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Division of Neuroscience, Oregon Regional Primate Research Center, Oregon Health Sciences University (G.A.D., D.F.H., M.E.C., S.R.O.), Beaverton, Oregon 97006-3448; and Center for Reproductive Biology, School of Molecular Bioscience, Washington State University (J.A.P., M.K.S.), Pullman, Washington 99164-4231
Address all correspondence and requests for reprints to: Dr. Gregory A. Dissen, Division Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006-3448. E-mail: disseng{at}ohsu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although NTs, and in particular NGF (9), are critical for development of the ovarian innervation, they also appear to exert direct actions on nonneuronal cells of the ovary as evidenced by the presence of trk tyrosine kinase receptors in these cells (6, 7). NGF is recognized by two different membrane-spanning receptor molecules, one displaying rapid dissociation kinetics, known as the low affinity NT receptor or p75NTR, and another with a slow dissociation rate, known as trkA (10, 11). Although p75NTR binds all other NTs, including BDNF, NT-3, and NT-4/5, with similar low affinity (4, 12), the trkA receptor binds NGF preferentially and with high affinity (13, 14).
The p75NTR potentiates the effects of trkA activation (12, 15), but also has an independent role in apoptosis, initiated by activation of a ceramide-dependent signaling pathway (16). TrkA receptors, on the other hand, are endowed with a tyrosine kinase domain similar to that of other receptor tyrosine kinases (17, 18) and mediate the biological effects of NGF via activation of signaling pathways similar to those activated by mitogenic receptor tyrosine kinases (19). Expression of trkA in the rat ovary increases dramatically in cells of the follicular wall during the preovulatory surge of gonadotropins (6). This increase is LH dependent and is accompanied by an increase in NGF messenger RNA (mRNA) levels. Immunological or pharmacological blockade of NGF action reduces the rate of ovulation (6), suggesting that activation of trkA receptors plays a role in this process. In the present study we considered the possibility that activation of trkA receptors in thecal compartment cells contributes to events known to occur during the hours antedating the first ovulation. Although trkA receptor gene expression can be induced by preovulatory levels of LH or by the cytokine interleukin-1ß (6), we chose not to use these agents to avoid potentially confounding effects unrelated to trkA activation. Experiments were, therefore, conducted in primary cultures of freshly plated purified thecal cells that contain endogenous trkA receptors or in thecal cells undergoing luteinization, transiently provided with the receptors via cationic lipid-mediated gene transfer.
Bovine thecal cells were chosen for these studies because they can be easily isolated in large quantities (20). In addition, the bovine ovary is monoovulatory and potentially similar to the human ovary in the mechanisms controlling follicular growth and differentiation. Because ovulation requires the intrafollicular action of progesterone (21) and the enhanced formation of PGE2 (22, 23), we examined the ability of NGF to stimulate the production of both progesterone and PGE2 from purified thecal cells. Because NGF binding to trkA receptors expressed in endocrine cells results in cell proliferation (24, 25, 26), and in view of the observations of thecal cell proliferation around the time of ovulation in the rat (27, 28, 29), studies were performed to determine whether NGF could affect the proliferative activity of cells cultured from the thecal compartment cells in culture. A partial report of these findings has appeared (30).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sprague Dawley rats (B&K Universal, Fremont, CA) were housed under controlled conditions of temperature (23-25 C) and light (14 h of light, 10 h of darkness; lights on from 0500–1900 h). They were provided ad libitum access to food (Purina laboratory chow, Ralston Purina Co., St. Louis, MO) and water. At 27 days of age (0900 h) the rats received a sc injection of either 8 IU PMSG in 0.2 ml saline or saline alone. Two days later hCG was administered sc to those rats that had been primed with PMSG, and the ovaries were collected 0, 4, 8, and 12 h later. Two hours before the scheduled time of tissue collection the rats were injected ip with 5-bromo-2'-deoxyuridine (BrdU; Roche, Indianapolis, IN; 50 µg/g BW) dissolved in saline solution-0.007 M NaOH.
Primary cell culture: freshly plated cells
The thecal compartment of bovine antral follicles was isolated
by microdissection. The theca externa was isolated from the theca
interna as previously described (20, 31). The theca
externa layer is generally isolated with the initial removal of theca
from the opened follicle. Adhering interstitial cells were scraped
away, and the theca interna was microdissected by pulling the intact
layer/shell of interna theca from the externa. This was done with two
small tweezers, which allowed the theca interna shell to stay generally
intact. The theca externa peeled away in pieces and was collected
separately. The cells were dispersed with 2 mg/ml collagenase
(Sigma, St. Louis, MO) in
Ca2+/Mg2+-free buffer as
previously described (31). The antral follicles were
classified as small (5 mm), medium (5–10 mm), or large (>10 mm)
before microdissection. Unless the size range of the follicles is
noted, the thecal cells were derived from a mixed-sized population of
antral follicles. Cell preparations obtained by this procedure have
negligible contamination from granulosa and stromal-interstitial cells
and show less than 3% contamination with endothelial cells
(31). Greater than 97% of the cells stain for
steroidogenic enzymes (Skinner, M. K., et al.,
unpublished observation). The thecal cells were immediately plated in
serum-free Ham’s F-12 medium (Sigma) containing 0.1% BSA
(Sigma) and were maintained at 37 C in a 5%
CO2 atmosphere. The cells were treated for
72 h with either NGF (25 ng/ml; Life Technologies, Inc., Grand Island, NY) or hCG (100 ng/ml; Sigma),
and androstenedione and progesterone levels were measured as previously
described (32). The sensitivities of the assays employed
are 0.01 ng/ml for both steroids. Data were normalized to the DNA
content per well as reported previously (32).
