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Reproductive Biology Unit, Departments of Obstetrics and Gynecology and Physiology (M.L, E.G.K., J.A.C., B.K.T.) and Department of Anatomy and Neurobiology (J.N.F.), University of Ottawa and the Ottawa Civic Hospital Loeb Research Institute (M.L., E.G.K., B.K.T.), Ottawa, Ontario, Canada KIY 4E9; and Departments of Medicine and Physiology (R.X., S.A.R.), McGill University, Montreal, Quebec, Canada and Centre for Food and Animal Research and Agriculture and Agri-food Canada (J.A.C.), Ontario, Canada KIA 0C6
Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Reproductive Biology Unit, Department of Obstetrics and Gynaecology, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada, K1Y 4E9.
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Introduction |
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2-antiplasmin
(6, 9). A urokinase receptor (uPAR), which binds specifically to uPA
and localizes the enzymatic activity to the vicinity of the cell,
has also been identified (10, 11, 12, 13). The two PA types have
different protein structures, tissue-specific expression and biological
activities and play distinct roles in different biological processes
(3). Whereas tPA has specific affinity for fibrin and produces
clot-restricted plasminogen activation, uPA lacks affinity for fibrin
and requires conversion from an inactive, single-chain precursor to a
catalytically active, two-chain enzyme (3). Complementary DNAs (cDNAs) encoding uPAR have been isolated and characterized and can encode a highly glycosylated receptor protein linked to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor (14, 15). Cleavage of this GPI anchor can lead to release of a soluble uPAR protein frequently present in biological fluids (16). The receptor binding region of uPA and pro-uPA is located at the amino-terminal fragment and shows sequence homology to epidermal growth factor (17). Binding of pro-uPA to cellular uPAR increases the rate of activation of the pro-enzyme both in vivo and in vitro by at least 20-fold (18, 19). Whereas uPA and pro-uPA alone are not internalized by the receptor, uPAR-uPA-inhibitor complexes are selectively internalized and degraded (20). It has been suggested that uPA-uPAR interactions stimulate cellular mitotic activity (21, 22, 23) and several growth factors modulate uPAR number and cell invasiveness in malignant tissues (24, 25, 26, 27). These findings indicate that cellular uPAR expression and regulation play a crucial role in localizing and promoting uPA action. Appropriate regulation of this multi-component system provides an adequate machinery for the controlled and targeted extracellular proteolytic activity that characterizes many biological and pathological processes (1, 2, 3, 5, 7, 27)
Ovarian follicular development is a consequence of follicle cell
proliferation and differentiation, oocyte maturation, and follicular
fluid accumulation. The expansion of the follicle requires tightly
controlled extracellular matrix synthesis and degradation. Plasmin has
been shown to decrease the tensile strength of the follicular wall (28)
and injection of tPA antibody and 2-antiplasmin into the
ovarian bursa suppresses the ovulatory rate (29). Both PA types have
been identified in the ovary but only tPA activity has been shown to be
hormonally regulated and to increase significantly during the ovulatory
period (30, 31). Although these findings clearly suggest that tPA is
involved in the ovulatory process, the expression of the various
components of the PA system during the early stages of follicular
development have not been examined. In addition, while we and others
(5, 7, 30, 31, 32, 33, 34) have proposed key roles for PAs and PAI in follicular
wall remodeling during follicular maturation, the presence and
regulation of uPAR in the ovary during follicular growth is unknown.
The purpose of the present study was to examine the follicular
localization of uPA and uPAR and the possible developmental changes in
their expression in relation to those of tPA and PAI during ovarian
follicular maturation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[32P]dCTP (250 µCi/µg) were obtained from Amersham
Life Science (Oakville, ON, Canada). Formamide was purchased from
Clonetech Laboratories Inc. (Palo Alto, CA). Hydroxyethylpiperazine
ethanesulfonic acid (HEPES), bromophenol blue, trizma base, SDS, xylene
and Zeta-probe blotting membrane were obtained from BDH (Toronto, ON,
Canada). Rat uPA and uPAR, rabbit antimouse uPA, antirat uPAR, and
antirat PAI-1 as well as mouse antihuman tPA were purchased from
American Diagnostica Inc. (Greenwich, CT). Isopropanol and
phenol:chloroform:isoamyl were obtained from Promega Corporation
(Madison, WI). Complementary DNA probes for tPA and PAI-1 were gifts
from Dr. X. Zhang (Department of Obstetrics and Gynecology, University
of Calgary, Calgary, AL, Canada) and Dr. T. D. Gelehrter (Department
of Human Genetics, University of Michigan, Ann Arbor, MI),
respectively.
