Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
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Autonomic Neuroscience: Basic and Clinical
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Prepubertal estrogen exposure modifies neurotrophin receptor expression in celiac
neurons and alters ovarian innervation
Gabriel Anesetti a,⁎, Paula Lombide a,b, Rebeca Chávez-Genaro a
a
b
Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, General Flores 2125, CP 11800, Uruguay
Departamento de Morfología y Desarrollo, Facultad de Veterinaria, Lasplaces 1550, CP 11600, Universidad de la República, Montevideo, Uruguay
a r t i c l e
i n f o
Article history:
Received 30 April 2008
Received in revised form 10 October 2008
Accepted 22 October 2008
Keywords:
Sympathetic neurons
Neurotrophins
Peripheral innervation
Ovary
a b s t r a c t
Estradiol is a key hormone in the regulation of reproductive processes acting both on peripheral organs and
sympathetic neurons associated to reproductive function. However, many of its regulatory effects on the
development and function on the sympathetic neurons have not been completely clarified. Sympathetic
neurons located in the celiac ganglion projects to visceral, vascular and glandular targets, and contribute to
ovarian innervation, being the main source of sympathetic fibers. In the present study, we analyze the effects
of elevated levels of exogenous estrogen during the prepubertal period in post-ganglionic sympathetic
neurons. Estrogen exposure induced a significant increase in sympathetic celiac neuronal size and modified
the expression of neurotrophin receptor p75. This change affected mainly small and medium size neurons.
The effect of estrogens on innervation of celiac target organs was heterogeneous, inducing a significant
increase in catecholaminergic innervation of the ovary, but not of the pyloric muscular layers. These findings
further support the role of estrogen as a modulator of neuronal plasticity and suggest that estrogen could
modify some features involved in the relation between sympathetic immature peripheral neurons and their
target organs throughout a neurotrophin-dependent mechanism.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
A functional interaction between reproductive and peripheral nervous
systems has been postulated by numerous investigations (Ojeda et al.,
1983; Aguado and Ojeda, 1984; Ahmed et al., 1986; Chavez and
Dominguez, 1994). The connection between both systems has shown
the celiac ganglion (CG) as part of a multisynaptic neural network
connecting the CNS and the ovary (Aguado, 2002; Gerendai et al., 2002).
The CG is localized on the ventral wall of the abdominal aorta just rostral to
the root of the superior mesenteric artery, and joins the superior
mesenteric ganglia to form the celiac superior mesenteric ganglion
complex. This ganglion contains the cell bodies of the postganglionic
sympathetic neurons projecting their axons toward the lower esophageal
sphincter, the stomach and the small intestine in addition to vascular and
glandular targets (Baljet and Drukker, 1979). The relation between
neuronal soma size and innervation target has been assumed after
many experimental approximations. In the CG, the largest neurons include
those projecting to the enteric plexuses and smallest neurons are related
mainly to vasoconstrictor pathways (Boyd et al.,1996; Jobling and Gibbins,
⁎ Corresponding author. Gabriel Anesetti, Departamento de Histología y Embriología,
Facultad de Medicina, Universidad de la República, General Flores 2125, CP 11800;
Montevideo, Uruguay. Tel.: +598 2 9243414x3481; fax: +598 2 9243414x3551.
E-mail address: [email protected] (G. Anesetti).
1566-0702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.autneu.2008.10.021
1999; Gibbins et al., 2000). CG contributes to ovarian innervation being the
main source of sympathetic fibers (Baljet and Drukker, 1979; Klein and
Burden, 1988a,b), modulating ovarian steroidogenesis and follicular
growth (Aguado and Ojeda, 1984; Mayerhofer et al., 1997).