Established cultures
NGF release. Bovine thecal cells collected from a
mixed-sized population of antral follicles were initially plated in
T-75 flasks. Three days later the cells were subcultured into 12-well
plates at 4,000, 10,000, and 20,000 cells/well in Ham’s F-12 medium
containing 0.1% calf serum (HyClone Laboratories, Inc.,
Logan, UT). The cells were incubated for 24 h, after which the
medium was collected and assayed for NGF using the NGF Emax Immunoassay
System (Promega Corp., Madison, WI).
PGE2 and steroid release. Three-day-old primary cultures of bovine thecal cells were subcultured into 6-well plates at a density of 500,000 cells/well in Ham’s F-12 medium containing 10% calf serum. The cells were allowed to recover for 72 h before transfecting them with an expression vector containing the coding region of rat trkA (pJM5) under the control of the cytomegalovirus promoter (33) or the plasmid alone (pCMV) as a control. The transfection employed 2 µg/ml of each plasmid and 5 µg/ml Lipofectamine (Life Technologies, Inc.) as the transfecting reagent in 1.0 ml OPTIMEM (Life Technologies, Inc.) (34). After 5 h, the transfection mixture was removed, and Ham’s F-12 medium containing 1% calf serum was added to the wells for 24 h to allow expression of the trkA receptor. The medium was then changed to serum-free Ham’s F-12 supplemented with 5 µg/ml insulin (Sigma), and the cells were treated with NGF (100 ng/ml), hCG (1 µg/ml), or hCG plus NGF for 1, 2, or 8 h. Each well received 2.5 ml medium to allow repeated sampling (0.5 ml) at the time points indicated. PGE2, progesterone, and androstenedione released into the medium were measured as previously described (35, 36, 37).
Cell proliferation. Three-day-old cultures of bovine thecal cells were subcultured into 24-well tissue culture plates at a density of 10,000 cells/well. Transfections were performed 3 days later with either pJM5 or pCMV, as outlined above, then the medium was changed to Ham’s F-12 containing 0.1% calf serum. The following day, growth factors [NGF, BDNF, NT-3,NT-4 (gift from G. Yancopoulos, Regeneron Pharmaceuticals, Inc., Tarrytown, NY), EGF, bFGF (Collaborative Research, Bedford, MA)] were added (100 ng/ml) in serum-free DMEM (Sigma). After 22 h, [3H]thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL) was added (2.5 µCi/well, in 0.5 ml medium), and the plates were incubated for 4 additional h at 37 C. At this time the cells were dissociated in a 0.5-ml solution containing 10 mM Tris (pH 7.5), 5 mM EDTA, 20 mM NaCl, and 0.25% trypsin at 37 C for 15 min. The samples were then frozen at -85 C until mechanical homogenization. The incorporated [3H]thymidine was bound to DE-81 filters, and the filters washed extensively and then counted in 10 ml scintillation fluid as previously reported (38).
Cell line culture
The NIH-3T3 cell line (39, 40) and NIH-3T3 cells
stably transfected with a CMV-driven trkA construct (trkA-3T3),
resulting in constitutive expression of the trkA receptor
(41), were used in this study. The cells were maintained
in DMEM containing 10% FCS (HyClone Laboratories, Inc.).
To determine the effect of NGF on PGE2 release, the cells were plated at 100,000 cells/well in 24-well plates containing 0.5 ml DMEM lacking serum or supplemented with 0.1% FCS. As there was no difference in PGE2 release between these two conditions the results obtained were pooled. The PGE2 concentration in the medium was determined by RIA as previously described (35).
