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Laboratory of Neurobiochemistry, Faculty of Chemistry and Pharmaceutical Sciences (H.E.L., A.P., J.L.F.), and Faculty of Medicine (V.L., H.F.), Universidad de Chile, Santiago, Chile; and the Division of Neuroscience, Oregon Regional Primate Research Center/Oregon Health Sciences University (G.A.D., S.R.O.), Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Dr. Sergio R. Ojeda, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies using a rodent model of the human syndrome of polycystic ovary (5) first suggested that an alteration in peripheral sympathetic activity may contribute to the etiology and/or progression of cystic ovarian disease. Polycystic ovarian syndrome (PCOS) is a complex disease characterized by ovulatory failure, amenorrhea, hyperandrogenemia, and variable levels of circulating gonadotropins (6, 7). PCOS is widely recognized as the most common cause of infertility in women. To date, the precise etiology of the disease remains unknown, even though it appears clear that its initiation and progression may be determined by a variety of interrelated factors (6, 7). The abnormalities detected in PCOS have been attributed to primary defects in the hypothalamic-pituitary unit, the ovarian microenvironment, the adrenal gland, and the insulin/insulin-like growth factor I metabolic regulatory system (7, 8). A very recent study used genetic linkage analysis to search for loci contributing to PCOS and identified follistatin as the gene product with the strongest linkage to the disorder (9). Despite this multiplicity of potential etiologies, a feature common to most forms of PCOS is the lack of dominant preovulatory follicles, which are replaced by multiple medium-size antral follicles containing an enlarged, androgen-producing, thecal layer (10).
The potential contribution of the peripheral sympathetic system to the syndrome has been suggested by several observations, including the increased density of catecholaminergic nerves detected by histofluorescence in patients suffering from the disease (11) and the effectiveness of ovarian wedge resection to restore ovulatory capacity, especially when it compromises the hilum (12, 13), the point of entry of nerves into the ovary. That PCOS may indeed be associated with an abnormal activation of the sympathetic nervous system is indicated by the results of a recent report showing an impaired metabolism of norepinephrine (NE) in adolescents suffering from the disease (14). Rats injected with a single dose of estradiol valerate (EV) develop an anovulatory condition that resembles in several, but not all, aspects the human syndrome (15). Using this rodent model, we demonstrated (5) that the appearance of ovarian cysts after EV administration is preceded by an increased activity of the sympathetic nerves arriving at the ovary via the superior ovarian nerve (SON). The SON carries the bulk of the noradrenergic innervation to the secretory cells of the ovary (16). Its transection in EV-treated rats resulted in restoration of cyclicity and ovulatory capacity (17), indicating that hyperactivation of the ovarian sympathetic nerves plays an important role in maintaining the EV-induced anovulatory condition.
The development and function of the ovarian sympathetic innervation depend on the ability of the ovary to produce nerve growth factor (NGF) (1), a target-derived neurotrophin required for the development of the peripheral sympathetic system (18). In the rat ovary, NGF is preferentially synthesized in cells of the follicular wall (19), which is a terminal field for the sympathetic neurons projecting to the ovary (1, 16). Transfer of NGF from its sites of production to the innervating fibers has been postulated to occur via binding of the neurotrophin to the low affinity neurotrophin receptor (20), known as p75 NGF receptor (p75 NGFR or p75 NTR). Thus, activation of this target-derived trophic system may be a factor involved in enhancing NE outflow to the gland in the EV-induced polycystic ovary. The results of the present study are consistent with this concept. They further indicate that an augmented intraovarian production of NGF and its low affinity receptor is an important component of the process by which follicular cysts, acyclicity, and anovulation are maintained in EV-treated rats. A preliminary report of these findings has appeared in abstract form (21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of tyrosine hydroxylase (TH) activity
To measure TH activity in the celiac ganglion, the main source
of sympathetic fibers reaching the ovary via the SON (24), the ganglion
was excised (along with adjacent tissue) from its location between the
abdominal aorta and the superior mesenteric and celiac arteries, as
described by Lawrence and Burden (16). The enzyme activity was
determined by the method of Waymire et al. (25), as
previously described (26). The procedure involves measuring the
14CO2 released from
1-[14C]tyrosine (SA, 52 mCi/mmol; NEN Life Science Products, Boston, MA) after hydroxylation by
endogenous TH, followed by DOPA decarboxylase-mediated decarboxylation.
