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Division of Neuroscience (G.A.D., D.F.H., M.E.C., S.R.O.), Oregon Regional Primate Research Center-Oregon Health Sciences University, Beaverton, Oregon 97006-3448; Laboratory of Neurobiochemistry, Faculty of Chemistry and Pharmaceutical Sciences (H.E.L., A.P.), Faculty of Medicine (V.L.),Universidad de Chile, Santiago Chile; and Center of Molecular Biology Severo Ochoa (A.M.S.), Autonomous University of Madrid, 28049 Cantoblanco, Spain
Address all correspondence and requests for reprints to: Gregory A. Dissen, Division Neuroscience, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006-3448. E-mail: disseng{at}ohsu.edu Address all requests for NGF cells to: Dr. A.
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Introduction |
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Abnormally elevated NGF levels may contribute to ovarian dysfunction as an activation of ovarian NGF synthesis and that of the low affinity NGF receptor (p75 NGFR) precedes the formation of follicular cysts induced by the administration of a single dose of estradiol valerate (EV) to rats (9). A cause-effect relationship between the elevated NGF/p75 NGFR levels and the alterations in follicular growth induced by EV was demonstrated by the restoration of normal populations of antral follicles observed after blockade of NGF actions and p75 NGFR synthesis in the ovaries of EV-treated rats (9).
Injection of a single dose of EV to cycling rats results in loss of estrous cyclicity, anovulation, and formation of follicular cysts (9, 10, 11). These changes are, in a broad sense, similar to some of the abnormalities seen in the human condition of polycystic ovarian syndrome (PCOS) (12, 13). PCOS is characterized by ovulatory failure, amenorrhea, hyperandrogenemia, and variable levels of circulating gonadotropins (12, 13) and is widely held as the most common cause of infertility in women (14). While the initiation and progression of the syndrome is determined by a variety of interrelated factors (12, 13), its precise etiology is unknown. The list of abnormalities associated with PCOS include defects in the hypothalamic-pituitary unit, the ovarian microenvironment, the adrenal gland and the insulin/IGF-I metabolic regulatory system (14, 15, 16). The unifying feature to most forms of PCOS is the presence of multiple medium-size antral follicles containing an enlarged thecal layer, and the occurrence of elevated serum androgen levels [especially androstenedione; (17, 18)]. A recent study implicated follistatin in the etiology of the syndrome, by showing evidence for genetic linkage of the follistatin gene to PCOS and hyperandrogenemia (19).
While this and other findings make it likely that human PCOS is determined by intraovarian defects not primarily dependent on changes in gonadotropin input (14), the nature of these defects has been only partially elucidated (18, 19). Within this framework, and in view of a potential involvement of NGF in the EV-induced cystic ovarian condition in rats (9), we set out to determine whether an abnormally elevated neurotrophic tone within the ovary is by itself capable of producing some of the ovarian abnormalities associated with the hormonal induction of ovarian cysts in this species. Such an effect would implicate an excessive production of NGF as one of the intragonadal factors contributing to the development of cystic ovarian dysfunction. The present study was undertaken to examine this hypothesis. To induce an ovary-specific increase in NGF production, without first affecting the hormonal input to the gland, we grafted into the ovary neural progenitor cells genetically engineered to produce NGF (20). Because in some PCOS patients the syndrome is associated with early initiation of puberty (16, 21), sexual precocity was induced in rats by treatment with PMSG and the NGF-producing cells were grafted into the ovary on the morning of the expected preovulatory surge of gonadotropins. The results indicate that a sustained elevation in intraovarian NGF production results in morphological and functional abnormalities resembling the early phases of EV-induced PCO.