Estrogens can modify the levels of expression of the enzyme tyrosine
hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, or alter the density of innervation of different organs. These changes
are well characterized at the uterus (Chavez-Genaro et al., 2002;
Krizsan-Agbas and Smith, 2002; Chavez-Genaro et al., 2006), where they
are more pronounced, but also take place in other reproductive organs
including the ovary (Shinohara et al., 1998; Lara et al., 2000) and the
vagina (Ting et al., 2004). An increase in the sympathetic innervation of
the ovary has been demonstrated in animal models of polycystic ovary
induced by a single estrogen injection. The human polycystic ovary
syndrome is characterized by follicular maturation arrest, hyperandrogenism, abdominal obesity, and insulin resistance (Nelson et al., 2001;
Barber et al., 2006). However, most of PCO animal models induced by
estradiol valerate results in acyclicity and ovarian morphology resembling PCO, but without the typical metabolic disturbances of human
PCOS (Schulster et al., 1984; Mannerås et al., 2007). A potential
contribution of the peripheral sympathetic system to the syndrome
has been suggested (Lara et al., 2002; Rosa et al., 2003). On the other
hand, prenatal exposure to diethylstilbestrol, a synthetic estrogen,
results in a decreased density of the sympathetic nerve network in the
ovaries and this decrease is associated with a reduction in the number of
celiac neurons (Shinohara et al., 1998; Shinohara et al., 2000). Estrogens
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G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
might act directly on ganglionic neurons or indirectly inducing changes
in the expression of growth factors in target tissues (Papka et al., 1997;
Bjorling et al., 2002). Nerve Growth Factor (NGF) and other neurotrophins are responsible for survival and differentiation of sympathetic and
sensory neurons. They also regulate cell fate decisions, axon growth, and
patterning of innervation and many aspects of neural function (LeviMontalcini, 1987; Huang and Reichardt, 2001). Nearly all postganglionic
sympathetic neurons are NGF responsive, without significant response
to others members of the neurotrophin family. NGF binds to two classes
of receptors: the low-affinity universal neurotrophin receptor p75
(p75NTR) that recognizes all members of neurotrophin family, and the
specific high affinity Trk receptor kinase (TrkA) that recognizes NGF and
in a lesser extent, Neurotrophin-3 (NT-3) (Lee et al., 2001). NGF
produced and released by target cells acts on TrkA and p75NTR
receptors located on axon terminals of innervating neurons. This factor
initiates locally biochemical signals within distal axons, and sends
information retrogradely to neuronal cell bodies and ultimately to their
nuclei (Ye et al., 2003), altering the expression of many of the genes that
codify proteins crucial for normal neuronal function (Campenot and
MacInnis 2004). Estrogen-responsive elements have been identified in
many of these genes (Toran-Allerand, 1996). Therefore, estrogen may
regulate neurite growth and differentiation indirectly by autocrine or
local paracrine mechanisms mediated by growth factors and their
receptors. Several studies showed that estrogen differentially regulate
p75NGFR and trkA expression, altering the ratio of the two NGF
receptors, and, consequently, neurotrophin responsivity (Miranda et al.,
1994; Richeri et al., 2005). On the other hand, long-term exposure to NGF
enhanced estrogen binding in PC12 cells (Sohrabji et al., 1994a). This
reciprocal regulation may affect peripheral neuronal sensitivity to both
estrogen and neurotrophins. Many of the regulatory effects of estrogens
on the development and function on the sympathetic neurons of the
peripheral nervous system have not been clarified yet. Since the celiac
neuronal population projects to gastrointestinal, vascular, and glandular
targets, alterations in neurotrophin receptor expression may have
important consequences. In the present study, we examined the effects
of elevated estrogen levels during the prepubertal period on several
phenotypic features of celiac sympathetic neurons using histological
and immunohistochemical approaches. We evaluated: 1) expression of
ERs; 2) neuronal morphology and neurochemical features; 3) neurotrophin receptor expression and 4) catecholaminergic innervation of
target organs. We hypothesized that prepubertal CG neurons are able to
respond to estrogen and that the type of response depends on target
organ identity.
2. Materials and methods
2.1. Animals and treatment
Studies were carried out on female Wistar-derived albino rats from
the breeding colony held at the Faculty of Medicine (Montevideo,
Uruguay). Animal care and protocols were in accordance with
international guides for use of laboratory animals and approved by
the Experimental Animal Committee (Comisión Honoraria de Experimentación Animal, CHEA) of the Universidad de la República,
Montevideo, Uruguay.
At birth, litters (n = 12) were culled to 1 male and 7 female pups per
litter and maintained with their dams until the end of experimental
period. Animals were housed in plastic cages with clear, hardwood
chips bedding, fed ad libitum and provided with tap water. The animal
room was maintained within a temperature range of 21–22 °C and
relative humidity of 50+/−10%. The dark–light cycle was 12:12 h (lights
on 07:00 a.m.–07:00 p.m.).
Each litter was assigned to control or treated groups. At fifteen
postnatal days, female rats from treated group were subcutaneously
injected with a single dose of 40 μg of β-estradiol-17-cypionate (ECPestradiol® Laboratorios König, Buenos Aires, Argentina) diluted with
peanut oil (Sigma, St. Louis, MO, USA) in a final volume of 0.1 ml.
Control rats from matched litters were injected with the same volume
of vehicle (peanut oil). Animals were assigned to one of the following
experimental groups: control animals killed 3 days after vehicle
injection (3d-C); control animals killed 7 days after vehicle injection
(7d-C); animals killed 3 days after estrogen injection (3d-E2); and
animals killed 7 days after estrogen injection (7d-E2). Body weight and
vaginal opening (VO) were daily monitored during the experiment.
Vaginal smears were taken daily from all rats after VO. Three different
litters were included in each experimental group.
2.2. Tissue preparation
At the end of the experimental period, animals were deeply
anesthetized with a combination of ketamine hydrochloride (Vetanarcol®, Laboratorios König, Buenos Aires, Argentina) and xylazine
(Sedomin®, Laboratorios König, Buenos Aires, Argentina) at doses of
100 mg/kg of body weight and 5 mg/kg respectively, and perfused
intracardially with 0.9% saline followed by a Zamboni's fixative
solution. Uteri were rapidly collected, stripped of adipose tissue,
luminal fluid removed by blotting onto absorbent paper, and weighed.
