2% nickel ammonium sulfate.
The intensity of the staining was estimated semiquantitatively at light
microscopy according to the following scale: +++, strong; ++, moderate;
+, weak; + -, very weak; and -, absent (see Table 1
).
Specificity of the antibody
According to the Santa Cruz Biotechnology, Inc.,
catalog, the ERß antibody is specific for ERß and does not
cross-react with ER, and the ER antibody is specific for ER
and does not cross-react with ERß. We confirmed the specificity of
these antibodies in the following experiments. 1) In a spot blot test
the antibodies were shown to recognize the blocking peptides, whereas
2) an adsorption test resulted in the blocking of the antibodies with
the peptide and elimination of the staining. 3) Staining of adjacent
sections with the antibody against the C-terminus of the ERß
(Santa Cruz Biotechnology, Inc., catalogue no. 6822) (23)
revealed the same pattern as with the antibody against the N-terminus
of the ERß used in the present study. 4) Human ovary and testis
samples were stained in alternating sections, because in both organs
the two ER subtypes (ERß and ER) are known to be expressed (23).
In the ovary, ERß cytoplasmic staining was observed in granulosa
cells and follicles, which is consistent with a recent study in the rat
(24), whereas both nuclear and cytoplasmic staining were found in the
adipose and connective tissues. In the testis, Leydig and connective
tissue cells showed nuclear ERß staining, whereas weak nuclear and
cytoplasmic staining was observed in Sertoli cells and spermatocytes.
In the pituitary, mainly weak cytoplasmic ERß staining was present.
Interestingly, staining with an ER antibody revealed a different
pattern of staining not only in the hypothalamus (Kruijver, F.
P. M., et al., in preparation), but also in the
pituitary, testis, ovary, and uterine tube. In the ovary, follicles and
stroma cells were stained more intensively with anti-ER. Also,
secretory cells of the Fallopian tube showed nuclear staining, which
was absent in ERß sections. In testis, Leydig cells showed weaker
nuclear and cytoplasmic staining for ER compared with ERß, whereas
no staining was observed in connective tissue cells. Sertoli cells and
some spermatocytes demonstrated cytoplasmic and nuclear stainings,
which were stronger than those for ERß. In the pituitary, with
anti-ER clear nuclear and more intense cytoplasmic staining was
observed, whereas only weak cytoplasmic staining was present in the
ERß-stained pituitary, which is in concordance with the study in the
rat pituitary, where ERß was expressed at a lower level than ER
(25). 5) The present study supports the specific staining of ERß and
ER, as a different pattern of staining was found for the two
receptors in the SON in relation to age and sex. 6) Recently, Western
blot analysis was successfully performed (24) for the ER antibody
that was used in the present study. 7) Staining without primary
antibody produced absolutely no staining. Taken together these results
demonstrate the specificity of the antibody used.
Image analysis
As 90–95% of SON cells are vasopressinergic (1), and the small
amount of OT cells could be distinguished on the basis of their dorsal
localization in adjacent sections stained for OT and their
cytoarchitectonic characteristics, AVP neurons were the main focus in
the present study. We analyzed the number of AVP cells that contained a
nucleolus, with nuclear or cytoplasmic ERß or ER staining as well
as the number of neurons negative for ERß or ER using an IBAS
image analysis system (Kontron Instruments Ltd., Zurich,
Switzerland; KAT-based system) (6). The image analysis system was
connected to a Sony XC-77CE black and white CCD camera (Tokyo, Japan)
equipped with a chalnycon tube mounted on a Carl Zeiss
microscope (New York, NY). All measurements were performed using a
560-nm pore size filter, which coincides with the maximum absorption of
the diaminobenzidine/nickel sulfate precipitate in the sections. Area
selection was performed as follows. In each section to be analyzed, an
area covering the SON (using the x2.5 objective of the microscope) was
loaded into the IBAS and displayed on the image analysis monitor. The
position of the section under the microscope was stored using the
x-y-z coordinates of the scanning stage. In this image the
contour of the dl-SON was outlined manually. To select a number of
fields in the SON, a grid that consisted of areas corresponding to the
image size at a x500 magnification (x40 objective) was superimposed
automatically over the SON area. From this grid all fields were
automatically selected. The position of each microscopic field
belonging to this sample was again expressed in the x-y-z
coordinates of the scanning stage. On the basis of these coordinates
the fields were retrieved for measurement. To determine the number of
cells displaying nuclear, cytoplasmic, or negative ERß and ER
staining; the x40 objective was positioned in the microscope; and the
scanning stage was moved to the previously defined positions of the
high magnification measuring areas.
