Paredes et al. Reproductive Biology and Endocrinology 2011, 9:66
http://www.rbej.com/content/9/1/66
RESEARCH
Open Access
Sympathetic nerve activity in normal and cystic
follicles from isolated bovine ovary: local effect of
beta-adrenergic stimulation on steroid secretion
Alfonso H Paredes1*, Natalia R Salvetti2, Ariel E Diaz 1, Bibiana E Dallard2, Hugo H Ortega2 and Hernan E Lara1
Abstract
Cystic ovarian disease (COD) is an important cause of abnormal estrous behavior and infertility in dairy cows. COD
is mainly observed in high-yielding dairy cows during the first months post-partum, a period of high stress. We
have previously reported that, in lower mammals, stress induces a cystic condition similar to the polycystic ovary
syndrome in humans and that stress is a definitive component in the human pathology. To know if COD in cows
is also associated with high sympathetic activity, we studied isolated small antral (5mm), preovulatory (10mm) and
cystic follicles (25mm). Cystic follicles which present an area 600 fold greater compared with preovulatory follicles
has only 10 times less concentration of NE as compared with small antral and preovulatory follicles but they had
10 times more NE in follicular fluid, suggesting a high efflux of neurotransmitter from the cyst wall. This suggestion
was reinforced by the high basal release of recently taken-up 3H-NE found in cystic follicles. While lower levels of
beta-adrenergic receptor were found in cystic follicles, there was a heightened response to the beta-adrenergic
agonist isoproterenol and to hCG, as measured by testosterone secretion. There was however an unexpected
capacity of the ovary in vitro to produce cortisol and to secrete it in response to hCG but not to isoproterenol.
These data suggest that, during COD, the bovine ovary is under high sympathetic nerve activity that in addition to
an increased response to hCG in cortisol secretion could participate in COD development.
Background
Cystic ovarian disease (COD) is an important cause of
abnormal estrous behavior and infertility in dairy cows.
The prevalence of COD in dairy herds has been
reported to vary from 5 to 30% [1] and this condition
may result in significant economic losses to the dairy
industry due to increased calving to conception and
inter-calving intervals [2] COD is mainly observed in
high-yielding dairy cows during the first month’s postpartum, as this is a period of high stress [2]. We have
previously reported that in lower mammals both the
steroid-induced increase in sympathetic nerve activity
and stress are able to induce a cystic condition similar
to the polycystic ovary syndrome (PCOS) in humans
[3-5]. Many possibilities have been involved in human
polycystic ovary syndrome (PCOS) development. It has
* Correspondence: aparedes@ciq.uchile.cl
1
Laboratory of Neurobiochemistry, Department of Biochemistry and
Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences,
Universidad de Chile, Santiago, Chile
Full list of author information is available at the end of the article
been demonstrated a correlation between high sympathetic nerve activity and PCOS in human [6]. Both
hypothalamic and local intraovarian mechanisms have
been suggested [7]. Our group has presented evidences
that the cold-stress which principally activates sympathetic nerve without modifying corticoids levels [8],
could be other of the mechanism involved in the development of follicular cyst in rats [9]. In addition, it has
been demonstrated that the effect of stress in the pathogenesis of polycystic ovary PCO is mediated by sympathetic discharge originating at the paraventricular
nucleus [10-12]. Therefore, changes in sympathetic
nerve activity are a major factor contributing to the
changes in sympathetic tone during stress. Regarding
this observation, it has also been proposed that sympathetic activity could be a component in bovine COD
because stress induces changes in beta-adrenergic receptors at the level of the hypophysis as well as that of the
ovary [13]. To analyze the hypothesis that sympathetic
nerves are also a principal component in cystic ovarian
disease in cows, and because sympathetic nerves
© 2011 Paredes et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Paredes et al. Reproductive Biology and Endocrinology 2011, 9:66
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penetrate to the mammalian ovary [14,15] to directly
innervate theca cells of follicles and the interstitial gland
[16], we used a recently published technique to isolate
follicles from the cow ovary [17]. We separated small
antral (SA), preovulatory (PO) and cystic follicles to analyze the in vitro NE release capacity of sympathetic
nerves arriving at specific follicular compartments in the
ovary and the related neurogenic-dependent steroidal
secretory response. Our results strongly suggest that
sympathetic activity is one of the components in maintaining the increased secretory activity associated with
the development of COD in cows.