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Materials and Methods |
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Grafting of NGF-producing neural progenitor cells
To increase NGF levels only in ovarian tissue, we used a gene
transfer-cell grafting approach previously employed by us (20, 22, 23)
and others (24, 25) to target gene overexpression to discrete regions
of the mammalian brain. Stable incorporation of the mouse NGF gene into
the genome of conditional immortalized embryonic day 16 rat
hippocampus-derived neural progenitor cells [HiB5; (26)] resulted in
the generation of a cell line (E8) that constitutively secretes mouse
NGF (20). The HiB5 cells were conditionally immortalized by the stable
expression of a temperature-sensitive mutant form of the large
T-antigen from SV40 virus (26). Thus, the cells proliferate at the
permissive temperature of 33 C and stop growing at 37 C. NGF
overexpression was achieved by infection with a retroviral vector
containing the coding region of the mouse NGF gene under the control of
the long terminal repeat of Moloney Murine Leukemia Virus (Fig. 1
). HiB5 cells infected with the same
retrovirus construct, but that failed to incorporate the transgene into
their genome (and thus, do not produce NGF) were used as controls
(clone D11). NGF release from HiB5 E8 cells (3 x
106 cells) cultured for 48 h in DMEM
containing 10% FBS was found to be greater than 4,000 pg/ml as
measured by a specific ELISA assay (see below). In contrast, the
control D11 cells did not secret detectable NGF levels into the medium
(Fig. 1). Both cell lines were grown at 33 C in DMEM plus 10% FBS,
10,000 U/ml penicillin and streptomycin, and 2 mM
glutamine. On the day of grafting, the cells were removed by mild
trypsinization (in PBS containing 0.05% trypsin/0.02% EDTA for 5
min), transferred into a centrifuge tube, collected by low speed
centrifugation, counted and diluted to a concentration of 40,000 cells
per µl of DMEM.
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NGF ELISA assay
NGF levels were measured in culture medium and whole ovaries by
an ELISA assay. The assays were performed using the NGF EMAX
Immunoassay system (Promega Corp., Madison, WI), according
to the manufacturer instructions. Before assay, the ovaries were
weighed and homogenized using a glass/glass homogenizer, in 300 µl of
buffer (100 mM Tris-HCl, pH 7.0, 0.4 M sodium chloride,
0.1% sodium azide, 2% BSA, 2% gelatin, 4 mM EDTA, 1
µM PMSF, and 0.1 U/ml aprotinin). Following a rinse with
an additional 50 µl buffer, the homogenates were microfuged for 5 min
at 4 C, and the supernatants were transferred to a clean tube.
Immunohistochemistry
Immunohistochemical detection of the p75 NGFR was performed on
14-µm cryostat sections from ovaries collected 60 days after
grafting. The ovaries were fixed by immersion in Zamboni’s fixative,
as described (27) and processed for p75 NGFR immunohistochemistry using
the monoclonal antibody 192 IgG (28). Tissue sections were incubated
overnight at 4 C with the antibody and the immunoreaction was developed
the next day using the diaminobenzidine procedure previously described
(6). Controls consisted of adjacent sections incubated without the p75
NGFR antibody (6).
RIA
Serum levels of ovarian steroids were measured as previously
described [estradiol (29), progesterone (30), testosterone (31), and
androstenedione (32)].
Histology
Sixty days after grafting, the ovaries carrying either
NGF-producing or control cells were immersed in Kahle’s fixative
solution, embedded in paraffin, serially sectioned at 8 µm, and were
stained with hematoxylin-eosin as previously reported (8). The number
of antral, type III, and atretic follicles were counted in every fifth
section (8). The criteria used to classify follicle types and the
morphometric analysis procedure used to quantitate the changes observed
are those described in detail in the companion paper (9).
Statistics
The differences among groups were analyzed using one-way ANOVA.
The Student-Neuman-Keuls test was used to analyze differences between
ovarian NGF levels achieved following grafting of control or
NGF-producing cells, in serum levels of sex steroids between the two
groups, and in the number and size of follicles and corpus lutea. The
number of days spent by each animal in a particular phase of the
estrous cycle was expressed as a percentage of the total days studied.
The arcsine transformation was then used to normalize the percentage
data before analysis by one-way ANOVA with LS means to differentiate
among groups. The 
2 test was used to analyze
differences in the incidence of cysts and type III follicles between
the ovaries receiving NGF-secreting or control cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Ovarian NGF
levels achieved were well above those reported in serum of rodents (33)
and humans (34).