The source of DOPA decarboxylase was a crude extract from pig kidneys.
Tissues were first homogenized in 10 vol 0.1 M
acetate buffer (pH 6.1) and 0.1% Triton X-100. The homogenates were
centrifuged at 20,000 x g for 10 min, and the
supernatants were used as samples. The assay was performed in the
presence of a saturating concentration of 1 mM
6-methyl-tetrahydrobiopterin (Sigma) as a cofactor for TH.
The enzymatic activity is expressed as nanomoles of
CO2 formed per 30 min.
Ribonuclease (RNase) protection assay
This assay was employed to measure the content of TH and class
IV ß-tubulin messenger RNA (mRNA) in the celiac ganglion and that of
NGF and its low affinity receptor p75 NGFR mRNA in the ovary. The
procedure used has been described in detail previously (27). A TH
complementary DNA (cDNA) corresponding to nucleotides (nt) 1241–1521
in rat TH mRNA (28) was subcloned into the riboprobe vector pGEM-3Z,
linearized with HindIII, and used as a template to
synthesize a 280-nt [32P]UTP-labeled TH
antisense RNA probe. To determine whether EV affects cytoplasmic
transport in celiac ganglion neurons, we measured the changes in mRNA
encoding class IV ß-tubulin (RBT2). Synthesis
of this tubulin subunit in the nervous system of the rat increases
after the major phase of neuronal differentiation and growth has ended
(29), and thus it may be more relevant to microtubule functions related
to axonal transport than to neurite elongation (29, 30). The
RBT2 DNA template used for transcription
(subcloned into the riboprobe vector SP64) spans 240 bp of the
3'-untranslated region of class IV ß-tubulin mRNA (29). Linearization
of the plasmid with EcoRI and use of SP6 polymerase as the
enzyme for transcription yielded the antisense RNA used for the assay.
To prepare an NGF complementary RNA (cRNA) probe, a 771-bp NGF cDNA was
subcloned into pGEM-3Z, linearized with TthIII1, and used to
synthesize a 324-nt antisense probe complementary to nt 702-1025 in rat
NGF mRNA (31). In the case of p75 NGFR mRNA, the DNA template was a
277-bp cDNA subcloned in pGEM-3Z and linearized with AvaI.
SP6 polymerase-directed transcription yielded a 275-bp antisense RNA
probe, complementary to nt 430–705 in rat p75 NGFR (32). In each case,
standards for mRNA quantitation were prepared from sense mRNAs
transcribed from the same DNA templates used for the preparation of
cRNAs, but in the opposite direction.
The tissue RNA samples (10–25 µg) and the RNA standards were hybridized to 500,000 cpm labeled probe for 15–17 h at 45 C. To correct for procedural variabilities, the RNA samples were simultaneously hybridized to a cyclophilin cRNA probe that recognizes the constitutively expressed cyclophilin gene (33). Cyclophilin mRNA levels remain unchanged throughout postnatal ovarian development (19, 34) and thus provide an adequate internal control for normalization of changes that may occur in the content of the mRNA of interest. The cyclophilin cRNA used is complementary to nt 338–469 in rat cyclophilin mRNA (33). After hybridization, the samples were treated with RNase A plus T1 to digest nonhybridizing species, and the protected fragments were isolated in a 5% polyacrylamide-7 M urea gel. After exposure to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -85 C, the autoradiographic signals were analyzed using an Agfa (Agfa, Bertronge, Belgium) flatbed scanner and the computer program NIH Image, as previously reported (19).
Combined in situ hybridization and retrograde fluorescent
tracing
This procedure was employed to determine whether the formation
of cysts induced by EV treatment is preceded by an increase in TH gene
expression in catecholaminergic neurons projecting from the celiac
ganglion to the ovary. Thirty days after EV injection, 3 µl of a 5%
fluorogold (Fluochrome, Englewood, CO) solution dissolved in 0.1
M cacodylic acid were pressure-injected into several sites
of the ovary of brevital-anesthetized animals. Both oil-injected
(n = 3) and EV-treated rats (n = 3) were similarly treated.