The ability of NGF to transregulate the PG endoperoxide synthase 2 (cyclooxygenase-II; COX-2) gene promoter (COX-2-P) (42) was examined in both native NIH-3T3 cells and trkA-3T3 cells. The cells were transfected with either the promoterless luciferase reporter vector pGL2-Basic (Promega Corp., Madison, WI) or pGL2-Basic containing 2.7 kb of the rat COX-2 gene 5'-flanking region (pCOX-2) (42). The cells were plated into 6-well plates at 350,000 cells/well in DMEM containing 10% FCS. The following day, they were transfected with the reporter pCOX-2 plasmid (at 100 ng/ml) or the vector alone (at 500 ng/ml), using Lipofectamine at 2.5 µl/µg DNA in 1 ml OPTIMEM. In the wells receiving pCOX-2, the total amount of DNA was maintained at 500 ng/ml by adding 400 ng vector alone. All wells also received 20 ng/ml of pCMV·SPORT-ßgal (Life Technologies, Inc.) to correct for transfection efficiency. After 5 h of transfection, the DNA mixture was replaced by DMEM containing 1% FCS. The following day growth factors (NGF, BDNF, and NT-3) were added at a concentration of 100 ng/ml. At different intervals thereafter (1, 2, and 24 h), the wells were rinsed with PBS, and the cells were removed into 160 µl cell lysis buffer for luciferase and ß-galactosidase assays (43). The luciferase values were normalized using the corresponding ß-galactosidase values.
Nucleic acid probes and complementary DNAs (cDNAs)
The antisense RNA probes used in these studies were
complementary to the bovine mRNAs encoding NGF, trkA,
p75NTR, and cyclophilin. Cyclophilin mRNA, which
is constitutively expressed in both the rat brain (44) and
ovary (6, 7), was used as an internal marker to normalize
the results of the ribonuclease (RNase) protection assay. Because no
bovine cyclophilin nucleotide sequence has been reported, rat primers
were used (see Table 1
) to produce a
350-bp bovine cDNA, henceforth referred to as cyclophilin 350. The
deduced amino acid sequence of this cDNA is identical to the first 93
amino acids of the known bovine cyclophilin protein (45).
Bovine-specific internal primers were then designed based on this
sequence (Table 1) and used in PCR to amplify a 107-bp cDNA template.
An NGF cDNA fragment was PCR amplified from a bovine NGF cDNA template
(provided by R. Heumann, Ruhr Universität, Bochum, Germany). To
obtain bovine trkA, p75NTR, and COX-2 cDNAs, the
cDNAs of two different species were aligned (Table 1), and conserved
sequences were selected to design amplifying primers (Table 1). Where
only one nonbovine species is listed in Table 1, these primers had been
synthesized for other purposes, but there was enough cross-species
similarity for the amplification of the bovine sequence. The sizes of
the bovine cDNAs generated by PCR are presented in Table 2, along with their accession numbers and
the percent similarity to the human and rat sequences.
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The PCR reactions were started with an initial denaturation at 95
C for 5 min (p75NTR and COX-2) or 7 min (all
others) of the template (either 2 µl RT reactions or 5–10 ng plasmid
NGF or cyclophilin 350 cDNAs) in the presence of 1 x
Taq buffer, 1.25 mM
MgCl2 (Promega Corp., Madison, WI),
and deoxynucleotides [200 µM each of deoxy
(d)-ATP, dCTP, dGTP, and dTTP; Amersham Pharmacia Biotech,
Inc., Piscataway, NJ]. After the initial denaturation step, the
primers (see Table 1
for sequence and amount) and Taq
polymerase (2.5 U; Promega Corp.) were added to the
reaction tube in a final reaction volume of 100 µl. There were three
PCR protocols used: 1) NGF and cyclophilin 107 (94 C for 4 min,
followed by 35 cycles of 94 C for 15 sec, 55 C for 1 min, and 72 C for
2 min), 2) trkA and cyclophilin 350 (95 C for 2 min, 55 C for 3 min,
and 72 C for 10 min; followed by 35 cycles of 92 C for 1 min, 55 C for
2 min, and 72 C for 3 min), and 3) p75NTR and
COX-2 (95 C for 45 sec, 57 C for 10 min, and 72 C-10 min, followed by
35 cycles of 95 C for 45 sec, 57 C for 2 min, and 72 C for 2 min; the
annealing temperature for COX-2 was 55 C). The PCR products were cloned
into the pGEM-T vector (Promega Corp.) and sequenced
(trkA, p75NTR, COX-2, and cyclophilin). In the
case of NGF three internal restriction enzyme sites were used to
confirm the identity of the cDNA.
As in rats and humans, bovine trkA appears to occur in two forms, either with or without an 18-bp insert in the extracellular domain (47). The neuronal isoform, which includes the insert (isoform II) (47), has been shown to exhibit greater responsiveness to NT-3 (48). The neuronal form of the receptor was cloned from thecal cells and used in these experiments. The complementary RNA (cRNA) transcripts generated from these templates were radiolabeled with [32P]UTP for RNase protection assay and with [35S]UTP for hybridization histochemistry (trkA and NGF). Preparation of templates for transcription and the transcription procedure itself were performed as previously reported (49, 50).
RNase protection assay (RPA)
Total RNA for RPA was prepared by the phenol-extraction method
for tissues (51) or cells (52). The RPA was
carried out according to the method of Gilman et al.