Methods
Collection and preparation of tissues
Ovaries with normal morphology (n = 16) and with
spontaneous cystic follicles (n = 16) were collected at a
local abattoir, within 20 min of death, from mixed
breeds of Bos taurus cows that were assessed visually as
being non-pregnant and without macroscopic abnormality in the reproductive system, Based on the records
contributed by the veterinary inspection, the animals
were in the second half of the lactation. The complete
ovaries were washed, refrigerated and transported
immediately to the laboratory. The surgical procedure
used to obtain the SA, PO and a cystic follicle has
recently been described [17]. During dissection, the follicular diameter was measured with calipers and follicular
fluid from each follicle was aspirated and stored separately at -20°C. Only one follicular structure type was
obtained from a single ovary collected per animal and
follicular health status was confirmed by measurement
of hormone concentrations in follicular fluid and morphological analysis [18,19].
Pieces of ovaries from SA, PO and cystic follicles were
fixed in 4% paraformaldehyde, embedded in paraffin and
cut into 6 μ m sections, then stained with hematoxylin
and eosin. The presence of preantral, small antral (SA),
preovulatory (PO) and cystic follicles was analyzed
according to [5]. Briefly, antral follicles were those in
which the nucleus of the oocyte could be visualized.
Preovulatory follicles have an average diameter of > 10
mm [1]. Ovarian cysts are defined as follicle-like structures of diameter greater than 25 mm in the absence of
a corpus luteum [20]. Our previous observations in rats
suggest the existence of a transitional stage between
healthy preovulatory follicles and the cystic follicles
described previously [5].
Measurement of NE content
We homogenized 50 mg pieces of follicular wall (SA,
PO and cystic) from the ovaries of each animal in 250 μ
l DPBS. The homogenized tissue was precipitated with 4
volumes of 0.25 N perchloric acid and centrifuged
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(15,000 × g, 15 min). We resuspended 50 μ l of follicular fluid in 150 μ L of DPBS, and 100 μ L from this suspension was precipitated with 4 volumes of 0.25 N
perchloric acid and centrifuged (15,000xg, 15 min). NE
was measured in the acid supernatant by HPLC coupled
with electrochemical detection as previously described
in [21]. Briefly, 20 μ l of the resulting supernatant (follicular wall or follicular fluid) were injected into a Waters
HPLC system equipped with a C18 reverse phase column (Lichrosphere, 60 RP-Select B, Merck, Darmstadt,
FR Germany) and an electrochemical detector (Waters
464). The mobile phase contained 0.1 M NaH2PO4,
0.42 mM octyl-sulphate, 0.02% EDTA and 1.5% acetonitrile (pH 2.5) with a 0.9 ml/min flow rate. The potential
of the amperometric detector was set to 0.7 V. Under
these experimental conditions, the retention time was 4
min for NE and 10 min for DHBA.
Uptake and release of NE
The procedure used, with some modifications, has previously been described [22,23]. The SA, PO or cystic
follicles wall pieces (about 50 mg) were rapidly
removed from the ovary and dissected as previously
described [17]. Tissues were preincubated for 20 min
in Krebs ringer bicarbonate buffer (KRB), pH 7.4, and
gassed with 95% O 2 - 5% CO 2 . Samples were then
incubated for 30 min at 37°C with 2 μ Ci 3HNE (New
England Nuclear Life Science Products, Boston, MA).