In two additional animals, the fluorogold was injected after section of
the ovarian nerves. After the injection, the site of needle entry was
sealed with a drop of superbonder (cyanoacrylate), and the ovary was
returned to the peritoneal cavity. Ten days later, the ipsilateral
celiac ganglion was dissected and fixed by immersion in 4%
paraformaldehyde-borate buffer, pH 9.5, for 18–20 h at 4 C (35).
Thereafter, the tissues were immersed in 10% sucrose in PBS for
24 h and frozen in OCT compound (Miles, Elkhart, IN), before
cryostat sectioning. Ten-micron sections were then hybridized, as
previously described (36), with a solution containing 1 x
107 cpm/ml of a
[35S]UTP-labeled TH cRNA probe. After
hybridization at 55 C for 18–20 h and washes to a final stringency of
0.1 x SSC at 65 C for 30 min, the sections were defatted and
dipped in NTB-2 emulsion. Ten days later, the reactions were developed,
and fluorogold-containing neurons were identified under a fluorescence
microscope equipped with darkfield illumination, using bands of UV
excitation (emission maximum, 408 nm; excitation maximum, 323 nm), as
previously recommended (37). Only sections containing
fluorogold-labeled neurons (at least three sections per ganglion) were
analyzed; microscopic images were photographed and digitized for
computer analysis, as previously reported (38). In brief, background
levels and individual grain size were first determined for each slide.
The grains over each neuron were then identified, captured, and
digitized. The number of grains per cell were calculated from the
number of positive digitized pixels, using as a reference the average
grain size, and the predetermined background density as a correction
factor. Neurons were considered positive when the corrected number of
grains exceeded the background density by a factor of 3. By estimating
the TH mRNA content in individual ganglion cells (as assessed by the
number of silver grains per cell) and identifying those cells
containing fluorogold (i.e. those that project to the
ovary), it was possible to assess differences in TH mRNA levels between
cells innervating the ovary and those sending their axons
elsewhere.
NGF two-site immunoassay and determination of p75 NGFR protein
content by cross-linking to [125I]NGF
Ovarian NGF content was measured by a highly sensitive two-site
fluorometric enzyme immunoassay (39), exactly as previously described
(40). Detection of p75 NGFR protein was achieved by cross-linking
[125I]NGF to ovarian membranes, followed by
immunoprecipitation of the NGF-p75 NGFR complex with a monoclonal
antibody (192 IgG) directed against the receptor (41) and separation of
the cross-linked species by SDS-PAGE. The procedure employed has been
described in detail previously (42).
Intraovarian administration of NGF antibodies and antisense
oligodeoxynucleotides to p75 NGFR mRNA
Rats were injected with EV as before and immediately implanted
sc with an Alzet miniosmotic pump (model 2ML4, Alza Corp.,
Palo Alto, CA) loaded with a mixture of polyclonal NGF antibodies
(diluted 1:100) to block NGF biological actions (1, 19), and a p75 NGFR
mRNA antisense phosphothionate oligodeoxynucleotide (5
µM) to reduce p75 NGFR synthesis (43). This mixture is
henceforth referred to as NGF Ab + p75 NGFR AS. The oligonucleotide
(5'-AGT-GGA-CTC-GCT-GCA-TAG-3') is directed against the 3'-region of
NGFR mRNA and has been shown to effectively block p75 NGFR synthesis
(43). Control animals were infused with preimmune serum (PIMS). As the
treatment was instituted to restore ovarian function, rather than to
disrupt it, control animals were not infused with a p75 NGFR scrambled
oligonucleotide sequence. Such a sequence has been previously shown to
be ineffective in reproducing the inhibitory effect of p75 NGFR AS on
kidney development (43).
The osmotic pump was connected to SILASTIC brand tubing (Dow Corning Corp., Midland, MI; id, 0.020 in.; od, 0.037 in.), which was then inserted underneath the bursa of the left ovary and kept in place by a drop of cyanoacrylate and sutures attached to the ipsilateral uterine horn. Delivering a flow rate of 2.5 µl/min, these pumps remain operational for 28 days. At the end of the first 28-day period, the pumps were changed to continue the treatment for a total of 56 days. Estrous cyclicity was monitored by daily vaginal lavages. At the end of the experiments, the rats were painlessly killed, the intrabursal location of the cannula’s tip was verified by visual inspection, and the ovaries were fixed for either histological analysis or immunohistochemistry (see below).