(53), as previously described (54). Sense RNA
standards were prepared using the templates described above. The sense
RNAs were transcribed, purified, and quantified according to published
procedures (54). The 32P-labeled
cRNAs of interest were simultaneously hybridized to total RNA extracted
from ovaries or cells. After RNase digestion, the protected species
were isolated by PAGE, visualized, and analyzed as previously reported
(54).
Hybridization histochemistry
The procedure employed was based on the method of Simmons
et al. (55) with modifications as previously
reported (56). Cellular expression of trkA and NGF mRNAs
was determined in ovaries collected at the abattoir and immediately
fixed by immersion in 4% paraformaldehyde-0.1 M
sodium borate buffer, pH 9.5 (overnight at 4 C). The ovaries were
embedded/frozen in OCT compound (Miles, Inc., Elkhart, IN). The
hybridization was performed on 10-µm cryostat sections
(56). Control sections were incubated with sense trkA or
NGF RNA probes.
Immunohistochemistry
TrkA and NGF. Immunohistochemical detection of trkA and NGF
was performed in 14-µm cryostat sections from ovaries collected and
fixed at the abattoir. The ovaries were fixed by immersion in
Zamboni’s fixative and embedded/frozen in OCT compound as previously
described (7) and processed for trkA and NGF
immunohistochemistry using the polyclonal antisera trk 763 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and K-596 (6, 9) to identify trkA and NGF, respectively. According to the
manufacturer, antibody trk 763 specifically recognizes trkA, without
cross-reacting with other trk receptors. We have previously shown that
antibody K-596 preferentially recognizes NGF (6). Tissue
sections were incubated overnight at 4 C with the antibodies at 2
µg/ml (trk 763) or at a 1:500 dilution (K-596), and the
immunoreaction was developed the next day with diaminobenzidine
(Sigma) as previously described (6).
BrdU
Rat ovaries were immersion-fixed in Carnoy’s fixative
(57) for 15–18 h, transferred to 70% ethanol, embedded
in paraffin, and serially sectioned at 4 µm. Paraffin was removed
from the sections by immersion in xylene, followed by rehydration in a
graded series of ethanol before a 30-min treatment at 37 C with 2
N HCl to increase the accessibility of the DNA to the
antibody (58). The sections were then incubated with a
monoclonal antibody to BrdU (diluted at 1:1000; Sigma)
overnight at 4 C, and the immunoreaction was developed the next day as
outlined above. The sections were lightly counterstained with Gill’s
hematoxylin.
Data analysis
The differences in mRNA levels, PGE2
release, and luciferase activity (relative light units) were analyzed
using one-way ANOVA and Student-Newman-Keuls multiple test for
individual means. Percentages were first subjected to the arcsine
transformation before applying a one-way ANOVA followed by the
Student-Newman-Keuls multiple test for individual means or the least
significant difference test for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, left panel). As expected,
the cerebral cortex showed no trkA mRNA, low levels of
p75NTR mRNA, and a NGF mRNA content similar to
that present in the basal forebrain.
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, right panel).
Thecal cells from small, medium, and large follicles showed
considerable variation in their content of trkA mRNA, whereas NGF and
p75NTR mRNAs were more uniformly expressed.
The neuronal form (form II) was detected as a 541-nucleotide band, and
the nonneuronal form (form I) as a 389-nucleotide fragment (Figs. 1 and 2). Surprisingly, not only thecal cells
but also granulosa cells contained trkA mRNA, which, as in thecal
cells, appeared to be predominantly expressed as the neuronal form
(Fig. 2). In contrast to trkA, NGF and p75NTR
were selectively expressed in thecal cells (Fig. 2). No apparent
differences in trkA, NGF, and p75NTR mRNA
expression related to either follicle size or the ovarian compartment
in which the mRNAs were detected (Figs. 1 and 2). Likewise, the three
mRNAs were equally expressed in the theca interna and externa of small,
medium, and large follicles (Fig. 2).
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, arrowheads) and the
presence of trkA mRNA in both thecal (arrowheads) and
granulosa cells (arrows; Fig. 3C). The expression of both
mRNAs did not appear to be limited to a subpopulation of thecal cells,
but instead it was diffusely present throughout the thecal compartment.
Adjacent sections incubated with the respective sense RNA probes showed
no specific hybridization (Fig. 3, B and D, respectively). In contrast
to the exclusive localization of NGF mRNA to thecal cells,
immunohistochemical detection of the NGF protein showed its presence in
both granulosa (arrows) and thecal cell
(arrowheads) compartments of antral follicles (Fig. 4A). Preabsorption of the antibody with
purified mouse NGF decreased, but did not eliminate, the
immunoreactivity (Fig. 4B). In agreement with the in situ
hybridization results, trkA immunoreactive material was detected in
both granulosa (arrows) and thecal cells
(arrowheads, Fig. 4C). Preabsorbing the trkA antibody with
the peptide used to generate it resulted in a substantial reduction in
immunoreactive material in both follicular compartments (Fig. 4D).