Animal preparation
Immature female Sprague-Dawley rats (50–60 g) were obtained
from Charles River Canada (Montreal, PQ, Canada) and fed prolab RMH
4018 (AGWAY Inc., C.G., Syracuse, NY) and water ad libitum.
A 14-h light, 10-h dark cycle was maintained with the light cycle
initiated at 0600 h. A group of rats (22 days old) were injected
sc daily for 3 days with 1 mg DES per day (in 0.1 ml sesame oil). They
were killed by cervical dislocation 24 h following the last
injection of DES. A second group of animals (23 days old) were injected
ip with 4 IU eCG and were killed 48 h later. A third group of rats
were injected ip with 4 IU eCG and 48 h later with 15 IU hCG.
These animals were killed 8 h post-hCG administration. The above
treatments synchronized ovarian follicular development at the
preantral/small antral, medium antral, and preovulatory stages,
respectively (35, 36).
Total RNA extraction and Northern blot analysis
Ovaries with follicles at different stages of development
were collected separately in ice-cold M199 supplemented with HEPES (25
mM; pH 7.4). Granulosa cells and residual ovarian tissues
were isolated as previously described (35, 37) but with minimal
modification. Briefly, granulosa cells were harvested in M199 by
follicle puncture with a 27-gauge hypodermic needle and centrifuged at
900 x g for 5 min. The supernatant was discarded and
the pellet immediately frozen on dry ice and stored at -80 C. The
residual ovaries (primarily theca-interstitial cells) were thoroughly
washed with M199 to release undissociated granulosa cells, transferred
into clean tubes, frozen on dry ice, and stored at -80 C. Total RNA
was isolated by homogenizing and solubilizing follicular cells in GTC
as previously described (38). The homogenate was then extracted with
acidified phenol and precipitated with isopropanol. The RNA pellet was
dissolved in DEPC-treated water, quantified spectrophotometrically at
260 nm, aliquoted, and stored at -80 C. The RNA samples (10–15 µg)
were size-fractioned by electrophoresis on formaldehyde-agarose gels
(1.1%) containing 1 µg/ml ethidium bromide to confirm even loading
of RNA samples and adequate separation of 28S and 18S ribosomal bands.
The RNAs bands were blotted onto a nylon membrane by capillary action
and cross-linked by UV light. Urokinase-type PA, uPAR, tPA, and PAI-1
probes were labeled using the Random Primed [-32P
dCTP]DNA Labeling Kit (Boehringer Manmhein, Germany). Membranes were
prehybridized in 50% formamide, saline sodium citrate (SSC; 750
mM NaCl, 75 mM Na citrate), 1x Denhardt’s
solution, 1% SDS, 4 mM EDTA and 100 µg/ml sheared salmon
sperm DNA for 4 h at 42 C. Hybridization was performed overnight
at 42 C with 20 million cpm of labeled probes added to the
prehybridization buffer. The membranes were then washed twice with SSC
(300 mM NaCl, 30 mM Na citrate) in 0.1% SDS
for 20 min at room temperature and twice with SSC (30 mM
NaCl, 3 mM sodium citrate) in 0.1% SDS for 20 min at 55 C,
sealed in a plastic bag and exposed to x-ray film at -80 C for
visualization. Following densitometric analysis of uPA, tPA uPAR, and
PAI-1 messenger RNA (mRNA) bands using a Molecular Dynamics
Phosphoimager (Bio-Rad, Mississauga, ON, Canada), the blots were
stripped of radioactivity (0.1% SDS) and reprobed with an 800 bp
BamHI restriction fragment of rat cyclophilin cDNA probe
(39). Data were normalized against cyclophilin mRNA to correct for
uneven sample loading. The probe for tPA was prepared by RT-PCR, using
total RNA from rat embryos as template. The RT-PCR product was 450 bp
in length and was cloned into pBluescript vector for sequencing to
verify its identity (40). The probe for PAI-1 was a PvuII
fragment (980 bp) of the rat full length cDNA (41). An EcoRI
fragment of rat uPA cDNA and an XbaI fragment of rat uPAR
cDNA were used to probe all blots (15, 42)
Immunocytochemistry
Ovaries from each experimental group (see Animal
preparation) were fixed in 4% paraformaldehyde in PBS at room
temperature for 12 h, dehydrated through a graded series of ethyl
alcohol (70–100%), and embedded in low-temperature paraffin, as
previously described but with slight modifications (43). Sections (5
µm thick) were mounted on poly-L-lysine-coated slides,
deparaffinized in xylene, and rehydrated in a series of ethyl alcohol
concentrations (100%–0%). After 2 min incubation in Tris-buffered
saline (TBS; 0.1 M Tris-base, 0.15 M NaCl, pH
7.4), the endogenous peroxidase activity was blocked with 2%
H2O2 in methanol for 30 min. The slides were
subsequently washed with TBS, blocked with 5% BSA (in TBS with 0.1%
Triton X-100) for 1 h to reduce the nonspecific binding, and
incubated overnight at room temperature with primary polyclonal
antibody (rabbit antimouse uPA, rabbit antirat uPAR, or rabbit antirat
PAI-1; 10 µg/ml) or monoclonal mouse antihuman tPA (30 µg/ml).