After incubation, tissues were washed six times,
10 min each, to eliminate radioactivity that was not
incorporated into the tissue. After washing the tissue
to remove non-incorporated radioactivity, the tissues
were placed in a multiwell plate with 24 flat-bottom
wells containing 2 ml of buffer per well. Tissues were
incubated for 2 min in each well. After 3 passages,
depolarization was effected by removal of the tissue to
another well with 80 mM K + KRB. After stimulation,
tissues were washed 3 times (2 min each). At the end
of the experiment, the follicular walls were homogenized in 0.4 N perchloric acid, and the level of [ 3 H]
catecholamines remaining in the tissue were determined by scintillation counting (Tri-Carb Liquid Scintillation Analyzer 1600TR Packard Instruments,
Meriden, CT); we obtained 72.5% efficiency for 3 H in
calculating the radioactivity remaining in the tissue
after the experiment. The radioactivity incorporated by
the tissue and the radioactivity released during stimulation were then calculated. The latter, which represents [ 3 H]NE overflow from tissue, was expressed as
fractional release, i.e., as a percentage of the total
radioactivity present in the tissue [23,24]. The total
amount of NE released under stimulation (net release)
was calculated as the area under the stimulation minus
the spontaneous release [23,25].
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b-adrenergic receptor binding
Membranes were prepared from ovarian follicular wall
(pieces of about 50 mg each) by differential centrifugation [3] with minor modifications. In brief, tissues were
homogenized in 0.02 M Tris/HCI and 0.25 M sucrose
(pH 7.4), and the homogenates were centrifuged at
30000 xg for 20 min. The resulting pellets were suspended in the same buffer and centrifuged as above.
This procedure was repeated again, and the pellets were
suspended in 0.02 M Tris/HC1 and 10 mM MgCl2 (pH
7.4, assay buffer) and then used in the radioreceptor
assay. The assay contained a 20 nM saturating concentration of 3H-dihydroalprenolol (92.0 Ci/mmol, Dupont/
NEN) with membranes (20 μ g of protein) in a total
volume of 0.2 ml. Non-specific binding was assessed in
tubes containing 10 -4 M DL-propranolol. Results are
expressed as femtomoles (fmol) of dihydroalprenolol
bound/milligram of protein per 30 min at 37ºC. Binding
was terminated by the addition of 10 volumes of assay
buffer and vacuum filtration through Whatman (Clifton,
NJ) GF/C fiberglass as described (Barria et al., 1993).
Radioactivity retained on the filters was determined by
scintillation counting (Tri-Carb Liquid Scintillation Analyzer 1600TR Packard Instruments, Meriden, CT) with a
72.5% efficiency for 3H.
“In vitro” testosterone and cortisol release
Steroid response to adrenergic and/or gonadotropin stimulation was done by incubating tissue in 2 ml KrebsRinger bicarbonate buffer, pH 7.4, for 3 h at 37ºC [26,27],
in the presence of D,L-isoproterenol-HCl (10-5 M; Sigma
Chem Co., St. Louis, MO), hCG (2.5 IU; Sigma Chem
Co., St Louis, MO), or with no stimulation (basal release).
The experimental design was such that all ovarian tissues
were simultaneously used; one served as a control, and
the other two were subjected to the different stimulatory
treatments. Testosterone and cortisol released into
the incubation medium were measured by ELISA, as
previously described [4].
Hormone assays
Follicular fluid and culture medium, testosterone and
cortisol were measured by ELISA kits (Testosterone
EIA, DSL-10-4000; Cortisol EIA, DSL 10-2000; Diagnostic Systems Laboratories, Webster, TX), according to the
manufacturer’s instructions. Testosterone and cortisol
concentrations in small follicles were not assayed due to
the insufficient volume of follicular fluid collected from
these follicles. The assay sensitivity was 0.04 ng/ml for
testosterone and 0.1 μ g/dl for cortisol.
The follicular health status was confirmed by measuring
the hormonal levels in the follicular fluid. Oestradiol and
progesterone in the follicular fluid were measured using
ELISA kits (Estradiol EIA, DSL-10-4300; Progesterone
Page 3 of 8
EIA, DSL-10-3900; Diagnostic Systems Laboratories,
Webster, Texas, USA), according to the manufacturer’s
instructions. The assay sensitivity was 7 pg/ml for oestradiol and 0.13 ng/ml for progesterone. All follicles were
categorized as oestrogen active without luteinization
(Table 1).
Statistical analysis
Differences between the different ages groups were analyzed by one-way ANOVA, followed by the StudentNewman-Keuls multiple comparison test for unequal
replication. The level of significance was set to P < 0.05.