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). Although the level of
p75NTR mRNA declined gradually after plating, it
was clearly detectable even after 18 days of culture. In contrast, NGF
mRNA remained at high levels throughout the duration of the culture (18
days; Fig. 5). The ability of thecal cells to make and
release NGF was demonstrated by its presence in the culture medium, as
measured by enzyme-linked immunosorbent assays. As shown in Fig. 6, the amount of NGF released to the
medium was directly proportional to the number of cells plated per
well.
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). The levels
remained elevated at 6 h, but returned to basal values by 24
h. In contrast to this increase, no up-regulation of NGF mRNA levels
was observed in the same cultures (Fig. 7). Likewise, hCG did not
influence the expression of p75NTR mRNA (not
shown).
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, A and C).
In contrast, cells plated at low density did not respond to NGF
treatment with steroid release (Fig. 8, B and D). Both high and low
density cultures responded equally well to hCG (Fig. 8). Thecal cells
treated with NGF or hCG for 8 and 24 h 6 days after plating failed
to release either progesterone or androgens in response to either
challenge (not shown).
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, left
panel). In contrast, the presence of the trkA receptor
resulted in a rapid increase in PGE2 release
after 1 h of treatment (Fig. 9A, right panel). The
effect was still evident by 2 h, but at 8 h basal
PGE2 release had increased to levels that were
not further modified by NGF treatment. Control cells responded to hCG
treatment with PGE2 release only after 8 h
of treatment, and NGF did not potentiate the hCG effect (Fig. 9B, left panel). In contrast, trkA-expressing cells responded to
the gonadotropin within 1 h. PG levels remained elevated for the
duration of the treatment (8 h; Fig. 9B, right panel). NGF
added in conjunction with hCG potentiated the early effect of hCG on
PGE2 release, i.e. after 1 h of
exposure, but not the effect at 2 and 8 h (Fig. 9B, right
panel).
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) demonstrated that the mRNA is present in thecal
cells as well as granulosa cells. Although the COX-2 mRNA detected in
thecal cells could be due to contamination with granulosa cells, the
presence of COX-2 mRNA in bovine thecal cells has been previously
reported by others (61).
To further characterize the role of trkA in NGF-induced
PGE2 release, NIH-3T3 cells ectopically
expressing trkA receptors (trkA-3T3) (41) were used. Like
thecal cells expressing trkA, trkA-3T3 cells responded to NGF with an
increase in PGE2 release within 1 h of
exposure (Fig. 10). To determine
whether this acute effect was accompanied by an NGF-dependent increase
in transcriptional activity of the COX-2 gene, native NIH-3T3 cells and
trkA-3T3 cells were transiently transfected with a luciferase reporter
construct driven by the rat COX-2 promoter (COX-2-P) or the
promoterless reporter plasmid alone (pGL2). Twenty-four hours later the
cells were exposed to NGF for a short (1 and 2 h) or a long (24 h)
period of time. As shown in Fig. 10, B and C, the COX-2-P was very
active in 3T3 cells, inducing more than a 10-fold increase in
luciferase activity 24 h after transfection and a 25-fold increase
at 48 h. The short-term exposure to NGF did not alter COX-2-P
activity. In contrast, after the 24-h exposure, basal COX-2-P activity
was significantly augmented by NGF. The increase in basal COX-2-P
activity may be due to endogenous NGF, which has been shown to be
produced by these cells (12). This effect was specific, as
it was obliterated by treating the cells with a neutralizing NGF
antiserum (Fig. 10C). The stimulatory effect of NGF on COX-2-P activity
was not seen in native NIH-3T3 cells (Fig. 10D) and was not reproduced
by either NT-3 or BDNF (Fig. 10D) in trkA-3T3 cells (Fig. 10D).
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, A and
B, arrowheads). Treatment with hCG inhibited this activity
for at least 8 h (Fig. 11, C and D). In contrast, a subset of
thecal compartment cells, which exhibited modest proliferative activity
in untreated rats (Fig. 11, A and B), become proliferative 10–12 h
after hCG administration, near the time of ovulation (Fig. 11, E and F,
arrows).
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, inset).
These cells responded to NGF or NT-3 with an increased rate of
[3H]thymidine incorporation (Fig. 12A). 2)
Cells in which transfection with the trkA expression vector (pJM5)
increased their basal rate of proliferation in the absence of added NGF
(Fig. 12B, both panels). These cells did not respond to either NGF or
NT-3 with a further increase in proliferation. Both sets of cells
responded equally well to EGF stimulation (Fig. 12A, left
and right panels), but failed to proliferate in response to
NT-4 or BDNF. As before, native NIH-3T3 cells did not respond to any NT
with proliferation (Fig. 12B). Both cell types responded equally well
to the mitogenic peptide bFGF (Fig. 12B, left panel).