Immunohistochemistry
After 18–20 h of fixation in Zamboni’s fixative, the ovaries
were transferred to PBS for 24 h, at 4 C and then to 20% sucrose
in PBS for an additional day. Thereafter, they were embedded in OCT
compound (Miles, Inc.), frozen on dry ice, and stored at -85 C until
cryostat sectioning. Ten-micron sections were subjected to
immunohistochemistry for p75 NGFR using monoclonal antibody 192 IgG
(41), according to a procedure previously reported (42).
Histology
The ovaries from oil-injected controls, EV-treated rats, and
EV-treated rats receiving an intrabursal infusion of NGF Ab + p75 NGFR
AS or PIMS were cleaned of adherent fat tissue, immersed in Bouin’s
fixative solution, embedded in paraffin, serially sectioned at 8 µm,
and stained with hematoxylin-eosin as previously reported (1). One
ovary per animal was subjected to morphometric analysis. When analyzing
the effect of NGF Ab + p75 NGFR AS on estrous cyclicity, the
contralateral noninfused ovary was also examined after 56 days of
treatment. The incidence of corpora lutea, expressed as the number of
corpora lutea per ovary, was used to determine whether ovulation had
occurred in the different groups of animals. The numbers of preantral,
antral, and atretic follicles were counted in every fifth section, as
previously described (1). The sizes of both healthy and atretic antral
follicles were also determined. Preantral follicles were defined as
follicles without any antral cavity and with two or more layers of
granulosa cells. Atretic follicles were defined as those follicles with
more than 5% of cells with pyknotic nuclei in the largest
cross-section, showing oocyte shrinkage and occasional germinal vesicle
breakdown (44, 45). Also included in this group were follicles showing
deformation (shrinkage or collapsing) or lacking the oocyte.
Cystic follicles were defined according to criteria proposed previously
(23) as those follicles devoid of oocytes, displaying a large antral
cavity, an enlarged thecal cell layer, and a thin (most frequently
monolayer) granulosa cell compartment containing apparently healthy
cells (Fig. 1
). Type III follicles were
also defined according to the criteria proposed by Brawer and
colleagues (46, 47). These follicles are large, devoid of oocytes,
contain four or five plicated layers of small, densely packed granulosa
cells surrounding a very large antrum, and display a seemingly normal
thecal compartment. Type III follicles may represent precystic
follicular structures (46, 47). Our observations suggest the existence
of a transitional stage between healthy preovulatory follicles and the
type III follicles described previously (46, 47). Follicles in this
transitional stage share all morphological characteristics previously
described for type III follicles, but in addition they contain a
healthy oocyte. Figure 1 illustrates the morphological differences
observed among normal preovulatory follicles (A), type III follicles
with oocyte (B), type III follicles without oocyte (C), and follicular
cysts (D).
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2 test for
the comparison of frequency distributions (48). |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3).
By day 15, the average corpora luteum size remained significantly
greater than that on day 0 (Figs. 2 and 3). Thirty days after EV
injection, both the number and size of the corpora lutea were
significantly decreased (Figs. 2 and 3) compared with those in
noninjected, day 0 animals. By day 60, corpora lutea were no longer
detected in the steroid-treated rats (Figs. 2 and 3). In other
experiments, however, corpora lutea were detected at this time in some
rats (three of seven rats, two to six corpora lutea per rat).
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), type III follicles
became apparent for the first time 30 days after EV injection (4
± 1.2/ovary; n = 3 rats). By day 60, cystic follicles were also
detected. They had an average size of 459 ± 38 µm (mean ±
SEM; n = 27) and were present at a frequency of
5.2 ± 1.5/ovary. No luteinized follicles were found. At this
time, type III follicles (750 ± 5 µm; n = 20) were
detected at a frequency of 1.1 ± 0.5/ovary in the ovaries of
EV-treated rats. Neither cysts nor type III follicles were detected in
the ovaries of untreated rats in this particular group of animals.