In the present study we defined, in addition to nuclear and cytoplasmic staining, a new category of ERs staining in neurons, i.e. perinuclear staining. This is the presence of a thin black band around the nucleus, probably corresponding to the perinuclear band of endoplasmic reticulum found around the nucleus in the neurosecretory neurons of the SON (26). After the image analysis was finished, the percentage of cells with the three different types of staining was calculated.
Statistical methods
The differences in the percentages of cells with the different types of staining between males and females in various age groups were tested using the two-way ANOVA and t test. To test the correlation between different parameters, such as fixation time, postmortem delay, age and sex of the subjects, and the mean percentage of ER-positive cells, linear regression analysis was used. P < 0.05 was considered to be significant.
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staining was observed in this group. In contrast, more intensely
nuclear and cytoplasmic ER-stained AVP neurons were observed in men
and in elderly women compared to young women. In some patients nucleoli
were stained for ER, but not for ERß. Muscle and endothelial cells
and probably pericytes of blood vessels also showed different patterns
of staining with the two antibodies. They were intensely stained for
ERß and less intensely or not at all stained for ER. Astrocytes
were stained for both ERs. AVP neurons
Only in young women (28–50 yr old) did a large proportion of AVP
neurons show both nuclear and cytoplasmic ERß immunoreactivity (Table 2 and Figs. 1 and 2D).
In the other three groups there was only a very small proportion of
ERß-positive neurons. The cells in these three groups showed
preferentially cytoplasmic staining for ERß and hardly any nuclear
staining. In young men (Table 1 and Fig. 2B) the majority of neurons
were negative for ERß. Elderly women (>50 yr old; Fig. 3D) showed almost exclusively cytoplasmic
staining for ERß, which was, moreover, weaker and present in fewer
neurons than in young women (Table 1). In contrast, in ER-stained
sections the intensity of nuclear staining in AVP neurons was higher in
elderly women and young men than in young women (Table 3 and Figs. 2 and 3). The intensity of
cytoplasmic staining for ER was also higher in elderly women and men
than in young women (Table 1). More negative cells were found in young
women than in any other group studied.
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) AVP
cells/patient were analyzed. Data for the young ovariectomized patient
(no. 80002, Table 1) were not included for the analysis. ERß
The two-way ANOVA test showed that both sex and age were important
for the percentage of AVP neurons staining for ERß in the nucleus
[main effects (ME): F(2, 32) = 5.016; P = 0.013;
the interaction effect between sex and age (IE): F(1, 32) = 6.968;
P = 0.013] and in the cytoplasm [ME: F(2, 32) =
3.773; P = 0.035; IE: F(1, 32) = 4.134;
P = 0.051] or not staining for ERß [ME:
F(2, 32) = 3.773; P = 0.035; IE: F(1, 32) =
4.134; P = 0.051]. The proportion of AVP neurons
staining for ERß in the nucleus was 50 times larger in young women
than in young men (P = 0.019) and 250 times greater in
young than in elderly women (P = 0.008; Table 2 and
Fig. 1). The percentage of cells expressing cytoplasmic ERß in young
women (97.9%) exceeded that in young men (78.3%; P =
0.007) and that in elderly women (88.8%; P = 0.048).
The proportion of AVP neurons negative for ERß was the highest in
young men (21.8%). In young women this percentage was 10 times lower
than in young men (P = 0.007) and 5 times lower than in
elderly women (P = 0.048; Table 2).