Uterine weight to body weight ratios were calculated for each animal.
Ovaries, pylori and celiac ganglia were quickly dissected and postfixed
in the same fixative for 6–8 h. Pylori and ovaries were chosen in order
to assess the effects of estrogen exposure on the peripheral CG neuron
endings. They were stored at 4 °C overnight in a 30% phosphatebuffered sucrose solution, and transferred to embedding medium
(Cryomatrix®, Shandon, Pittsburgh, PA, USA) frozen and sectioned at
40 μm thickness using a cryostat and used as free floating sections for
immunohistochemistry. Celiac ganglia were transferred to 70%
ethanol before processing into paraffin using standard methods.
Three or four ganglia obtained from each litter were pooled,
dehydrated in increasing concentrations of ethanol and embedded
in a single paraffin block. Five micrometer thick sections were
obtained and mounted on silane-coated slides. Every fifteenth
sections from each block were mounted together on slides destined
to immunohistochemical procedures as explained below. Ten to
twelve slides containing 6–8 sections of each group of ganglia were
obtained by this procedure. Remaining sections were deparaffinized
in xylene, rehydrated and stained with hematoxylin–eosin (H–E).
2.3. Immunohistochemistry
For celiac ganglia immunohistochemistry, sections were deparaffinized, rehydrated and rinsed in distilled water. Antigen retrieval was
achieved with microwave treatment at 160 W in 10 mM sodium citrate,
pH 6.0, for 30 min, cooling down 20 min. The sections were washed
three times (10 min each) in phosphate buffered saline (PBS) and
incubated for 20 min in 0.3% hydrogen peroxide in PBS to inhibit
endogenous peroxidases. Sections were rinsed again three times in PBS
followed by a 30-min incubation at room temperature in PBS containing
0.5% Triton X-100. The sections were blocked in PBS containing 0.5%
Triton X-100 and 2% bovine serum albumin for 1 h and incubated
overnight at 4 °C with the primary antibody diluted in blocking solution.
Sections were then rinsed in PBS and incubated with the corresponding
biotinylated secondary antibody (1:300 in blocking solution) at room
temperature for 1 h. To visualize the immunoreactive cells, the sections
were incubated for 1 h at room temperature with avidin–biotin complex
(Vector Laboratories, Burlingame, USA), washed in PBS and then
exposed to a 0.02% 3,3′-diaminobenzidine (Sigma Chemical Co, St.
Louis, USA) solution. The reaction was stopped in tap water and some
sections were counterstained in hematoxylin (20 s) dehydrated in
ethanol, cleared in xylene, coverslipped and examined on a Nikon
photomicroscope. The primary antibodies used have been extensively
characterized. To recognize the presence of estrogen receptors, the
following antibodies were used: a polyclonal rabbit anti-estrogen
G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
37
receptor α (ERα, MC-20; SC-542 no. K0402, Santa Cruz Biotech, Santa
Cruz, USA; 1:400) that recognize the C-terminus of ERα (Pendaries et al.,
2002) and a rabbit polyclonal antiserum against a recombinant protein
corresponding to the amino acids 1–150 mapping at the N terminus of
ERβ (ERβ, H-150; SC-8974 no. K1102, Santa Cruz Biotech, Santa Cruz,
USA; 1:100), (Tsukahara and Yamanouchi 2002; Chen et al., 2004). To
identify the presence of the low affinity Nerve Growth Factor-Receptor
(p75), a monoclonal anti-Nerve Growth Factor Receptor antibody
(p75NTR, clone 192-IgG, no.MAB365, Chemicon, Temecula, USA; 1:50)
that recognize the extracellular domain of the rat low affinity Nerve
Growth Factor-Receptor (p75) was used. Its specificity has been shown
in previous studies (Chandler et al., 1984; Mukai et al., 2000). The
presence of tyrosine hydroxylase was assessed using a monoclonal antityrosine hydroxylase antibody, (NCL-TH, Novocastra Laboratories, Newcastle, UK; 1:400). The specificity of this antibody was assessed
previously (Robertson et al., 2004).
Each immunoreaction was performed on at least three slides from
each set of ganglia belonging to a litter and repeated at least 3 times on
three litters from the same experimental group. To confirm the
specificity of the staining, selected sections were incubated without
primary or secondary antibodies with positive and negative controls
being included.
For ovary and pylorus, free floating sections were treated with a
0.3% H2O2 in PBS for 1 h to quench endogenous peroxidase activity, and
then washed in PBS. Sections were permeabilized and blocked in PBS
containing 0.5% Triton X-100 and 4% BSA for 1 h. Next, tissues were
incubated in the same solution containing monoclonal mouse anti-TH
IgG (NCL-TH, Novocastra Laboratories, Newcastle, UK; 1:200) for 48 h
at room temperature. Tissues were subsequently washed thoroughly
and then treated with blocking solution containing biotinylated horse
anti-mouse IgG (1:300 dilution; Vector Laboratories, CA) for 1 h,
washed thoroughly and then incubated in a solution of avidin and
biotin complex (ABC Elite; Vector Laboratories, Burlingame, USA) for
1 h. After three rinses in PBS, tissues were incubated in a solution
containing 0.02% 3-3′ diaminobenzidine in PBS. Sections were
mounted onto gelatine-coated slides, air-dried, dehydrated through a
graded series of ethanol, cleared, and coverslipped.