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Discussion |
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A potential involvement of trkA receptors in ovulation was initially suggested by studies performed in rats, which demonstrated that trkA receptors are localized to the thecal-interstitial compartment of periovulatory follicles and that their expression increases more than 100-fold during the hours preceding the first ovulation (6). The ability of hCG to rapidly increase trkA receptor expression in ovarian cell dispersates (6) indicated that a significant fraction of the preovulatory increase in trkA mRNA abundance is a LH-dependent phenomenon. Activation of ovarian trkA receptors appears to contribute to the cascade of events leading to ovulatory rupture, as pharmacological blockade of trkA signaling or immunoneutralization of NGF actions reduced the incidence of ovulation in response to gonadotropins (6).
By showing that NGF can act directly on purified thecal cells containing trkA receptors to activate signaling pathways important for ovulation, the present results offer additional evidence in support of a facilitatory role for NGF in the ovulatory process. They are also in harmony with the view that the ovarian thecal compartment represents a physiological, nonneuronal target for NGF action (6, 34, 63) and emphasize the emerging concept that NTs exert pleiotropic actions throughout the organism, and that, within the endocrine system, they target discrete subpopulations of endocrine cells (5).
In keeping with previous observations made in rat ovaries (6), bovine thecal cells were found to contain both trkA mRNA and trkA immunoreactive material. Separation of the thecal compartment into theca interna and externa revealed that both subcompartments of the follicular wall contain the receptors; RNase protection assays, complemented by in situ hybridization experiments, revealed the presence of trkA receptors in antral follicles of all sizes collected. Although this may indicate the existence of a fundamental difference between bovine and rat ovaries, in which trkA mRNA becomes abundant only during the hours preceding ovulation (6), we were not able to analyze bovine periovulatory dominant follicles in which such an increase would have been apparent. Thus, antral bovine follicles may simply express higher trkA receptor levels during development than rat follicles and still show a substantial increase at some point during the 22–30 h window elapsing between the LH surge and ovulatory rupture in this species (60, 61, 64). A surprising finding was the detection of trkA mRNA and immunoreactive trkA protein in granulosa cells of bovine follicles, and the presence of the receptors in the granulosa cell compartment of all size follicles examined. This localization, verified by RPA, in situ hybridization, and immunohistochemistry, is in contrast to the highly localized expression of trkA in the thecal-interstitial compartment of the rat ovary (6) and the apparent absence of trkA mRNA expression in granulosa cells of the rhesus monkey ovary, assessed by the sensitive method of RT-PCR (Dissen, G. A., C. L. Chaffin, and S. R. Ojeda, unpublished data). We do not know what the functions of NGF in bovine granulosa cells might be, but it is possible that they are particular to this species. NGF acting via trkA receptors in bovine granulosa cells may affect functions not specifically related to ovulation and/or required for ovulation to occur. Although it could be argued that trkA in granulosa cells may be relevant to the fact that cows are a monoovulatory species, nonhuman primates are also monoovulatory, and yet their ovarian granulosa cells do not contain the receptor. Alternatively, trkA may play a role in processes such as those underlying the regulation of granulosa cell differentiation by thecal cells in this species (65) and/or the maintenance of basal levels of PG synthesis within antral follicles (60). Further studies are required to resolve this issue, which is made more intriguing by the unexpected finding that the predominant trkA form expressed in bovine granulosa and thecal cells is the neuronal isoform, which has been shown in neuronal systems to confer the receptor responsiveness to NT-3 (47). The presence of this form in the bovine ovary is in contrast to the predominant expression of the nonneuronal trkA isoform in rat ovaries (6, 63, 66).
Regarding NGF itself, our findings show the presence of both NGF mRNA
and NGF immunoreactive protein in most cells of the thecal compartment.
We were not able to detect a high content of either TrkA or NGF in the
theca interna, which would have suggested the preferential expression
of this NT signaling complex in thecal steroidogenic cells. This
localization, similar to that described in the rat ovary
(6), suggests that NGF acts on thecal cells via
paracrine/autocrine mechanisms to affect cellular functions. Although
granulosa cells showed an abundance of NGF-like immunoreactivity, no
NGF mRNA was detected in these cells, indicating that the
immunoreactive material is either NGF bound to its trkA receptor or a
cross-reacting protein. We favor the latter possibility, as it would be
unusual for a ligand to remain so tightly bound to its high affinity
receptor throughout the process to which the tissue is subjected before
imunohistochemical analysis. The polyclonal antibodies to NGF were
generated using a purified preparation of mouse 2.5S NGFß extracted
from mouse submaxillary glands, which was shown to be devoid of renin
contamination (9). It is possible, however, that this
preparation may have contained traces of NGF
, a lysine-specific
serine proteinase of the kallikrein family that reversibly associates
with NGF (67). Submaxillary glands secrete a noncovalent
multimeric complex composed of three polypeptides, NGF
, the NT
NGFß, and NGF (68). Whereas the functions of NGF
remain to be elucidated, NGFß is the NT shown to support the survival
of neurons in the central and peripheral nervous system. NGF, on the
other hand, has been implicated in the processing of NGFß precursors
(69, 70) and, more recently, in the cleavage of
urokinase-type plasminogen activator (71); other functions
have not been characterized. In considering that granulosa cells are a
rich source of proteinases (72), it is conceivable that
the NGF-like immunoreactivity detected in bovine granulosa cells
represents cross-reaction of the antibodies with an NGF-related
proteinase, probably of the kallikrein family, that may be produced in
granulosa cells (73). Obviously, the epitopes recognized
by our antiserum in such a proteinase would necessarily have to be
different from those present in proteases synthesized by rat granulosa
cells, because in no instance have we detected NGF-like material in
these cells (6).