After extensive rinsing with TBS, sections were incubated with
biotinylated goat antirabbit IgG or biotinylated rabbit antimouse IgG
(secondary antibody; 1:100 in TBS with 5% BSA and 1% Triton X-100;
1 h). The slides were then washed with TBS and incubated with
avidin-D-conjugated horse radish peroxidase (1:100 in TBS containing
5% BSA and 0.1% Triton X-100; 1 h.). The reaction was visualized
with DAB (1 mg/ml) and 0.03% H2O2 and enhanced
with 0.5% lithium chloride. Control sections were incubated in the
absence of primary antibody or with or without normal rabbit IgG or
normal mouse IgG (in the case of tPA). Six animals were used in each
experimental group.
ELISA
The uPA and uPAR protein expression of ovarian tissues harvested
from animals receiving different hormonal treatment was determined by
indirect enzyme-linked immunosorbent assay (ELISA) using Triton X-100
and plasma membrane fractions respectively (15). Briefly, 0.2 ml of
sample was added to individual wells on a 96-well ELISA plate and
incubated overnight at 4 C in a humidified box. After aspiration of
culture medium, wells were washed 3 times with 0.3 ml of PBS-Tween 20
(PBS containing 0.05% Tween-20, 1% BSA and 0.02% sodium azide) and
then incubated with 0.3 ml of 3% BSA in 0.05 M
Na2CO3 buffer (pH 9.6) containing 0.02% sodium
azide for 1 h at 37 C. Subsequently, wells were incubated with 0.2
ml of antimouse uPA monoclonal antibody or antirat uPAR antibody
(American Diagnostica Inc., Greenwich, CT) for 24 h followed by
incubation of 0.2 ml of goat antirabbit IgG conjugated to alkaline
phosphatase (Sigma) diluted 1:1000 for 2 h at 37 C. Finally, wells
were incubated with 0.2 ml of p-nitrophenyl-phosphate (1 mg/ml) in 0.05
M Na2CO3 buffer (pH 9.6) containing
0.5 mM MgCl2 for 45 min at 37 C. ELISA plates
were read by Microplate Reader (model 3550, Bio-Rad). The concentration
of uPA and uPAR in each sample was calculated by reading against a
standard curve generated with rat uPA and uPAR (American Diagnostica
Inc., Greenwich, CT) and expressed in ng of uPA or uPAR/mg of total
protein present in the plasma membrane and Triton-100 fractions
(42).
Statistical analysis
Results are presented as mean ± SEM of four
replicate experiments and were analyzed by multiple-way ANOVA.
Differences between groups were determined by the least significant
difference multiple range test. Statistical difference was inferred at
P < 0.05. Immunohistochemical studies were repeated
five to six times, and photographic records of representative
experiments are presented.
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Results |
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, 2, 3, and 4), immature female rats were injected with DES, eCG, or
eCG + hCG, as described in the Materials and Methods
section, for the synchronization of follicular maturation at the
preantral/small antral, medium antral, and preovulatory stages,
respectively. Intense immunostaining for uPA was uniformly distributed
within the granulosa and theca interna layers during early follicular
development (Fig. 1C vs. 1A and 1B). However, only scattered
cellular staining for uPA was detected in either cell compartment in
response to follicular maturation.
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). While
immunostaining appeared to be uniformly distributed between the
granulosa and theca-interna cell compartments, some very intensely
stained cells were scattered throughout the theca interna layer (Fig. 2E). A ring of immunoreactivity outlining each cell was characteristic
of many of the uPAR positive granulosa cells (Fig. 2F, arrows). In contrast, minimal staining was observed in the
preantral as well as the small and medium antral follicles (Fig. 2, C
and D). Control sections (omission of primary antibody or replacement
of primary antibody with normal rabbit IgG: Fig. 2, A and B,
respectively) showed no immunostaining.