For whole-mount TH immunohistochemistry, the protocol of
Enomoto et al (2001) was used with minor modifications. Fixed
tissues were dehydrated through a graded series of methanol
solutions (50–80%) and incubated overnight in 20% dimethylsulfoxide
(DMSO)/80% methanol solution containing 3% H2O2 to quench
endogenous peroxidase activity. Tissues were then re-hydrated,
incubated overnight in blocking solution (4% BSA/1% Triton X-100 in
PBS) and incubated for 48–72 h at 4 °C with monoclonal anti-TH
antibodies (1:200 in blocking solution). After several rinses in PBS,
tissues were incubated for 24 h with biotinylated anti-mouse Ig
antibodies (1:250 in blocking solution). The signal was detected using
diaminobenzidine after treatment of the tissues with avidin–biotin
complex. Tissues were re-fixed, dehydrated by methanol series and
cleared with benzyl benzoate/benzyl alcohol (2:1 mixture) to allow
visualization of staining inside the tissue. For a better quantification,
pieces were included in paraffin and 40 μm thick sections were
obtained, dewaxed and mounted with DPX.
particular section. Samples include animals from all litters in each
group.
For densitometric measurement of p75NTR immunoreactivity,
twenty fields of each ganglion taken to 40× were sampled and all
neurons showing nuclear profiles were used for densitometric studies.
All images were converted to 8-bit grayscale, the cytoplasmic outlines of
neurons were delineated with the cursor and then the average optical
density (OD) within that area was obtained. The OD of the stained
material was then determined by subtracting the OD of an unstained
section. OD value within the selection is reported in calibrated units (e.g.,
optical density ranging from 0 to 3.6, where 0 correspond to 255 gray
value and 3.6 to 0 gray value). Since modifications on the size of celiac
neurons may be expected, integrated optical density per cell profile
(IOD = OD × cytoplasmic area) was also determined. Samples from
animals of all litters in each group were included and at least 900
neurons of each group were measured.
To determine density of innervation, whole-mounts preparations
were initially used. Although stained axons were easily identifiable,
background staining and field depth disturbs quantitative analysis.
Then, we performed this analysis on cryostat sections in order to obtain
more accurate measures. Density of innervation was determined in low
magnification images (4×) from sections of the ovaries and pylori from
control and treated animals (four animals of each group). In pyloric
sections, the density of immunoreactive fibers was assessed in the
muscular layer. This procedure was carried out accordingly to Maney
et al (2005) through threshold filtering each image to display darker
immunoreactive units in the field using the Image J thresholding feature,
which automatically determines a threshold based on contrast. Sections
were analyzed by comparing the overall area occupied by immunoreactive axons in a defined area, resulting in a density of immunoreactivity that was expressed as percentage.
2.4. Image analysis and quantification
3.1. Effects of estrogen treatment on vaginal opening (VO), body and
uterine weights
Microphotographies were obtained using a 4× or 40× objective lens
as necessary, via a Nikon DS-2 M digital camera (Nikon, USA) attached
to a Nikon Eclipse E200 microscope. Morphometric quantification
analysis was carried out using a computer-based image analysis
system (NIH Image J software v1.37; National Institute of Health, USA).
Neuron size was determined in H–E stained sections taken to 40× by
manually tracing around the perimeter of the soma and measuring the
cell body cross-sectional area. Only those neurons in which the cell
nucleus could be clearly identified were considered for counts in a
2.5. Determination of estrogen plasma levels
Estrogen plasma levels were measured in control and estrogentreated rats at 3 and 7 days after treatment. After 2-h clotting at room
temperature, trunk blood was centrifuged for 10 min at 5000 rpm and
the recovered serum stored at −20 °C until assay. Serum samples were
assayed in the Laboratory of Nuclear Techniques, Veterinary Faculty,
Montevideo, Uruguay. 17β Estradiol was determined by a solid phase
radioimmunoassay (RIA) using DPC kits (Diagnostic Product Co., Los
Angeles, CA, USA). Samples were analyzed in duplicate in the same
assay. The sensitivity of the assay was 14.9 pg/ml. The intra-assay
coefficients of variation for low (20 pg/ml), medium (139 pg/ml) and
high (512 pg/ml) controls were 22.3%, 7.7% and 6.4%, respectively.
2.6. Statistical analysis
Results are expressed as the mean ± S.E.M. Means were compared
with the Student t-test, while medians of strongly skewed data were
compared with Mann–Whitney Rank Sum Test. Values of P b 0.05 were
considered statistically significant.