Neurotrophin changes in the ovary after EV administration
These experiments were performed to determine whether the changes
in NE outflow that occur in the ovary 30 days after EV administration
(5) are accompanied by an activation of NGF production in the ovary
and/or its low affinity receptor p75 NGFR. This receptor is thought to
facilitate the transference of NGF from its sites of production to the
innervating fibers (20, 49). Ovarian p75 NGFR mRNA levels, measured by
RNase protection assay, increased noticeably after EV administration
(Fig. 4A). The first significant increase
was observed 15 days postinjection, with values reaching maximal levels
by 30 days. At this time, there was a 5-fold increase in message levels
with respect to control values; values declined by 60 days, but still
remained significantly elevated over control levels. In contrast to p75
NGFR, there was no change in the levels of the mRNA encoding the high
affinity NGF receptor, TrkA. NGF mRNA levels, on the other hand, were
also increased by EV, but only at 30 days (Fig. 4C), a time at which
the NGF mRNA content was 6-fold higher than that in oil-treated
controls. Sixty days after EV administration, NGF mRNA values had
almost returned to control levels.
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). NGF
content increased 2-fold by 30 days (Fig. 4D), decreasing to
intermediate values thereafter.
EV administration activates noradrenergic neuronal output
The prevertebral celiac ganglion is a prominent site of
noradrenergic neurons innervating the ovary (16, 24). To determine
whether EV administration affects the activity of noradrenergic neurons
in the celiac ganglion and whether such changes precede and/or
accompany the appearance of ovarian cysts, TH mRNA content was measured
30 and 60 days after EV injection. The results demonstrated that TH
mRNA levels were modestly, but significantly, increased at both time
points (Fig. 5, upper panel).
In contrast to these changes in TH mRNA levels, TH activity in the
ganglion decreased markedly 30 days after EV, returning to basal values
by 60 days (Fig. 5, middle panel). As ovarian TH activity
increases at these same intervals after EV injection (5), the
possibility was considered that the drop in TH activity in the
ganglion is related to an increase in axonal transport of the
enzyme to the nerve terminals. TH is transported to nerve endings by
slow axonal transport (50). Measurement of ß-tubulin type IV
(RBT2) mRNA levels as an indirect index of axonal
transport (29, 30) demonstrated a substantial increase in
RBT2 mRNA content in the celiac ganglion (Fig. 5, lower panel) at the two intervals examined after EV
injection. The increase was more prominent 30 days after EV (4-fold),
but was still clearly evident by 60 days (3-fold).
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, upper panels). Computerized
assessment of cellular TH mRNA levels (determined by the number of
silver grains per cell) was used to estimate the relative changes in TH
mRNA abundance per cell in control and EV-treated groups. As some of
the cells were also fluorescent (indicating that they innervate the
ovary), it was possible to assess differences in TH mRNA levels between
cells innervating the ovary and those sending their axons elsewhere.
All measurements were made 30 days after EV administration,
i.e. before the appearance of cysts. In agreement with the
results of the RNase protection assays, we found that EV treatment
resulted in a small, but significant (P < 0.05),
increase in TH mRNA content in catecholaminergic cells not projecting
to the ovary (Fig. 6, lower panel). A much more robust
(40%; P < 0.01) increase in TH mRNA content was
observed in catecholaminergic cells projecting to the ovary
(i.e. cells containing both TH mRNA and fluorogold; Fig. 6, lower panel).
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, upper
panel). Continuation of the infusion for an additional 28-day
period by replacement of the minipump with a fresh mixture of
inhibitors led to the initiation of irregular estrous cyclicity in four
of seven animals (Fig. 7, upper panel). Control EV-treated
animals receiving an infusion of PIMS behaved like noninfused
EV-treated rats, i.e. remained in constant estrus throughout
the duration of the experiment (Fig. 7, lower panel).
Infusion of NGF Ab + p75 NGFR AS for only the first 28 days after EV
injection failed to restore estrous cyclicity in all eight rats,
whereas initiation of the infusion 30 days after EV resulted in
restoration of estrous cyclicity in four of eight animals (not shown).
As in the case of the prolonged 56-day treatment (Fig. 7), the pattern
of estrous cyclicity in these short term treated rats did not have the
regularity of that in normal animals.
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illustrates
these findings. Figure 8A shows the ovary from a normal cycling adult
rat. Figure 8B shows the ovary of an EV-treated rat infused with PIMS.
Figure 8C demonstrates the presence of corpora lutea in the ovary of a
rat treated with EV and infused with the NGF Ab + p75 NGFR AS
solution.