The intensity of staining for tPA protein increased with follicular
development (Fig. 3). Whereas minimal staining was
observed in the granulosa layer of preantral follicles (Fig. 3C), the
staining intensity increased steadily during maturation to the antral
and preovulatory stages (Fig. 3, D–F). Likewise, although tPA positive
cells were occasionally detected in the theca-interstitial cell
compartment at the two early stages of follicular maturation, more
intensely stained theca and interstitial cells were observed by the
preovulatory stage (Fig. 3, E and F). Indeed, the overall staining
intensity in this layer appeared more pronounced by the later
developmental stage.
Similarly, the intensity of PAI-1 staining increased with
follicular development, reaching peak levels in the preovulatory
period. Indeed, compared with control sections incubated in the absence
of primary antibody (Fig. 4A), low but detectable
immunoreactivity for PAI-1 was observed in preantral/small antral as
well as medium antral follicles (ovarian sections of DES- and
eCG-treated rats, respectively; Fig. 4, B and C) and the intensity of
this staining increased by the preovulatory stage of development (Fig. 4D). In general, PAI-1 immunostaining was distributed fairly uniformly
between the granulosa and theca interna cell compartments (Fig. 4, D
and E). The oocytes showed significant immunoreactivity for uPA, uPAR,
tPA and PAI-1, but the intensities of this immunostaining were
independent of the stage of follicular maturation (
Figs. 1–4).
Changes in mRNA abundance during follicular maturation
The distribution of uPA mRNA in follicular cells, as determined by
Northern blot analysis, followed a pattern similar to that for uPA
protein observed by immunolocalization (Fig. 1). Urokinase-type PA
transcript levels in the two compartments decreased with follicular
maturation and were similar irrespective of the developmental stage
(Fig. 5). Likewise, comparable levels of uPAR transcript
were detected in both granulosa and residual ovarian preparations from
ovaries of rats pretreated with DES (preantral/small antral follicles),
eCG (medium antral follicles) and eCG + hCG (preovulatory follicles)
(Fig. 5). While the residual ovarian tissue showed a developmental
increase in uPAR mRNA levels, high levels of this transcript in
granulosa cells were observed only during the preovulatory period (eCG
+ hCG-treated group).
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). Tissue-type PA mRNA levels in both cell
types were low but detectable in preantral/small antral follicles (DES
group) and medium antral follicles (eCG group) but markedly increased
(P < 0.05) with follicular maturation (eCG + hCG
group). Moreover, the expression of tPA mRNA was significantly higher
(> 3-fold) in granulosa cells than in residual ovaries during the
preovulatory stage (eCG + hCG group; Fig. 5). PAI-1 transcript levels
were also highest in granulosa cells and residual ovaries during this
latter period but no significant difference in mRNA abundance for the
inhibitor was noted between the granulosa and residual ovarian
preparations (Fig. 5).
Urokinase PA and uPAR content in granulosa and theca-interstitial
cells during follicular maturation
Urokinase-type PA protein content was higher in granulosa
cells compared with the residual ovaries as determined by ELISA.
Consistent with its mRNA abundance, the levels of uPA in follicular
cells were maximal at early stages of follicular development and
decreased markedly (45–50%) as the follicle approached ovulation
(Fig. 6). Urokinase PAR protein content was similar in
both granulosa and residual ovarian preparations and increased
(P < 0.05) with follicular maturation, reaching
maximal levels (4-fold) at the preovulatory stage (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of uPAR in the ovary have not been previously examined. Urokinase PAR is a membrane-bound protein with the same affinity for uPA and pro-uPA. The conversion of pro-uPA to the active enzyme is enhanced over 20-fold during uPAR-pro-uPA interaction. Receptor-bound uPA dissociates slowly from the cell surface and is not protected from the inhibitory action of PAI (18, 19, 20, 47, 48). High levels of uPA and its receptor have been correlated with cellular invasion and tissue involution during normal physiological processes and during metastasis of tumor cells (1, 2, 4, 46, 49). These observations suggest the primary role of uPAR is to localize uPA activity to specific extracellular sites rather than to modulate its proteolytic activity. Results from the present studies indicate that uPAR is expressed in both granulosa and residual ovarian preparations in a developmentally dependent manner. Whereas significant levels of uPAR mRNA and protein were detected at early stages of follicular growth, greater uPAR expression was observed during the preovulatory period. Although the physiological significance of this apparent dichotomy in uPA and uPAR expression is presently unknown, it is possible that uPA down-regulates the expression of its own receptor in follicular cells as has been demonstrated in a colon cancer cell line (11) to keep the levels of uPA activity in check during follicular maturation and ovulation. Moreover, active receptor-bound uPA is not internalized nor degraded until it has bound PAI and formed a uPAR-uPA-PAI complex. After internalization, the uPA-PAI component is degraded and uPARs are recycled and distributed to new locations on the cell surface (20). Thus, it is possible that the coincidental rise in PAI-1 and uPAR mRNA and protein observed during the preovulatory period could facilitate uPAR in clearing uPA and PAI-1 from the sites of action, thus permitting a more active tPA (29, 31, 36, 50) for the ovulatory process. In contrast, the low level of PAI-1 at the early stages of follicular development could be insufficient to neutralize the high levels of uPA produced at this time, an observation consistent with an important role for urokinase in extracellular matrix remodeling during folliculogenesis.