3. Results
VO occurred 72–96 h after estrogen exposure, with a persistent
presence of cornified cells in the vaginal smear until end of the
experimental period. Control animals did not show VO during the
same period. Then, only rats showing VO were included in 3d-E2
group. The growth rate of the animals was unaffected by the estrogen
treatment and uterine weight was significantly increased after three
or seven days of estrogen exposure. Hormone assays showed that in
rats killed three or seven days after treatment, estrogen levels were
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G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
Table 1
Body weight, relative uterine weight and plasma levels of estrogen (mean ± S.E.M) in
control (C) and estrogen (E2) exposed rats killed at three (3d-C and 3d-E2) or seven days
(7d-C and 7d-E2) after treatment
3d-C
3d-E2
7d-C
7d-E2
Body weight (g)
Uterus weight (mg/100 g bw)
Estrogen (pg/ml)
35.67 ± 1.95 (21)
32.07 ± 1.53 (21)
45.15 ± 3.95 (21)
43.14 ± 0.88 (21)
87.68 ± 4.08 (21)
275.90 ± 44.93 (21)a
80.41 ± 7.85 (21)
231.56 ± 8.59 (21)b
41.62 ± 8.54 (9)
1318.81 ± 296.11 (9)a
32.70 ± 5.58 (9)
135.28 ± 13.33 (9)b,c
The number of animals used for each determination is given in parentheses.
a
p ≤ 0.05 vs 3d-C.
b
p ≤ 0.05 vs 7d-C.
c
p ≤ 0.05 vs 3d-E2.
fifteen and four times higher, respectively than in control animals
(Table 1).
3.2. Effects of estrogen treatment on celiac ganglion cell population
Quantification of the cross-sectional area of sympathetic somata
revealed that the ganglionic population in estrogen-treated rats
displayed an increase in cell size over those in age-matched control
rats. This increase was significantly different at seven days after
treatment (Table 2). Most of somata (38.1%) ranged in size between
300 and 400 μm2 after estrogen exposure whereas in control condition
the greatest group (41.7%) ranged between 200 and 300 μm2, (Fig. 1).
Then we performed TH immunodetection in the sympathetic
somata of both control and treated rats to analyze the changes in
neurochemical profile. However no differences were observed
between both groups (data not shown).
3.3. Expression of ERα and ERβ
Immunohistochemical staining revealed that ER expression is a
common feature of celiac sympathetic neurons during prepubertal
period. Control and treated rat CG neurons showed immunoreactivity
for both ER receptors (Fig. 2). All neurons displayed a strong ERα
signal, while ERβ staining was in general weak but heterogeneous,
with a small population displaying a more intense signal. Most
neurons exhibited cytoplasmic ER immunoreactivity with only few
cells displaying the classical nuclear localization (less than 10% in all
groups). Estrogen exposure did not modify the distribution and
staining intensity of ERs immunoreactivity.
3.4. Expression of p75NTR in celiac neurons
Most of CG neurons displayed heterogeneous p75NTR immunoreactivity in both, control and treated rats (Fig. 3). Following three days
of estrogen exposure, optical density of p75NTR immunoreactivity
was slightly reduced (13%). On the other hand, seven days after
estrogen treatment we detected a 34% increase in p75NTR levels
Table 2
Quantitative assessment of neuronal soma size (mean± S.E.M) in CG of control (C) and
estrogen (E2) exposed rats killed at three (3d-C and 3d-E2) or seven days (7d-C and 7d-E2)
after treatment
3d-C
3d-E2
7d-C
7d-E2
Cross sectional area (μm2)
n
268.93 ± 11.68
298.48 ± 7.76
266.06 ± 8.83
329.65 ± 14.11a
905 (9)
910 (9)
904 (9)
954 (9)
n shows the total number of neurons measured and the number of animals used for
each determination is given in parentheses.
a
p ≤ 0.05 vs 7d-C.
Fig. 1. Distribution of cross-sectional areas of sympathetic somata in the CGs of control
(C) and estrogen (E2) treated rats, three (A) or seven (B) days after exposure. Somata
were sorted into groups at 100 μm2 intervals (x axis), according to their cross-sectional
area and expressed as percentage of total number of neurons; quantified somata were
collected from ganglia of 9 animals from 3 different litters.
(Table 3). Since our results showed modifications in the size of celiac
neurons after treatment, these size changes were taken into account
for alterations in receptor expression. Although the OD (per unit area
of soma) was reduced after three days of estrogen exposure, no
significant changes in the IOD (equivalent to the total receptor density
per neuron) of p75NTR were observed. Both the OD and the IOD of
p75NTR were significantly increased after seven days treatment
(Table 3).
In an attempt to discriminate the heterogeneous changes in
p75NTR expression, we created neuronal categories according to
cytoplasmic area. Somata were sorted into groups at 100 μm2
intervals. Results for IOD for each category are summarized in Fig. 4.
Significant differences occurred in small and medium size neurons
after seven days of estrogen exposure, whereas the larger neurons did
not seem to undergo modifications in p75NTR expression.