The percentage of perinuclear stained neurons was also dependent on age and sex [ME: F(2, 32) = 44.026; P < 0.0001; IE: F(1, 32) = 32.748; P < 0.0001]. It was 7 times higher in young women (54%) than in young men (8%; P = 0.643) and in elderly women (8%, P = 0.107) and was significantly higher in young men than in elderly men (P = 0.033) and in elderly women than in elderly men (P = 0.05). The pattern for the four groups followed that of cytoplasmic staining.
Because the subdivision of the groups at the age of 50 yr is arbitrary, we also performed linear regression analysis that demonstrated a correlation between the percentage of nuclear ERß-positive AVP cells and age (r = 0.509; P = 0.002) and between the percentage of cytoplasm positive and negative cells and sex (r = 0.456; P = 0.008). After subdivision into males and females, the correlation between age and the percentage of nuclear (r = 0.682; P = 0.002), cytoplasmic (r = 0.571; P = 0.013), and negative (r = 0.571; P = 0.013) cells was present only in females and was absent in males (r = 0.288; P = 0.279 and r = 0.255; P = 0.341, respectively).
ER
Age and sex had no significant effect on the proportion of
ER-stained neurons (Table 3 and Fig. 1), but a significant IE
between these two factors was present for the percentage of nuclear
ER-positive AVP neurons [IE: F(1, 32) = 4.703;
P = 0.038]. The proportion of nuclear ER-positive
neurons was higher in young men than in young women (P
= 0.018) and higher in young men than in elderly men (P
= 0.015). Linear regression analysis showed a significant relationship
between the proportion of cytoplasmic ER-positive AVP neurons and
age (r = 0.419; P = 0.015) and between the
percentage of ER-negative neurons and age (r = 0.419;
P = 0.015) in the whole group. After subdivision into
males and females, the above-mentioned relationships (r = 0.610;
P = 0.007) and the correlation between the percentage
of nuclear ER-positive cells and age (r = 0.548;
P = 0.019) were found again only in women and not in
men (r = 0.348; P = 0.204 and r = 0.980;
P = 0.727, respectively).
OT neurons
OT neurons were identified in the SON on the basis of their
position (a small number of cells in the cap of the dl-SON) (1) and
their cytoarchitectonic characteristics, i.e. smaller size
and OT staining in adjacent sections. All subjects demonstrated
cytoplasmic staining of OT neurons for ERß, whereas only a few OT
cells showed nuclear staining (patients 94040 and 92047). In some
patients the intensity of the cytoplasmic ERß staining of the OT
neurons was stronger than that in AVP neurons (Table 1). It did not
show clear sex differences, but decreased during the course of aging.
In addition, OT cells in the SON, when stained with anti-ER
antibody, showed moderate cytoplasmic staining regardless of age and
sex.
Pathological parameters
Fixation time was not correlated to the percentage of 1) nuclei
staining for ERß (P = 0.305) or ER
(P = 0.608), 2) cells with positive cytoplasm for ERß
(P = 0.235) or ER (P = 0.717), and
3) ERß (P = 0.235)- or ER (P =
0.717)-negative cells. Postmortem delay appeared to be significantly
correlated to the number of cells showing ERß (P =
0.017) and ER (P = 0.012) nuclear staining. This
correlation could, however, be fully explained by the accumulation of
subjects with long postmortem times in the younger age group. As there
was no difference in postmortem delay between young males and young
females (P = 0.876), whereas only young women showed a
prominent nuclear ERß staining, this difference cannot have
influenced the sex differences in our data.
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Discussion |
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in AVP neurons of the human SON and their differential
expression in relation to age and sex. As nuclear staining of ERs is
considered to be bound and stimulating, cytoplasmic to be unbound and
not active (see below), it is of great interest that nuclear and
cytoplasm ERß-positive AVP neurons were found predominantly in young
women, whereas in young men and elderly women more nuclear
ER-positive AVP cells were observed. In our previous studies (6, 7)
we showed that AVP neurons in young women are less active than those in
young men or elderly women. Our current data show that ERß
immunoreactivity is stronger, and the proportion of ERß-positive AVP
neurons is highest in young women and lowest in young men. Elderly
women showed decreased expression of ERß in the SON. This indicates
that the inhibitory role of estrogens in AVP neuron activity is
probably mediated via the increase in ERß and the decrease in ER
(6, 7). Activation of female AVP cells in aging, as shown previously
(6, 7), may thus occur as a result of the drop in estrogen levels after
the menopause (19), a subsequent loss of ERß in AVP neurons, and an
increase in ER, resulting in diminished inhibition on these neurons.