Our data on follicular tPA expression during ovarian development are consistent with previous findings that tPA mRNA and protein expression by granulosa cells and the residual ovaries increased markedly during the preovulatory period and that granulosa cell-derived tPA accounted for 70–80% of the total tPA produced during this period (50, 51). A physiological role for these developmental changes in tPA message is supported by the current demonstration of high levels of tPA immunoreactivity in granulosa and theca-interstitial cells from preovulatory follicles (eCG + hCG group) and by an earlier observation that tPA activity significantly increased in ovarian homogenates as well as in granulosa and theca-interstitial cell compartments following hCG treatment in vivo (50). Thus, the high level of tPA during the preovulatory period and the induction of tPA activity by gonadotropin in cultured granulosa cells (33, 36, 50, 52) suggest that this protease may be involved in the cascade of biochemical events leading to follicular wall rupture and expulsion of the oocyte. In addition, changes in PAI-1 mRNA abundance and protein content during follicular maturation followed a temporal pattern very similar to that of tPA. Whereas the expression of PAI-1 mRNA and protein were low in both granulosa and residual ovarian tissues in early stages of development, they were markedly increased during the preovulatory period. Because interaction between tPA and PAI-1 results in the formation of tPA-PAI-1 complexes and the neutralization of the protease activity, synchrony in the synthesis of these proteins may be important in focusing the tPA action to primarily the site of follicle rupture as well as keeping the functional proteolytic activity low and in preventing premature rupture of the ovulatory follicle until the expected time of ovulation when PAI-1 expression and activity are down-regulated (50).
It is possible that the mRNA abundance for the various components of the PA system in the the residual ovarian preparations may be contributed in part by granulosa cell contamination. However, the presence of cells that are highly immunoreactive within the theca-interstitial compartment, as demonstrated in the present studies, and the detection of tPA and PAI-1 mRNAs in the theca-interstitial layer by in situ hybridization (51) suggest local synthesis of these molecules. Whether these substances are produced by the theca cells or other cell types present in this compartment (e.g. endothelial cells, macrophages, and fibroblasts) remains to be determined. In addition, the present studies demonstrate heavy immunostaining of the oocyte for all components of the PA system irrespective of the stage of follicular maturation. Although uPA and tPA activities have been shown to be present in cultures of oocyte-cumulus complexes (53) and oocytes are capable of secreting various factors having important regulatory roles in the function of its support cells (54), the present observations must be interpreted with caution since oocytes are known to be notoriously nonspecific in this type of assay.
In conclusion, we have demonstrated that uPA and uPAR are expressed in granulosa cells and residual ovarian preparations in a follicular stage dependent manner. The developmental expression ovarian uPAR may play an important role in controlling and targeting proteolytic activity during follicular development. The reciprocal expression of uPA and tPA and the temporal correlation in tPA and PAI-1 production during follicular development further support the concept that the proteolytic activity involved in tissue remodelling during folliculogenesis is tightly controlled and that the two PA types have different physiological functions during follicular maturation and ovulation. Whereas tPA may be involved in matrix degradation during the ovulatory process, uPA may be essential for the proteolytic activity associated with cell proliferation and migration and overall growth of the developing follicles.
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Acknowledgments |
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Footnotes |
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2 Visiting scholar from Foshan College of Science and Technology,
Guangdong, Peoples’ Republic of China.
Received November 8, 1996.
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