3.5. Effect of estrogen treatment on TH-immunoreactive innervation of
the ovary and pylorus
In control ovaries, TH-immunoreactive nerve fibers were mainly
distributed around blood vessels and associated to follicular walls.
Some intrinsic neurons were also identified in the hilum. The
distribution pattern of catecholaminergic fibers was not modified
by estrogen treatment (Fig. 5A, B). The percentage of ovarian tissue
occupied by immunoreactive fibers in estrogen treated animals was
significantly greater than in control animals (Fig. 5C). In both animal
groups, TH-immunoreactive fibers were distributed as free bundles,
G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
39
Fig. 2. Immunohistochemical detection of ERα in CG neurons of control rats at postnatal day 21 (A) or after seven days of estrogen exposure (B). In both cases, ER showed a
cytoplasmic localization. Expression of ERβ in CG neurons of control (C) and treated rats (7d after estrogen exposure) (D). Most neurons showed weak immunoreactivity for ERβ,
generally with a cytoplasmic localization. In few cases, a classical nuclear localization was seen (arrow). Control section of CG incubated in the absence of primary antibody, showing
no stained structures (E). Positive control sections of endometrium (F) for ERα and ovary (G) for ERβ. In both, the immunoreactivity was limited to a nuclear localization. Sections
were slightly counterstained with hematoxylin. Scale bars: 50 μm.
isolated fibers or accompanying blood vessels within the pyloric
muscular layers and the estrogen exposure did not modify the
density of catecholaminergic innervation (Fig. 5C).
4. Discussion
In the present study, we characterized the response of celiac
sympathetic neurons to estrogen administration in the prepubertal
rat. Both ERα and ERβ were expressed in these neurons, and the
response to estrogen exposure induced a significant soma size
increase with temporary changes in the expression of p75NTR.
These effects were associated with a significant increase in density
of sympathetic innervation of the ovary.
Previous studies demonstrated the presence of ERs in neurons of
both peripheral and central sexually dimorphic structures associated
with the reproductive system, such as the pelvic ganglion (Keast,
2006), the spinal nucleus of the bulbocavernosus and the dorsolateral
nucleus motoneuron populations (Breedlove and Arnold, 1981;
Zuloaga et al., 2007) or the medial amygdala (Cooke, 2006). In most
of these neurons, castration provokes a striking reduction of the size of
neuronal somata, while steroid administration reverses this effect.
Many of these studies focused on adult animals, and little is known
about the effects of steroids on neuronal systems of prepubertal
animals. Our results showed that the size of neuronal somata in CG
increases after 7d-E2 treatment. However, the serum estradiol levels
were significantly higher, even after 3d-E2 treatment more than those
showed during estrous in normal cycling female rats of the same
strain. The lack of changes in neuronal somata size after 3 days of
hormonal administration suggests that a prolonged exposure time is
required to identify estrogen action on this parameter. Furthermore,
since the effects on uterus and vaginal opening were observed around
72–96 hrs, it is possible to suggest a non direct activation pathway at
CG. Other reports (Richeri et al 2005) analyzing the soma size of
neurons located in the sympathetic chain (T7 to L2) or neurons of the
superior cervical ganglion (SCG), did not show changes in soma size
after three consecutive doses of estrogen. In the same report, authors
described a reduction of the neuronal soma size during late
pregnancy, when levels of estradiol increased and degeneration of
intrauterine sympathetic nerves was observed. These results allow us
to suggest different responses between sympathetic ganglia populations. We propose that the neuronal vulnerability to changes in the
hormonal milieu could also be influenced by the hormone doses
administrated and the physiological status of animals (prepubertal,
adult cyclic or adult pregnant rat), such as has been observed in other
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G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
Fig. 3. Immunohistochemical detection of p75NTR. Sections of CGs from control rats at postnatal days 18 (A) and 21 (B) and age-matched estrogen exposed rats (C–D) stained
immunohistochemically for p75NTR. The ganglia of control animals (upper row) displayed somata with variable immunostaining for p75NTR. In treated rats, a similar situation was
seen in the ganglia after three day estrogen exposure (C). However, a notable increase in staining intensity for p75NTR immunoreactivity appeared after seven days of estrogen
exposure (D). Note the granular appearance of the reaction product for p75NTR immunostaining in the cytoplasm of sympathetic somata. Arrows indicate p75-positive nerve
bundles. Sections were slightly counterstained with hematoxylin. Scale bars: 50 μm.
experimental procedures (Chavez-Genaro et al., 2002; Chavez-Genaro
et al., 2006). In summary, our results further support the idea that
peripheral neurons respond to estrogen administration in different
ways and this fact could be associated with the role and target of
sympathetic neurons.
Endogenous estradiol levels of control prepubertal animals, were
similar to those found during estrous in cycling female rats (SavoyMoore et al., 1980), but these estradiol levels did not induce
reproductive changes. This fact could be attributable to the presence of alpha-fetoprotein, which binds up the estrogens available
(Mizejewski, 2004). It is generally accepted that estrogen is inactive
when bound to alpha-fetoprotein. This protein is known to be of
fetal origin and its concentration, which is very high in the rat fetus,
decreases with age reaching zero level at postnatal day 29 (Raynaud,
1973; Noda et al., 2002). It could be possible that in treated animals,
serum estradiol levels exceeded the protective influence of alphafetoprotein, inducing VO and uterine weight increase 3–4 days after
treatment.