Results
Morphological aspects of small antral (SA), preovulatory
(PO) and cystic follicles
The morphological characteristics of the ovarian follicles
from normal ovulating cows and those with ovarian cystic disease are shown in Figure 1. Small antral (SA) follicles (diameter < 5 mm) and preovulatory (PO) follicles
(diameter > 10 mm) have a well-defined granulosa cell
layer and internal theca cell layer. Cystic follicles (diameter > 25 mm) obtained from the ovaries of nonovulatory cows presented a small granulose cell layer,
which was basically a monolayer, and a much higher
diameter (and thus more volume of follicular fluid) than
PO and SA follicles. In a typical experiment, we
obtained 30 μ l from each SA, 1 ml from PO follicles
and 3 ml from cystic follicles. Due to the substantial difference in the amount of follicular fluid, we decided to
analyze all results in terms of concentration and amount
separately for each follicular type.
NE concentration in follicular fluid and in the walls of SA,
PO and cystic follicles
We measured NE concentration in each type of follicle
(Figure 2B). Although there was no difference between
SA and PO follicles, there was a > 90% decrease in NE
concentration in the walls of the follicular cysts. To
determine whether this difference was due to a change
in the compartmentalization of NE, we also measured
the NE concentration in the follicular fluid (Figure 2A).
There was no difference in NE concentration (expressed
as ng NE/μ l of follicular fluid) in follicular liquid
obtained from SA, PO and cystic follicles.
Table 1 Estradiol and progesterone in the follicular fluid
Steroid (ng/mL)
SA
PO
Cyst
Estradiol
97.31 ± 6.65
282.41 ± 22.1*
313.09 ± 18.62*
Progesterone
62.12 ± 9.01
81.52 ± 9.48
57.28 ± 13.53
Results correspond to Mean ± SEM concentrations of 4 determinations for
estradiol and progesterone in the follicular fluid of SA, PO and cystic follicles.
* = P < 0.05 vs. SA).
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Page 4 of 8
in PO follicles; cystic follicles presented the lowest
number.
Changes in the in vitro release of testosterone and its
relation to intrafollicular testosterone concentration
Figure 1 Microphotography of the follicular wall. Panel a and d
correspond to low and higher magnification of the small antral
follicle wall. Panels b and e represent preovulatory follicle wall.
Panels c and f are representatives images of the cystic follicle wall.
In a, b, and c, bars correspond to 50 μ m, and for d, e and f, bars =
20 μ m.
Cysts displayed the highest basal release of testosterone
as compared with SA and PO follicles (Figure 5A). Cysts
also had the highest responsiveness to both hCG and
isoproterenol as measured by testosterone release capacity, in comparison to both SA and PO follicles. To verify whether the increased basal release of testosterone
reflects the local synthesis of testosterone, we measured
the concentration of testosterone in follicular fluid
(Figure 5B). The highest concentration of testosterone
was found in follicular cysts; the lowest concentration
was observed in SA follicles.
3
H-NE uptake and its induced release from SA, PO and
cystic follicles
Changes in the in vitro release of cortisol and its
relationship to intrafollicular cortisol concentration
No changes in the amount of 3H-NE incorporated and
retained by the pieces of follicular wall of the different
types of follicles were found (Figure 3A) ). The three
types of follicles were able to incorporate NE, and they
were also able to release NE when a depolarizing stimulus was applied to the preparation (Figure 3B). There
was however a decrease of 54% in the amount of 3H-NE
released from cystic follicles as compared with SA and
PO follicles. It is also interesting to note that basal
release, i.e., 3 H-NE released spontaneously without
stimulation, was 37% higher than in PO follicles.
To verify whether the ovary is strongly influenced by
cortisol following stress, we measured the intrinsic capacity of the ovary in vitro to produce cortisol, and we
also measured the cortisol concentration in follicular
fluid. The highest concentration of cortisol in the follicular fluid was found in cystic follicles; this concentration was twice that found in PO follicles (Figure 6A).
There was no change in basal cortisol release among the
various follicle types (Figure 6B). PO and especially cystic follicles are highly responsive to hCG stimulation in
vitro; however, isoproterenol was not able to elicit any
secretory cortisol response from the three types of follicles (Figure 6B).