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Consistent with earlier findings (6), p75 NGFR immunoreactive material
was present in nerve fibers surrounding blood vessels and coursing
across the interstitial tissue, as well as in fibers associated with
follicles in different stages of development (Fig. 3). As before, p75 NGFR immunoreactivity
was also observed in thecal cells of antral and preantral follicles.
Control sections incubated without primary antibody did not show any
specific staining. The ovaries grafted with NGF-producing cells
exhibited cells containing p75 NGFR immunoreactive material in the
interstitial tissue (Fig. 3, E–G), and—unexpectedly—infiltrating the
granulosa cell compartment of large type III follicles (Fig. 3D). The
morphological aspect of these cells was variable. In some cases, when
located in the interstitial compartment, they exhibit a neuronal aspect
with multiple, single or bipolar processes (Fig. 3, E—G). When
detected in the granulosa cell layer their aspect appeared more
astrocyte-like (Fig. 3D). As no such cells were detected in control
nongrafted ovaries, they must represent two morphological variations of
the grafted neural progenitor cells.
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). In a 4-day cycle, it is
expected that 25% of the time the rats would be in proestrus, 25% in
estrus, and 50% in either diestrus 1 or 2. The rats receiving the
control (D11) cells exhibited cycles in which 19% of the days the rats
were in proestrus, 31% of the days they were in estrus and 39% of the
days the rats were in diestrus. There were a few days in which some
animals were in transitional phases of either proestrus/estrus or
estrus/diestrus (Fig. 5, A and B). In
contrast to this profile, estrous cyclicity was rapidly interrupted in
those rats grafted with NGF-producing cells (Fig. 4). These rats showed
a significantly (P < 0.05) greater number of days in
estrus than in proestrus or diestrus (Fig. 5A). In addition, their
vaginal lavages persistently showed the presence of cornified
epithelial cells, resulting in a greater number of transitional days
(both proestrus/estrus and estrus/diestrus; Fig. 5B). Overall, the
total percentage of days wherein cornified epithelial cells were
present, was doubled in the rats grafted with NGF-producing cells than
in control animals receiving cells unable to produce NGF (87 ±
2% vs. 42 ± 3%, P < 0.0001; Fig. 5C).
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). This decrease was not
accompanied by a similar reduction in the total number of antral
follicles (Fig. 6A), likely because of a slightly increased number of
antral atretic follicles (Fig. 6C). In addition to the selective
decrease in the number of healthy antral follicles, the ovaries grafted
with NGF-producing cells showed an increased size of both healthy and
atretic antral follicles (Fig. 6, D and E).
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). They did, however, exhibit a significant
increase in the incidence of precystic, Type III antral follicles
(Table 1). While they also showed a
doubling in the incidence of follicular cysts, this difference did not
achieve statistical significance (Table 1). Noteworthy, the ovaries of
rats injected with control cells also showed some cysts and type III
follicles, a feature that may be, at least in part, related to the
trauma of the grafting procedure.
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). Though mildly elevated, serum
estradiol and testosterone levels were not statistically greater than
in control rats (Fig. 7, A and B). Serum progesterone levels were also
similar in the two groups.
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Discussion |
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One important component of the neurogenic makeup of a tissue is the
regulatory system of neurotrophins and their receptors. Like in other
tissues innervated by the peripheral nervous system, neurotrophins are
required for the development of the ovarian innervation and for
maintaining the mature function of the innervating neurons (8).
Neurotrophins also enhance the production of neurotransmitters, in
addition to maintaining the functional integrity of the neurons
themselves (37). NGF is the prototypic member of the neurotrophin
family (38). NGF signals via two receptors: one, termed p75 NGFR (also
known as neurotrophin receptor), is a member of the tumor necrosis
factor receptor/FAS/Apo-1/CD95 family (39); the second known as trkA
(40), is a member of the trk family of tyrosine kinase receptors. The
trkA receptor can by itself mediate NGF signaling via the
ligand-dependent activation of its tyrosine kinase intracellular domain
(41, 42, 43). The p75 NGFR on the other hand, can interact with the trkA
receptor to enhance the actions initiated by low levels of NGF (44, 45). In addition, p75 NGFR can effect independent signaling as its
ligand-dependent activation has been shown to stimulate sphingomyelin
hydrolysis leading to an elevation in ceramide levels (46, 47),
increased synthesis of the NF
-B transcription factor (48), and
apoptosis (47, 49).