Table 3
Effects of estrogen exposure on cytoplasmic area, optical density and integrated optical
density (mean ± S.E.M) for p75NTR in sympathetic neurons of the rat CG of control (C)
and estrogen (E2) exposed rats killed at three (3d-C and 3d-E2) or seven days (7d-C and
7d-E2) after treatment
3d-C
3d-E2
7d-C
7d-E2
n
Cytoplasmic area (μm2)
Optical density
Integrated Optical Density
954 (9)
923 (9)
937 (9)
962 (9)
174.02 ± 6.74
199.85 ± 3.58a
230.85 ± 2.86a
284.46 ± 7.36b
0.30 ± 0.007
0.26 ± 0.008a
0.29 ± 0.007
0.39 ± 0.006b
51.52 ± 1.60
50.86 ± 1.02
67.57 ± 1.03a
108.89 ± 2.10b
n shows the total number of neurons measured and the number of animals used for
each determination is given in parentheses.
a
p ≤ 0.05 vs. 3d-C.
b
p ≤ 0.05 vs. 7d-C.
Our immunohistochemical results showed that CG neurons
expressed both ERs. Evidence of the literature has showed that almost
all sympathetic neurons express ERβ, whereas approximately one
third expresses ERα (Papka et al., 1997; Papka et al., 2001; Shughrue
and Merchenthaler, 2001). These receptors are located in the nuclei of
neurons and sometimes immunoreactivity is present uniformly
throughout the nucleus and cytoplasm. Notably while previous studies
showed a selective nuclear location of the ERs, our findings showed
that in prepubertal CG neurons both ERs were mainly distributed in the
cytoplasm, as has been reported by Zoubina and Smith (2002) in
lumbar paravertebral and prevertebral sympathetic ganglia of adult
female rats. Naturally truncated isoforms of ERα showing a cytoplasmic localization were described during normal pituitary development.
These isoforms are generated through differential splicing of the ERα
primary transcript and may act antagonistically with full-length
receptors, inhibiting the estrogen responsiveness mediated by them
(Pasqualini et al., 2001). In vitro studies using well characterized cell
lines have shown that proteins antigenically related to ERα could also
reside at intracellular membranes (Monje et al., 2001). It remains to
determine whether cytoplasmic ER immunoreactivity found in our
study corresponds to truncated forms.
Our results also showed a significant increase in p75NTR expression in small and medium size celiac neurons induced by increased
estrogen levels in prepubertal rats. Several neuronal types display
modifications when exposed to natural or artificial estrogen levels.
Sensory neurons show an up-regulation of p75NTR mRNA expression
during proestrus (Sohrabji et al., 1994b), while a transient downregulation of p75NTR mRNA has been observed in sympathetic
neuron-like PC12 cells after exposure to estrogen and NGF (Sohrabji
et al., 1994a). Moreover, sympathetic neurons of SCG of adult
ovariectomized rats exposed to acute steroid hormone did not show
modifications in neurotrophin receptor expression, or when the
animals were exposed for seven days to high levels of estrogen (Hasan
et al., 2005). A significant reduction in the number of neurons
G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
41
Fig. 4. Integrated optical density (IOD) for p75NTR in GC neurons of control (C) and estrogen (E2) exposed rats killed at three (3d-C and 3d-E2) or seven days (7d-C and 7d-E2) after
treatment. Quantified somata were collected from ganglia of 9 animals from 3 different litters and divided into groups according to cytoplasmic area. Data from image analysis are
presented as box plots where 50% of the values are within the box and the median is marked with a line. The bars represent the 5th and 95th percentiles. Values of p ≤ 0.05 were
considered statistically significant.
displaying p75NTR protein was observed at the end of pregnancy,
when estrogen levels are increased (Richeri et al., 2005). The
differences between diverse studies and our results contribute to
support the idea that sympathetic ganglia could be part of a
heterogeneous population of neurons displaying diverse morphological and functional properties. The response to different stimuli could
be related to the neuronal peripheral projections and therefore closely
associated to the neuronal localization. Moreover, taking into account
that we found a selective increase in p75NTR immunoreactivity
mainly affecting small and medium size neurons, and that organs
innervated by celiac neurons show clear differences in response to
estrogens, we could expect that modifications in target innervation
could be heterogeneous. Density of sympathetic innervation was
modified in both pylori and ovaries after seven days of estrogen
exposure but only in the ovaries was significant. Adult rats exposed to
a single dose of estradiol valerate—a long-acting estrogen-, showed
significant changes in the content of norepinephrine in the ovary
within thirty days of hormone administration and even more
pronounced after sixty days (Lara et al., 1993; Lara et al. 2000). This
finding was interpreted as a hyperactivation of sympathetic neurons
that innervate the ovary associated with the development of a
polycystic condition. Whether increase of ovarian density found in our
study is associated to polycystic condition development remains to be
determined. Preliminary results of follicular population in ovaries
from 7d-E2 animal group showed an increase in the number of
growing follicles (Anesetti et al., 2006), and similar effects has been
reported for PCO condition development (Franks et al., 2008).