Changes in b-adrenergic binding sites in small antral,
preovulatory and cystic follicles
We determined the total amount of binding sites by
incubating tissue with a 20 nM saturating concentration
of 3H-dihydroalprenolol (Figure 4). The highest number
of binding sites (expressed per mg of protein) was found
Discussion
In this paper, we wanted to analyze the participation of
sympathetic nerve activity in the formation and steroid
secretory activity of ovarian cysts in dairy cows. As it
Figure 2 Concentration of noradrenaline in ovarian follicular fluid and follicular wall. In A is shown the concentration of the noradrenaline
content in follicular fluid and B is shown the concentration in the follicular walls, as well as in the small antral (SA), preovulatory (PO) and cystic
follicular wall (cystic). Values represent mean value ± SEM of the number of samples with four individual follicles per experimental group, *, p <
0.05 vs. SA and PO.
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Figure 5 Testosterone concentration in the ovarian follicular
fluid and testosterone secreted in vitro in follicular wall. In A is
shown the testosterone concentration in the follicular fluid and in B
is shown testosterone secreted following stimulation with a breceptor agonist (isoproterenol) and with hCG (B). Follicular wall
pieces were incubated for 3 h in 2 ml of Krebs-Ringer bicarbonate
buffer (basal), 10 μ M isoproterenol (ISO) or 5 IU hCG. Results are
expressed as ng testosterone per ml of incubation medium per mg
of tissue during 3 h. Each bar represents the mean ± SEM of four
independent observations per group. *= p < 0.01 vs Small antral
(SA); **= p < 0.01 vs Preoulatory follicles (PO). a, b = p < 0.05 vs.
basal (SA, PO and Cystic).
Figure 3 Incorporation and release of 3H-NE from small antral
(SA), preovulatory (PO) and cystic follicular wall. In A is shown
the amount of 3H-NE incorporated in follicle wall, In B is shown the
release of 3H-NE induced by high potassium depolarization (black
rectangles). In A results are expressed as dpm per 50 mg tissue and
in B the release is expressed as a percentage of 3H-NE retained in
the tissue at each interval studied. The first row upper numbers
represent the total release of 3H-NE induced by potassium
depolarization and the lower row represents the spontaneous
release as percentage of radioactivity released before the
depolarization stimulus. *= p < 0.05 vs Small antral (SA) and
Preovulatory; & = p < 0.05 vs cystic,
Figure 4 Concentration of b-adrenergic receptor from small
antral (SA), preovulatory (PO) and cystic follicular wall. The breceptor concentration is expressed as fmol of dihydroalprenolol
bound/mg of membrane protein. Each bar represents the mean ±
SEM of 4 experiments, for each group. * = p < 0.01 vs. preovulatory
follicular wall.
has described previously in other species, including
humans [3,4,6]), we found an increase in the capacity of
the cyst to release NE recently taken up and also in the
basal NE release suggesting that it could be associated
to an increased sympathetic nerve activity and be a
component in the COD found in dairy cows. If it is
assumed that the changes in the uptake and basal and
induced release of NE represent nerve activity, thus the
changes in these experimental values could be related to
changes sympathetic nerve activity to correlate to ovary
function [9]. Many similarities have appeared when
comparing rats, human and cows regarding morphometry of ovarian cysts. Morphological aspects of small,
Figure 6 Cortisol concentration in the ovarian follicular fluid
and secreted in vitro in follicular wall. In A is shown the cortisol
concentration in follicular fluid and In B is shown of cortisol
secreted following stimulation with b-receptor agonist
(isoproterenol, 10 uM) and with 5 IU hCG. Follicular wall slides were
incubated for 3 h in Krebs-Ringer bicarbonate buffer (basal), 10 uM
isoproterenol (ISO) or 5 IU hCG. Results are expressed as ng cortisol
per ml of incubation medium per mg of tissue. Each bar represents
the mean ± SEM of four independent observations per group. * = p
< 0.01 vs Small antral (SA); **= p < 0.01 vs Preoulatory follicles (PO).
a= p < 0.05 vs. basal (PO and Cystic).