A form of cystic ovary that exhibits some of the features of the human syndrome can be induced in rats by a single injection of EV (10, 11). As discussed in the companion paper (9), the EV treated rat—while not a faithful model of PCOS—has been useful to identify new attributes of the pathology in rodents. For instance, it has been shown that before the appearance of the cysts there is an activation of the sympathetic innervation to the ovary, as demonstrated by an increased release of norepinephrine in response to electrical stimulation, increased ovarian norepinephrine content and tyrosine hydroxylase enzymatic activity and an enhanced uptake of the catecholamine by the nerves of the dysfunctional gland (50). The elevated activity of the sympathetic neurons reaching the ovary was demonstrated to contribute to the maintenance of the anovulatory state in EV-treated animals, when it was shown that transection of the superior ovarian nerve [the primary source of sympathetic innervation to the ovary (51)] resulted in prompt resumption of cyclicity and ovulatory capacity (52).
This elevated sympathetic activity is related to an intraovarian increase in the production of NGF and p75 NGFR (9). Both the increase in NGF/p75 NGFR synthesis and the activation of the sympathetic input to the ovary precede by several weeks the appearance of follicular cysts, suggesting a cause-effect relationship (9). Though inhibition of this abnormally elevated NGF tone brought ovarian morphology and function toward normalcy, these studies did not rule out the possibility that the actions of NGF/p75 NGFR (or the result of their pharmacological inhibition) require an estrogen-dependent background provided by the initial administration of EV. This is an important issue, because an EV-independent effect of NGF on ovarian function, mimicking all or part of the morphological and functional consequences of EV treatment, would implicate this neurotrophin as one of the intragonadal factors that contributes to the development of a cystic ovarian dysfunction, in the absence of primary alterations in hormonal regulatory control.
The present results provide evidence for this concept. An ovary-specific increase in NGF availability, obtained via grafting of neural progenitor cells genetically engineered to produce NGF, led to disruption of the estrous cycle, and to changes in follicular dynamics similar to some of the alterations produced by EV treatment. The most obvious similarities between the two experimental situations was the decrease in the number of healthy antral follicles and the development of precystic, type III follicles. These are large antral follicles containing four or five plicated layers of densely packed, but healthy, granulosa cells surrounding a large antrum (9, 53). Their thecal cell compartment does not appear hypertrophied. Because type III follicles contain a normal complement of LH receptors (53) and ovulate in response to LH, they may be the only follicles able to ovulate in EV-treated rats (53, 54). At the same time, however, they are considered as precystic structures, which, despite their size are unable to ovulate or luteinize in the presence of basal LH levels. Type III follicles appear to become developmentally arrested before turning into a cyst (53, 54).
Type III follicles are the first abnormal ovarian structures detected 30 days after EV injection, preceding the formation of follicular cysts (9, 53). The present results indicate that they also represent the most predominant structural follicular abnormality seen in ovaries grafted with NGF-producing cells. This finding and the elimination of type III follicles caused by the inhibition of NGF actions in EV-treated rats (9), suggest that an abnormally elevated intraovarian NGF tone contributes significantly to the genesis of these precystic structures.
The intraovarian grafting of NGF-secreting cells did not significantly increase the number of follicular cysts in the ovary. This outcome may have related to the small sample size, the relatively small (2-fold) increase in intraovarian NGF levels caused by the cells, and/or the variable and unpredictable distribution and survival of the grafted cells within the ovary. If the assumption is made that none of these factors is sufficiently important to explain the relative inability of NGF to induce cyst formation, the inevitable conclusion is that an excess of NGF alone cannot fully reproduce the ovarian abnormalities caused by EV. Intuitively, we would suspect that such is, indeed, the case. Nevertheless, it would also appear evident that the use of a transgenic approach to achieve elevated NGF levels affecting a greater fraction of the ovarian follicular population is required to provide a definitive answer to this question.