According to Lara et al (2000), cyst develop after at least 30 days
post-estrogen exposure, and histological inspection of ovaries from
our animals seven days after treatment did not detected ovarian cystic
structures. Furthermore, an augmented intraovarian production of
NGF and its low affinity receptor is an important component in the
maintenance of the polycystic condition. Therefore, it is possible that
estrogens act on peripheral tissue increasing neurotrophic factor
availability (Lara et al., 1993; Lara et al., 2000; Rosa et al., 2003).
Nonetheless, a direct effect of estradiol on celiac neurons cannot be
ruled out. Estrogen exposure seems to induce age-dependent effects
on sympathetic neurons. A prenatal estrogen exposure induces a
reduction in the number of neurons displaying ERα in the adult CG,
associated with a reduction in the catecholaminergic ovarian
innervation (Shinohara et al., 1998; Shinohara et al., 2000). However,
estrogen administration in prepubertal or adult rats induces an
increase in ovarian norepinephrine concentration (Lara et al., 1993;
Rosa et al., 2003). In these animals, the effects were reversed by the
section of superior ovarian nerve, the principal sympathetic pathway
between the CG and the ovary. If these findings can be applied to
reproductive function in women is not clear. The effectiveness of
ovarian wedge resection (Donesky and Adashi, 1995; Campo, 1998) or
laparoscopic laser cauterization (Balen and Jacobs, 1994) to increase
ovulatory response in women with PCOS, raise the possibility that
superior ovarian nerve input may play a role in the development of
polycystic ovaries in PCOS women. However, ovulatory menstrual
cycles, pregnancy, labor and delivery seems not to be modified in
women after traumatic spinal cord injury (Reame, 1992; Cross et al.,
1992).
Our results suggest that prepubertal CG neurons are able to
respond to estrogen exposure. This could be a direct response if ERs
found in neuronal cytoplasm can be activated and induce transcription. In fact, putative estrogen response elements have been identified
in the NGF, BDNF, trkA and p75NTR genes (Toran-Allerand, 1996).
Then, transcription of p75NTR induced by estrogen could explain the
rise in immunoreactivity found in our study. On the other hand, an
indirect response cannot be discarded. Several reports showed that
estrogen acting on peripheral organs might modify neurotrophic
factors production. Estrogen treatment decreased NGF and TH protein
in the SCG and its targets, as was shown by quantitative immunoblot
and immunohistochemistry (Kaur et al., 2007). However, estrogen
treatment induces an increased synthesis of NGF in the ovary and is
postulated as a possible mechanism underlying the development of
polycystic ovaries (Lara et al., 2000). Björling et al (2002) indicate that
estrogen and progesterone can increase synthesis of NGF mRNA and
protein in peripheral organs as uterus and submandibular salivary
gland, but not in heart or bladder. Then, since different target organs
may respond in different ways to estrogen, this could contribute to
explain the differences between our work and other studies.
In conclusion, our evidence further support the idea of a
reproductive peripheral nervous system whose function can be
modulated by hormonal status (Papka et al., 2001; Krizsan-Agbas
and Smith, 2002; Zoubina and Smith, 2002). Estrogens modify
sympathetic neuronal features through mechanisms that in turn
modulate neurotrophin receptor expression. This effect could be
related to changes in the density of target innervation, suggesting a
critical role for estrogen as a mediator of neurotrophin-dependent
mechanisms of neuronal survival, plasticity, and repair. More studies
are required to understand the impact of these findings on
reproductive performance and health.
Acknowledgements
We are grateful for the support given by Luis Abad and Mariela
Santos (Animal Facility, Facultad de Medicina, Montevideo) and
Mariela Gonzalez Quijano for technical assistance. This work was
42
G. Anesetti et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 35–43
Fig. 5. Changes in density of innervation as shown in ovary whole mount preparations
processed for in toto TH immunoreactivity. Ovary of a control rat at postnatal days 21
(A; 7d-C) and an age-matched estrogen exposed rat (B; 7d-E2). An increase of
immunoreactive fibers was evident in ovaries from treated animals. Scale bar: 100 μm.
(C) Quantitative assessment of the effect of estrogen exposure on the percentage area
occupied by TH-immunoreactive nerve fibers in the ovary and pyloric muscular layer in
control and estrogen exposed rats killed 7 days after treatment. Results are represented
as the mean ± S.E.M. Data were collected from 4 animals of each group. Data were
compared using the Student t-test. Values of p ≤ 0.05 were considered statistically
significant. (⁎) Significant difference with ovary control.
partially supported by the PEDECIBA, Universidad de la República,
Montevideo, Uruguay.
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