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preovulatory and cystic bovine follicles are similar to the
ones described for others species such as rats and
humans [5,7]. In dairy cows, preovulatory follicles have
an average diameter of 16-19 mm [1]. Ovarian cysts are
defined as follicle-like structures of diameter greater than
25 mm persisting for at least 10 days in the absence of a
corpus luteum [20]. A cystic follicle is also characterized
by a large accumulation of fluid; the wall of the cyst has a
rich vasculature, even more so than that of healthy preovulatory follicles [28]. Recently we used [17] a surgical
method to isolate ovarian follicles of different diameters
and hence different stages of maturation. Notably, there
was a substantial difference in size between the different
follicles, especially the cystic follicles. Not only the
amount of follicular liquid inside cystic follicles is many
times that found in SA follicles, but also the area of follicular wall is highly increased in cystic follicles. Thus, is
important to consider these variables to understand the
relative contribution of nerve terminals in the piece of
follicular wall used in the study. Cyst follicles could be a
reservoir of substances secreted by granulose or theca
cells, as well as substances permeating from plasma.
The measurement of NE concentration in follicular
wall represents the amount of neurotransmitter in close
contact to beta-receptors present in both theca and
granulosa cells [29,30]. The follicular walls from SA and
PO follicles did not differ in NE concentration, but the
cyst wall presented a much lower concentration of NE
as compared with both SA and PO follicles. As discussed above, the relative decrease in NE concentration
could be the result of the big increase in the area of cystic follicles. According to diameter of each type of follicles and because we used follicular pieces of similar size,
the relative area used from each follicle is highly variable. (0.25, 25 and 15,625 mm2 from SA, PO and cystic
follicles respectively), thus an increase of almost 600
fold between PO and cystic follicles, suggesting not only
a higher release activity of the nerves but also an
increased efflux of NE from cystic wall to follicular fluid
as compared to the efflux existing in SA and PO follicles. In support of this hypothesis, there was a 10-fold
increase in the total amount of NE present in follicular
fluid obtained from cysts as compared with the follicular
fluid derived from PO follicles. Thus it was evident that
total NE release from the cystic wall was increased as
compared with SA and PO follicles. It was also evident
that the cystic wall presented the highest spontaneous
release of NE without a change in the capacity to incorporate NE into the tissue. If we consider the increase in
spontaneous release together with the induced release,
we get a higher release capacity from the cystic follicles
as compared to the follicular wall from PO follicles.
Despite of the higher total release of NE (after correction by the increase in the size of cyst), the amount of
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NE in the cystic follicular fluid seems to exceed the
capacity to release NE from the follicular wall of cyst.
This contrasts with the polycystic condition induced in
rats after estradiol valerate administration in which
there is no increase in the size of the ovary and there is
a direct correlation between NE amount and release
after estradiol treatment [31]. In cows, there is likely an
additional factor that it is not present in the estradiolinduced rat model that decreased the stimulus-induced
release capacity of the nerve terminals associated with
cysts. Stress-induced cortisol release could be one of
these factors, as it has been previously described for the
rat under a combined cold and restraint stress procedure, in which there is a increase in corticosterone
plasma levels [32]. As we discussed below, cystic follicles
present an increased capacity to secrete cortisol, either
basal or induced by hCG and this hormone could act
locally to decrease NE release [32]. Although, the cystic
follicles posses few granulosa cells, we have previously
reported that granulosa cells have the capacity to incorporate and release NE as a neuron-like cells [33] and
thus they could participate as a intrafollicular regulatory
compartment for NE. This mechanism required futures
studies. Unpublished data give us information that in
the rat ovary interstitials cells participate in NE uptake
too. The relative contribution of each compartment it is
not exactly known but denervation studies [15], strongly
suggest that extrinsic innervations (nerve terminals
located close to theca cells and in interstitial tissue) correspond at least 80% of total NE. Whatever the underlying mechanism, the continuous non-regulated outflow
of NE to the follicular fluid likely induced a decrease in
the number of b-adrenergic receptors in the cystic wall
as a consequence of a ligand-induced downregulation of
the receptor [3,25,32,34]. It has been well documented
that b-receptor number in many tissues, including the
rat ovary under polycystic conditions [3], is downregulated by increased amounts of NE in the synaptic space,
as observed in the follicular cyst. Odore et al., [35] were
the first to demonstrate a decrease in b-adrenergic
receptor number in follicular cysts from dairy cows.