Previous studies demonstrated the ability of gonadotropins and growth
factors to affect the steroidogenic output of isolated bovine thecal
cells, using the same culture system we employed in the present
experiments (20, 74). An important conclusion of these
studies was that the response of thecal cells to either gonadotropins
or growth factors is determined by the cytodifferentiation stage of the
cells in vitro. Thus, androgen production was elevated
during the first 3 days in culture and increased readily in response to
hCG stimulation; androgens declined thereafter, and the cells became
unresponsive to the gonadotropin (20). In contrast,
progesterone secretion increased with time in culture and in response
to hCG, at a time when the androgen response had been lost. A similar
picture emerged from studies examining the effect of transforming
growth factor- (TGF) and TGFß on thecal cell steroidogenesis.
TGF suppressed androgen and progesterone secretion during the first
3 days in culture, but had no effect thereafter (74).
Conversely, TGFß stimulated progesterone secretion during the earlier
days in culture, but also became ineffective at later times, as basal
progesterone secretion increased (74). These and other
observations led to the suggestion that thecal cells in culture undergo
a differentiation (luteinization) process that profoundly affects their
steroidogenic response to both gonadotropins and intraovarian growth
factor regulators (74). The present observations are
consistent with this interpretation, as they show the ability of NGF to
stimulate androstenedione and progesterone secretion during the first
72 h after seeding, but not after a week in culture. Because trkA
receptor expression declines precipitously 1 day after plating, the
ineffectiveness of NGF to stimulate steroidogenesis in established
cultures may be attributed to the rapid in vitro loss of
functional high affinity receptors. This explanation is not, however,
supported by the inability of NGF to affect steroidogenesis in thecal
cells transfected with a trkA-encoding plasmid, a procedure that
allowed the cells to respond to the NT with PG release and
proliferation. It would then appear that NGF may facilitate thecal cell
steroidogenesis, and thereby the biochemical differentiation of these
cells, only before their in vitro steroidogenic capability
becomes further differentiated. As shown in other cell systems
(12, 41), the differentiating actions of NGF on thecal
cells appear to be mutually exclusive with its proliferative effects.
Thus, when applied to freshly seeded cells, NGF stimulated
steroidogenesis in high density, presumably nonproliferating cultures,
but not from low density, rapidly proliferating cells. Conversely, when
administered to established 1-week-old cultures, it induced
proliferation, but failed to affect steroidogenesis. Taken into an
in vivo context, these findings suggests that NGF may
contribute to increased thecal steroidogenic output during the early
phases of the preovulatory period, particularly because at this time
the ovulatory increase in LH secretion acts to simultaneously inhibit
ovarian cell proliferation and stimulate steroidogenesis
(75).
The results of experiments in which proliferation of follicular cells was examined in rats sequentially treated with PMSG and hCG confirmed the reported ability of hCG to inhibit cell proliferation (75) and suggested that this effect may occur very rapidly (within 4 h) after its administration. They also provided supportive evidence to the earlier observations that shortly before ovulatory rupture thecal fibroblasts of the follicular wall increase their proliferative activity (76, 77). Although we did not perform a detailed morphological analysis or a morphometric quantitation of these changes, BrdU-positive cells were consistently observed in the follicular wall 10–12 h after hCG. Interestingly, their spatial pattern of distribution indicated that this preovulatory increase in thecal cell proliferation is not limited to the apex of the follicular wall (76). That at least part of this enhanced proliferative activity may be due to an NGF-initiated, trkA-mediated signaling process was suggested by the ability of NGF to stimulate proliferation in isolated bovine thecal cells containing trkA receptors, but not from those lacking them. Unexpectedly, thecal compartment cells transfected with the trkA-encoding plasmid behaved in two different ways; in some experiments, basal proliferation remained at control values 48 h after transfection. These cells responded to NGF with a significant increase in proliferative activity, as defined by an increase in [3H]thymidine incorporation. In other experiments, there was an increase in [3H]thymidine incorporation after transfection, in the absence of NGF treatment; these cells did not respond to NGF with a further increase in proliferation. Because thecal compartment cells produce NGF, we interpret these findings as indicative of differences in responsiveness to endogenous NGF (which may be determined by the relative composition on small, medium, and large follicles in each preparation tested).