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), infusion
of NGF Ab + p75 NGFR AS reversed this effect, resulting in a number of
antral follicles per ovary similar to that in control untreated rats
(Fig. 9A). These changes were not particular to either healthy or
atretic antral follicles, as the number of both types of follicles
decreased with EV and increased toward control values after blockade of
NGF/p75 NGFR actions (Fig. 9, B and C). Likewise, they were observed in
both rats that responded to the NGF Ab + p75 NGFR AS treatment with
restoration of cyclicity and those that remained in constant estrous
(see Fig. 7). No changes in the number of preantral follicles were
detected with either treatment (not shown). As observed in initial
experiments (Fig. 3), EV injection followed by PIMS infusion did not
significantly increase the average size of healthy antral follicles
(Fig. 9D). Infusion of NGF Ab + p75 NGFR AS was also without effect.
EV-treated rats showed a small increase in the size of atretic antral
follicles (from 299 ± 15 to 361 ± 27 µm), a change that
was obliterated by the inhibition of NGF/p75 NGFR actions (301 ±
27 µm; Fig. 9E). Importantly, the ovaries from EV-treated animals
that reinitiated estrous cyclicity after infusion with NGF Ab + p75
NGFR AS (see Fig. 7) had a greater number of corpora lutea than rats
treated with EV and infused with PIMS (13.3 ± 4.4 vs.
3.8 ± 1.7; Fig. 9F). The contralateral, noninfused ovaries showed
similar differences (not shown).
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). Surprisingly, five
of the six untreated controls also had cysts, but at a frequency much
lower than that in EV-treated rats and similar to that detected in NGF
Ab + p75 NGFR AS-treated rats (Table 1). In addition to reducing the
incidence of follicular cysts, blockade of NGF/p75 NGFR actions
eliminated type III follicles (Table 1), making the overall follicular
population of the ovary similar to that in untreated rats.
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) and nerve fibers
(Fig. 10A1) of ovaries from EV-treated rats infused with PIMS. Thecal
p75 NGFR immunoreactivity was decreased in ovaries infused with NGF Ab
+ p75 NGFR AS (Fig. 10B). In contrast to this effect on thecal cells,
the infusion was ineffective in decreasing the levels of p75 NGFR
immunoreactivity in ovarian nerves (Fig. 10, A1 and B1).
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Discussion |
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Hallmarks of the polycystic ovarian syndrome, the most common endocrine disorder of women in reproductive age (8, 51), are the presence of multiple, medium-sized, nonatretic, antral follicles in the ovary (6) and augmented androgen secretion (6, 51, 52, 53). To date, an animal model that thoroughly mimics all of the abnormalities underlying the human pathology has not been described. Nevertheless, administration of a single injection of EV to adult rats has been shown to reproduce several of the ovarian abnormalities in PCOS, including the formation of ovarian cysts, hyperthecosis, and increased androgen production in addition to anovulation and loss of estrous cyclicity (17, 23, 46). Using this rodent model, an alteration of the neurogenic control of the ovary, inferentially suspected by others to contribute to the human syndrome (11, 14), was found to be a distinct component of the EV-induced ovarian pathology (5, 17). Specifically, these studies showed that preceding the appearance of the cysts, there was an activation of the sympathetic innervation to the ovary, as revealed by an increased release of newly incorporated NE from ovarian nerves, augmented ovarian NE content and TH enzymatic activity, and enhanced incorporation of labeled NE into ovarian tissue (5). That this abnormally high sympathetic activity contributes to maintaining the anovulatory state in EV-treated animals was indicated by the prompt resumption of cyclicity and ovulatory capacity in animals subjected to transection of the superior ovarian nerve (17), the main source of sympathetic fibers innervating ovarian follicles (16).
EV may enhance peripheral sympathetic activity by acting on neuronal systems that directly (16, 24) or transsynaptically innervate the ovary (54, 55) and/or on the ovarian system of target-derived trophic factors that supports the sympathetic innervation of the gland (56). Without contesting the potential importance of the former site of action, the present results suggest the existence of the latter. Together, they support two related concepts: 1) that the increased noradrenergic flow to the ovary observed in animals with EV-induced cystic ovary (5) is, to a significant extent, caused by an increased production of NGF and the low affinity p75 NGFR in the ovary; and 2) that a deranged expression of this neurotrophic signaling complex is an integral component of the mechanism by which EV induces anovulation in rats.