Independently of the decrease in cystic follicle b-receptor number found in this work, we sought to determine
whether there is a physiological coupling between badrenergic stimulation and steroid secretion from the
ovary. The follicular cyst presented a higher capacity to
spontaneously release testosterone when incubated in
vitro. This characteristic could be in line with an
increased sensitivity to NE stimulation derived from
hypersensitivity among the low number of b-adrenergic
receptors present in the wall of follicular cysts. The continuous stimulation by NE present in the follicular fluid
could be responsible for this spontaneous secretion of
testosterone. The increased capacity of the piece of
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follicular cyst to respond to isoproterenol strongly suggests that follicular cysts in dairy cows are highly
responsive to b-adrenergic stimulation and induce an
elevation in testosterone secretion. We have demonstrated previously that, not only in rats but also in the
human ovary [3,9], b-adrenergic stimulation controls
androgen secretion and thus participates in the control
of steroid secretion. The relationship between increased
steroid secretion and stress has been demonstrated in
rats as well as in humans [22,36]. In humans it has been
demonstrated that women with PCOS present a higher
stress level and higher levels of sympathetic nerve activity [6,22]. Anxiety could be also a component of this
response to stress [37] and cortisol regulate this
response. The higher concentration of cortisol in follicular cysts as compared to SA or PO follicles and the
increased response to hCG, introduces the interesting
possibility that intrafollicular cortisol could represent an
index for the plasma levels of corticoids or even local
production by the follicular wall. The in vitro incubation
of follicles under stimulation by hCG or isoproterenol
clearly demonstrates that hCG but not ß-adrenergic
agonist, induced the synthesis of cortisol, leading to the
concept that cortisol could be preferentially regulated by
a hormonal way (i.e. gonadotropins) and testosterone
secretion is regulated by both hormonal and neural stimulation. More studies are needed to characterize the
role of cortisol in the context of ovarian function.
Follicular cysts from the ovaries of cows and their
pathophysiology are of increasing interest due to the use
of b 2 -adrenergic compounds to improve the performance of meat-producing animals [38-40]. b2-adrenergic agonists are powerful compounds that, following
long-term administration at high doses, cause a significant repartition of feed energy, thus increasing protein
accretion at the muscular level, which results in lipolytic
effects. Independent of the possible effects of residues
consumed by humans, b-agonists could also be dangerous for the animal’s welfare.
Acknowledgements
Grant support: CONICYT-SECyT-2005-7-162 (HEL, HHO) and CONICYTMINCyT2008-133 (AHP, HHO). Chile-Argentina, Fondecyt 1090036 (HEL),
Fondecyt 1090159 (AHP),
Author details
1
Laboratory of Neurobiochemistry, Department of Biochemistry and
Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences,
Universidad de Chile, Santiago, Chile. 2Morphological Sciences Department,
Faculty of Veterinary Sciences, Universidad Nacional del Litoral (FCV-UNL),
Esperanza, Santa Fe, Argentina & National Council for Science and
Technology (CONICET), Argentina.
Authors’ contributions
NRS: participated in ovarian morphology and quantification of steroid
hormone. AED: chromatographic determination of noradrenaline in ovarian
follicular fluid and wall. BED: determination of testosterone concentration
Page 7 of 8
and secretion study in vitro. HHO: participated in the incorporation and
release noradrenaline, secretion study in vitro, determination of cortisol an
testosterone concentration. HL: discussion and manuscript preparation. AP:
participated in the incorporation and release noradrenaline, determination of
β-adrenergic receptor in the follicular wall and contributed to the
development, design, coordination of the research, manuscript preparation.
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 7 December 2010 Accepted: 16 May 2011
Published: 16 May 2011
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doi:10.1186/1477-7827-9-66
Cite this article as: Paredes et al.: Sympathetic nerve activity in normal
and cystic follicles from isolated bovine ovary: local effect of betaadrenergic stimulation on steroid secretion. Reproductive Biology and
Endocrinology 2011 9:66.
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