A stimulatory effect of NGF on thecal cell proliferation is in keeping with the early observation that ectopic expression of trkA receptors in fibroblastic cells did not result in differentiation or enhanced cell survival, but led to a proliferative response instead (12, 78). In fact, it is now well documented that NGF acts on nonneuronal and, in particular, endocrine cells to induce proliferation. Such an action has been observed in a variety of cellular systems, including normal undifferentiated hemopoietic cells (79), normal keratinocytes (80), thyroid (24) and pancreatic (25) cell lines, adrenal chromaffin cells (81), and more recently in a prostate adenocarcinoma cell line (26). The ability of NGF to induce proliferation in thecal cells expressing trkA receptors, the activation of thecal cell proliferation that occurs near the time of ovulation (Refs. 76 and 77 and present study) and the striking increase in trkA receptor expression that takes place during the hours preceding the ovulatory rupture (6) suggest that activation of trkA receptors plays a significant role in inducing the proliferative activity of thecal compartment cells at the time of ovulation.
At least in rodents, an increase in intraovarian synthesis of
PGE2 is required for ovulation to occur
(82). Mice carrying a null mutation of the COX-2 gene,
which encodes the rate-limiting enzyme in PG synthesis, fail to ovulate
in response to gonadotropins (23). The demonstration that
the response is restored by the administration of
PGE2 (83) makes it evident that
PGE2 is the critical PG implicated in the
ovulatory process in this species. Furthermore, mice lacking the
PGE2 receptor EP2 exhibited
a decrease in ovulation rate (84), whereas deletion of the
PGF2 receptor did not interfere with ovulation
(85). Whether PGE2 or
PGF2 is the most important PG involved in the
ovulatory process of the ruminant ovary (86) remains to be
established. Our results show that NGF induces
PGE2 release from isolated bovine thecal cells
containing trkA receptors, but not from cells lacking the receptors.
The effect is rapid, as it is already evident after 1 h of
exposure to the NT, and is capable of amplifying the early increase in
PGE2 release elicited by hCG. As this
amplification was no longer apparent after 2 h of simultaneous
exposure to both secretagogues, it would appear that the initial
amplification may reflect an NGF-dependent priming effect on the
signaling pathway used by LH, preceding the productive coupling of LH
receptors to this pathway.
The early stimulation of PGE2 release by NGF was not accompanied by an increase in transcriptional activity of the COX-2 gene promoter, examined in 3T3 fibroblasts transfected with a trkA-encoding plasmid. Instead, the promoter activity was increased after 24 h of exposure to the NT (the earlier time assessed after the initial 2 h of exposure). That this increase is NGF dependent and NGF specific was demonstrated by the effectiveness of neutralizing antibodies to NGF to obliterate the increase and the ineffectiveness of related NTs such as NT-3 and BDNF to reproduce the effect of NGF. In all, the dynamics of the changes induced by NGF on PGE2 release and COX-2 promoter activity in the present study are entirely consistent with that reported for the NT in neuronal cells. Exposure of PC-12 cells to NGF results in rapid (<1 h) stimulation of arachidonic acid metabolism (87), but causes a delayed activation of the gene encoding the constitutive cyclooxygenase isoform, COX-1 (88). It is plausible that this delayed genomic response represents a replenishment mechanism used by different cells to maintain an adequate level of COX proteins ready to be used upon ligand-induced stimulation of their enzymatic activity.
Inhibition of ovarian trkA tyrosine kinase-dependent signaling as well as immunoneutralization of ovarian NGF action have been shown to inhibit ovulation in rats (6). The present findings suggest that the actions of NGF in thecal cells at the time of ovulation involve at least three different components thought to contribute to the ovulatory process: stimulation of progesterone secretion, stimulation of PGE2 production, and cell proliferation before ovulatory rupture. Although the exact contribution of trkA activation to the ovulatory cascade remains to be elucidated, it is important to note that in addition to PGE2, several molecules recently shown to be required for ovulation were originally described as signaling molecules regulated by NGF, via activation of trkA receptors. Prominent among them are C/EBP-ß and NGF-IA, two members of the basic leucine zipper family of transcriptional regulators. Both of them were identified in neuronal cells as direct downstream targets of the NGF high affinity receptor (89, 90) and shown to be present in preovulatory follicles, where they play an essential role in the ovulatory process (91, 92, 93, 94).
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Footnotes |
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Received March 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TGF) and its receptor in the hypothalamus of female rhesus
macaques. Neuroendocrinology 60:346–359[Medline]
receptor in bovine preovulatory follicles. Endocrinology 137:3348–3355[Abstract]
activates soluble and receptor-bound single
chain urokinase-type plasminogen activator. J Biol Chem 268:16327–16331 and -ß differentially regulate growth and steroidogenesis
of bovine thecal cells during antral follicle development.
Endocrinology 129:2041–2048 (C/EBP) in the rat
ovary: implications for follicular development and ovulation. Dev Biol 179:288–296[CrossRef][Medline]
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