Although EV-induced follicular cysts become first detected around 60 days after the EV injection (Ref. 46 and this study), activation of the sympathetic innervation to the ovary precedes the formation of follicular cysts by at least a month (5). In turn, synthesis of p75 NGFR increases as early as 15 days after EV administration and is followed shortly by augmentation of NGF synthesis, indicating that activation of this ligand/receptor module is an early event in the process by which EV administration disrupts ovarian function. An increased neurotrophic support to the sympathetic neurons projecting to the ovary is likely to play a significant role in enhancing the sympathetic outflow to the ovary in EV-treated rats. TH mRNA levels were selectively augmented in noradrenergic celiac neurons projecting to the ovary, an effect that can hardly be attributed to estrogen itself, as the steroid inhibits, rather than stimulates, TH synthesis (38, 57, 58). In contrast, NGF is a potent inducer of both TH gene expression (59, 60) and TH enzymatic activity (59, 61). In the ovary, NGF and both of its receptors, p75 NGFR and the high affinity TrkA tyrosine kinase receptor, are synthesized in thecal cells (19, 42), a prominent terminal field of the sympathetic neurons innervating the ovary (42, 62, 63). Neonatal immunoneutralization of NGF actions inhibits the development of ovarian sympathetic innervation and delays follicular growth (1), highlighting both the critical importance of the trophic factor in supporting the innervation of the ovary and the facilitatory role of sympathetic nerves on follicular development.
The early activation of ovarian p75 NGFR synthesis after EV suggests that there is also an early increase in NGF availability to the innervating neurons. The p75 NGFR not only facilitates transfer of NGF from its sites of production to NGF-sensitive fibers (20, 64), but can collaborate with the high affinity TrkA receptor to potentiate cellular responses to the neurotrophin (65). The subsequent increase in NGF synthesis detected 30 days after EV would ensure a continuously supply of the polypeptide to the nerve terminals and, hence, to the projecting neurons.
The increase in NGF and p75 NGFR synthesis that follows EV administration raises the question of the mechanism(s) by which EV causes such an increase. Previous studies have shown an increase in ovarian NGF gene expression (19), but not in p75 NGFR (42), in the afternoon and evening of the first proestrus, i.e. at the time of the preovulatory surge of gonadotropins. No alterations in either NGF or p75 NGFR mRNA levels were detected at times preceding the LH surge, when plasma estrogen levels are most elevated, suggesting that the preovulatory changes in NGF expression are not due to estrogen per se, but to gonadotropins instead. It is doubtful that the dual up-regulatory effect of EV on NGF and p75 NGFR expression is due to an increase in basal gonadotropin release caused by a centrally mediated effect of the steroid. Both plasma LH and FSH levels decrease within 2 weeks after EV injection, remaining below control levels for at least 60 days (15, 23). An EV-dependent defect in the ovarian receptor system mediating LH actions remains a possibility, as transgenic animals overexpressing the LH gene develop a PCO-like condition in the absence of any functional changes reflecting LH receptor down-regulation by the inappropriately high plasma LH levels (66). Another possibility is a direct up-regulatory effect of EV on ovarian p75 NGFR and NGF gene expression. Such an effect appears unlikely, as estrogen has been shown to down-regulate, rather than up-regulate, p75 NGFR mRNA levels in other tissues (67). Moreover, we observed that EV injection is followed 24 h later by some increase in p75 NGFR and a marked, but transient, increase in ovarian TrkA mRNA content (coinciding with an ovulatory LH surge), with no change in NGF mRNA levels (data not shown). Thus, there must be an intermediate event responsible for the delayed (7–30 days) p75 NGFR and NGF responses to EV. Further studies are required to resolve this issue.
The intrabursal administration of NGF antibodies and a p75 NGFR AS decreased the incidence of precystic structures (type III follicles) and follicular cysts and reversed, in a majority of animals, the anovulatory condition imposed by EV. This effectiveness strongly suggests that the increase in ovarian NGF and/or p75 NGFR production that follows EV administration is a contributing factor to both the process by which the ovary becomes anovulatory in response to EV, and the mechanism that leads to cyst formation. Supporting this idea is the finding that a 2-fold increase in intraovarian production of NGF, via genetically engineered cells grafted into the ovaries of otherwise normal animals, disrupted estrous cyclicity and resulted in an increased incidence of precystic type III follicles (68).