The decrease in the number of healthy antral follicles observed in ovaries grafted with NGF-producing cells is consistent with the observation that the reduction in the number of these follicles caused by EV-treatment is partially antagonized by intraovarian blockade of NGF actions (9). Noteworthy, NGF secreting cells did not reduce the number of atretic follicles as EV did, and surprisingly, caused an increase in the size of both healthy antral and atretic follicles. The most plausible explanation for these discrepancies between the two experimental paradigms is that NGF has an overall stimulatory effect on follicular development, and that this effect can only be fully manifested in the absence of those profound hormonal alterations resulting from the EV treatment. The delayed follicular development observed in rats subjected to neonatal immunoneutralization of NGF actions (8) supports this notion.
The increased size of antral follicles and the higher incidence of type III follicles observed in ovaries receiving NGF-producing cells may contribute to explaining the peculiar alterations in estrous cyclicity observed in the treated animals. A small, but persistent increase in estrogen levels by these enlarged follicles would account for both the interruption of cyclicity and the almost ever-present background of cornified cells detected in the vaginal lavages of rats carrying an intraovarian graft of NGF-producing cells. Likewise, the availability of both relatively large, healthy antral follicles and LH-responsive type III follicles may have allowed the occurrence of irregular, but still functional ovulatory cycles. The presence of a similar number of corpora lutea in the ovaries from control and NGF-cell-recipient animals indicate that, this was, indeed, the case. Because we did not attempt to estimate the morphological/functional differences among corpora luteal populations, we cannot be certain of their actual age-related composition.
Rats carrying NGF-producing cells showed significantly elevated serum androstenedione levels, suggesting that an excess of NGF may significantly contribute to the hyperandrogenemia of cystic ovarian disease. NGF may enhance ovarian androgen production via both direct and indirect mechanisms. On the one hand, it may directly stimulate androgen production from thecal cells; on the other, it may do so via its trophic effects on the ovarian noradrenergic innervation. Support for the first possibility comes from the finding that NGF is able to stimulate androstenedione secretion from purified bovine thecal cells (Dissen, G. A., J. A. Parrot, M. J. Skinner, D. F. Hill, S. R. Ojeda, in preparation). An indirect, complementary effect mediated by catecholamines is supported by the well known ability of norepinephrine to stimulate ovarian androgen secretion (55, 56).
An aspect of the present study that deserves comment is the behavior of the grafted cells. Although they produce and release NGF both in vitro [(57), this study], and after grafting into host tissues [(57, 58), this study], it has been difficult to identify them in the host tissue by standard immunohistochemical techniques [(57), this study]. This appears to be a common feature of NGF-producing neural cells as they do not accumulate significant amounts of the peptide (57). In a previous study using the same HiB5 neural progenitor cells employed in the present experiments, they were identified by prelabeling them with bromodeoxyuridine or 3H-thymidine (57). In our study, we took advantage of the finding that HiB5 cells, which contain p75 NGFR in culture, continue to express the receptor when grafted into the ovary, so they can be identified by immunohistochemistry using monoclonal antibodies to the receptor. Instead of remaining clustered near the site of injection, the cells dispersed throughout the ovary with some of them infiltrating the granulosa cells compartment of antral follicles. We do not know if this location resulted from the direct deposition of the cells inside some of these follicles at the time of injection.
Taken altogether the results demonstrate that a primary alteration in NGF availability to the peripubertal ovary results in morphological and functional abnormalities similar to some of those detected in cystic ovaries induced by EV treatment. The ability of chronically increased intraovarian levels of NGF to disrupt estrous cyclicity, induce formation of precystic follicular structures, and stimulate androgen production, suggests that an exaggerated intraovarian NGF tone may contribute to both the etiology and maintenance of cystic ovarian dysfunction in the rat. Whether these effects of NGF are due to a direct action on the ovary and/or an increase in the sympathetic outflow to the gland remains to be determined. An NGF contribution to human PCOS, and the potential relationship that may exist between this signaling system and those genes implicated in the syndrome by linkage analysis (19, 59) also need clarification.
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Acknowledgments |
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Footnotes |
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Received August 30, 1999.
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References |
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