Indeed, it was previously shown that ovariectomy in the rat caused an
increase in plasma AVP levels (27) and in the neurosecretory activity
of SON neurons (28) in females. One case in our study (no. 80002), a
46-yr-old woman who underwent a bilateral ovariectomy 22 months before
her death and, hence, had low estrogen levels, deserves special
attention. No nuclear staining of ERß was found in that patient,
whereas prominent cytoplasmic ERß staining was marked in both AVP and
OT neurons. Interestingly, when stained for ER, this patient showed
the strongest nuclear staining. This observation supports the idea of
ERß-mediated inhibition and ER-mediated stimulation of AVP cells
by estrogens acting at the genomic level. The observed sex differences
in ERß expression support the previous reports (6, 7) that the
activity of AVP neurons in young women may be suppressed directly by
estrogens via ERß when small amounts of ER are present. It was
demonstrated in vitro in a GH3 cell
line that estrogens up-regulate ERß expression (25) and down-regulate
ER in some rat brain areas (29), indicating that the effects of
estrogen on ER expression are region specific (29). Moreover, it has
been shown recently that in the same region (in rat dorsal root
ganglion neurons) long-term estrogen treatment of ovariectomized rats
down-regulates the levels of ER mRNA and up-regulates the levels of
ERß mRNA (30), which is in line with our data about the differential
expression of ER subtypes in relation to age and sex. In the rat an
alternative trans-synaptic regulation of SON neurons by
estrogens from lamina terminalis and preoptic area projecting to the
SON has, in addition, been proposed (31). Whether a similar mechanism
of SON regulation also operates in the human hypothalamus is not known
at present.
In contrast with ERß, a totally different pattern of ER staining
was observed in the AVP cell population. Men and elderly women showed
more nuclear and cytoplasmic positive neurons than young women. More
negative ER cells were observed in young women than in any other
group studied. Our data are fully in agreement with the proposed
antagonistic roles of ERß and ER in HeLa cells in
vitro, where ER-activated and ERß-inhibited transcription
(15). Thus, in young women in whom ERß immunoreactivity is high,
ER expression is significantly lower, while in elderly women and
young men, in whom ERß immunoreactivity is negligible, ER is
abundantly expressed. A large body of evidence in animal experiments
suggested differential roles of ERß and ER. Thus, in the rat,
mRNAs and peptides of these two ER subtypes appeared to be
differentially localized not only in the hypothalamus (32, 33), but
also in the ovary and uterus (34, 35, 36). Moreover, the content of ERß
in female rhesus macaques was higher than that in males (37). It was
further suggested that ERß and ER may differ in transcriptional
activities (38).
Nuclear/cytoplasmic ER staining
In the SON of rat (8, 9) and monkey (11), ER immunoreactivity was
described in both the nucleus and the cytoplasm. The presence of
nuclear ERs in neurosecretory cells in animals (8, 9, 11, 17, 18) and
humans, as appears from the present study, suggests a direct genomic
regulatory effect of estrogens in AVP neurons. It has been well
demonstrated that both liganded and unliganded ERs are localized in the
nucleus of the neurons (39) and that unliganded ERs are present in both
the nucleus and the cytoplasm of neurons, including dendrites and
axonal terminals (40, 41, 42). Cytoplasmic immunostaining was eliminated
1 h after 17ß-estradiol administration, probably due to
conformational changes in the receptor (43). This means that if
estrogens strongly affect cell function they are mainly present in the
nucleus and to a much lesser degree in the cytoplasm. Steroid receptors
continuously shuttle between the nucleus and cytoplasm by both
diffusion and active transport (44). In addition, it was shown in the
rat that high levels of ERs coincide with the preovulatory estrogen
level surge (45), suggesting ER (probably ER) up-regulation in the
brain tissue by estrogens. All observations to date indicate that
binding of the appropriate hormonal ligand to the receptor activates
the receptor by phosphorylation, resulting in its movement from the
cytoplasm to the nucleus (44). This sequence of events fully agrees
with our observation of a high proportion of SON neuronal nuclei
staining for ERß exclusively in young females. The existence of a sex
difference may, in principle, be due either to organizational effects
during development or to activational effects of sex steroids in
adulthood (46). Our data show an inhibitory effect of estrogens on AVP
neurons depending on circulating levels of estrogens in adulthood and
possibly mediated by ERß (Refs. 6, 7 and the present study). The
reported sex difference in ERß and should thus be interpreted as
"activational inhibitory" effects in adulthood.