Two potential mechanisms may underlie the recovery of ovulatory capacity that follows attenuation of NGF/p75 NGFR function in EV-treated rats. One is a reduction in the abnormally high sympathetic tone to the ovary, as transection of the superior ovarian nerve in EV-treated rats results is a similar recovery (17). The other may involve correction of an exaggerated influence of NGF on follicular homeostasis, as NGF appears to exert a direct effect on follicular growth (69). The increased number of antral follicles and the reduction in precystic and cystic structures observed in the ovaries of EV-treated rats infused with NGF Ab + p75 NGFR AS suggest that in the absence of an abnormal NGF influence, more normal follicles become able to reinitiate growth and eventually succeed in ovulating. This interpretation is supported by the findings presented in the companion paper (68) showing that sustained induction of abnormally high intraovarian NGF levels results in a reduction in the number of antral follicles per ovary and a greater incidence of follicle type III formation. That is, exposure of the ovary to an abnormally high NGF influence leads to alterations in follicular dynamics resembling some of those caused by EV.
The subpopulation of follicles that ovulate in response to the normalization of NGF/neurogenic influences may be recruited not only from normal, antral follicles, but also from transitional type III follicles, i.e. those containing a healthy oocyte. Earlier findings indicated that even though type III follicles may represent a precystic condition, they might be the only follicles capable of ovulation in EV-treated rats (46, 47). Type III follicles bind labeled hCG as intensely as a preovulatory follicle and yet do not ovulate or luteinize unless exposed to preovulatory LH levels. As the incidence of type III follicles decreased in rats infused with NGF Ab + p75 NGFR AS and increases in rats subjected to an isolated increase in intraovarian NGF levels (68), it may be inferred that the amelioration of an abnormally increased NGF/p75 NGFR function in EV-treated rats leads to dismissal of these structures via ovulation.
With respect to the hormonal mechanisms responsible for the recovery of ovulatory competence and estrous cyclicity seen in NGF Ab + p75 NGFR-treated rats, a decrease in androgen production, and, consequently, in androgen negative feedback, needs to be considered as a contributing factor. While the ovaries of EV-treated rats exhibit a profound androgenic hyperresponsiveness to both hCG and ß-adrenergic stimulation (17), rats carrying intraovarian grafts of NGF-producing cells have elevated serum levels of androstenedione (68). Thus, the normalization of NGF/p75 NGFR function in EV-treated rats may allow restoration of ovulatory capacity by disrupting an abnormal NGF influence on both ovarian androgen output and follicular growth. Not all animals responded to NGF Ab + NGFR AS with restoration of estrous cyclicity and/or ovulation despite showing changes in follicular dynamics similar to those seen in rats that reinitiated cyclicity. This partial recovery may be related to inefficient delivery of the test substances to the ovary and/or to the likely need of correcting the availability of additional factors to achieve full restoration of normal ovarian function.
A surprising observation made in the course of these studies was the recovery of ovulatory capacity of both the ovary infused with NGF Ab + p75 NGFR AS and the contralateral, untreated gland. An early study (47) described a similar phenomenon after hemiovariectomy of rats with EV-induced PCO and implicated an increase in pulsatile LH release in the process by which the remaining ovary became able to ovulate. We infer that in the present study the loss of an abnormally high intraovarian NGF-p75 NGFR signaling activity during the infusion with NGF Ab + p75 NGFR AS results in changes in pulsatile gonadotropin levels able to stimulate both ovaries and thus reverse the anovulatory condition. Support for this idea comes from the finding that hemiovariectomy of EV-treated animals results in an ovulatory rate (47) comparable to that detected in ovaries with inhibited NGF/p75 NGFR function.
In summary, the present results demonstrate the contribution of a target-derived neurotrophic component to the physiopathological process underlying EV-induced PCO in rats. In conjunction with the results presented in the companion paper (68), this study identifies NGF, signaling through the p75 NGFR, as a neurotrophic factor relevant to the process. The potential relationship that this signaling system may have to the intragonadal production of follistatin, a candidate gene product recently identified by linkage analysis as involved in the etiology of human PCOS (9) remains to be determined.
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Received August 30, 1999.
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