Perinuclear and nucleolar staining
In 8–54% of the SON neurons we noticed a clear ERß perinuclear
staining. This staining followed the pattern observed in cytoplasmic
staining concerning its sex and age differences. According to the
observations of Eneström (26), in the rat this band might be the
perinuclear part of the granular endoplasmic reticulum (nuclear
envelope), which is consistently proliferating, and its outer leaf
invaginates into the perikaryon. Indeed, we observed a similar
perinuclear band in thionine-stained sections in the SON of the
patients studied, suggesting that the nucleus of the AVP neurons in the
human dl-SON is also surrounded by the perinuclear part of the
endoplasmic reticulum. Fewer (1–9%) ER-stained neurons showed
perinuclear staining.
Interestingly, we observed nucleolar staining in ER-stained cells in
several cases, predominantly in young men and elderly women. This
localization is in agreement with the study in human breast cancer
epithelial cell lines, where nucleolar staining was also found with
ER and was suggested to be a consequence of the mechanism involved
in ER down-regulation (47), and is thus consistent with our data
showing lower ER expression in the SON in young women compared to
other groups.
ERß and ER in OT neurons
In OT neurons, very prominent ERß cytoplasmic staining without
nuclear staining was generally present. Only in two cases was nuclear
staining found for ERß. We cannot speculate on the reason for the
weak nuclear staining in patient 92047, but in the case of patient
94040 reanimation was performed with high doses of vasoactive drugs
followed by a cardiogenic shock that may have influenced these SON
cells. In a few cases OT neurons in the SON stained more intensively
for ERß than AVP cells. We did not find a clear sex difference in
ERß immunoreactivity in OT neurons, whereas a gradual decrease in
ERß cytoplasmic staining was observed in aging. ER expression in
the SON OT cells showed only moderate cytoplasmic staining regardless
of age or sex. This was unexpected, because in animals OT neurons were
found to express both ERß and ER immunoreactivities (17, 18, 48),
and the OT gene contains estrogen-responsive elements in the rat (49)
and human (50). The large body of experimental evidence suggests that
estrogens up-regulate OT production in the rat (51, 52, 53, 54). It was,
however, further suggested that estrogens not only directly regulate
genes present in OT neurons via estrogen receptors (9, 17), but also
exert their action at the OT cell membrane level (55), which may
explain the absence of sex differences in ER staining in the OT neurons
of the SON. It should also be noted that OT neurons of the SON
represent only a small number per case, and it is well known that the
majority of OT neurons are located in the paraventricular nucleus (1),
which is the subject of future study.
In summary, our results demonstrate for the first time differential
expression of ERß and ER in the human SON that is strongly
influenced by age and sex in an antagonistic way. The decreased ERß
and increased ER staining in postmenopausal women are probably
essential parts of the mechanism of activation of AVP neurons in this
group of subjects. The activation of AVP neurons in postmenopausal
women may be at least a part of the explanation for the frequent
occurrence of hypertension and other cardiovascular diseases in this
group of people.
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Acknowledgments |
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Footnotes |
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Received November 22, 1999.
Revised June 1, 2000.
Accepted June 7, 2000.
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References |
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- and -ß-mRNA in the
rat central nervous system. J Comp Neurol. 388:507–525.[CrossRef][Medline]
and ERß at
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