0013-7227/99/$03.00/0
Endocrinology
Copyright © 1999 by The Endocrine Society
Vol. 140, No. 11
Printed in U.S.A.
Ovulation in Plasminogen-Deficient Mice*
ANNELII NY, GÖRAN LEONARDSSON, ANNA-CARIN HÄGGLUND,
PETER HÄGGLÖF, VICTORIA A. PLOPLIS, PETER CARMELIET, AND TOR NY
Department of Medical Biosciences, Medical Biochemistry, Umeå University (A.N., G.L., A.-C.H., P.H.,
T.N.), S-901 87 Umeå, Sweden; the Department of Chemistry and Biochemistry, University of Notre
Dame (V.A.P.), Notre Dame, Indiana 46556-5670; and the Center for Transgene Technology and Gene
Therapy, Vlaams Interuniversitair Instituut voor Biotechnologie (P.C.), B-3000 Leuven, Belgium
ABSTRACT
Many different studies suggest that plasmin generated from plasminogen plays a crucial role in the degradation of the follicular wall
at the time of ovulation. We have assessed the physiological relevance
of plasmin on ovulation by studying plasminogen-deficient mice. Ovulation efficiency (mean number of ova released per mouse) was determined both in a standardized ovulation model in which 25-day-old
immature mice were injected with finite amounts of gonadotropins to
induce ovulation and during physiological ovulation using adult normally cycling mice. Our results revealed that the temporal onset of
follicular wall rupture (first ova observed in bursa or oviduct) was not
delayed in plasminogen-deficient mice during gonadotropin-induced
E
XTRACELLULAR proteolysis provided by the active
serine protease plasmin has been associated with many
physiological and pathological processes such as ovulation,
embryo implantation and embryogenesis, mammary involution, fibrinolysis, angiogenesis, arteriosclerosis, inflammation, and tumor invasion (1–3). Ovulation is a well defined
physiological process regulated by hormones and growth
factors that results in liberation of the mature ovum from the
preovulatory follicle into the periovulatory space (4 – 6). For
the ovum to escape from the mature follicle, a tightly regulated proteolytic degradation of basement membranes and
the connective tissue that constitutes the follicle wall is required. A number of correlation studies suggest that plasmin
together with matrix metalloproteinases play a role in follicular rupture (7–11).
The plasminogen activator (PA) system is comprised of
plasminogen, which is activated to plasmin by either of two
physiological PAs, tissue-type PA (tPA) or urokinase-type
PA (uPA). Activation of this system is initiated by the release
of tPA or uPA by specific cells in response to external signals
and results in local extracellular proteolytic activity (2, 12).
The system is also regulated by specific inhibitors directed
against PAs and plasmin (2, 13). Both PAs and PA inhibitors
have been identified in ovarian cells (14) and were found to
be coordinately regulated by gonadotropins in a manner that
Received April 28, 1999.
Address all correspondence and requests for reprints to: Dr. Tor Ny,
Department of Medical Biosciences, Medical Biochemistry, Umeå University, S-901 87 Umeå, Sweden. E-mail: [email protected].
* This work was supported by the Swedish Medical Research Council
(K97–13X-09709 – 07A), the Swedish Cancer Society (3912-B97– 01XAB),
Cancerforskningsfonden in Umeå (LP 1177/95), the J. C. Kempes Foundation in Umeå, and the Human Frontiers of Science Program (RG
363/95).
ovulation. However, there was a trend toward slightly reduced ovulation efficiency in the plasminogen-deficient mice. This reduction
was only 13% and not statistically significant (P 5 0.084) and may be
connected to a delayed maturation of these mice manifested in reduced body and ovary weights. During physiological ovulation adult
plasminogen-deficient mice had normal ovulation efficiency compared with plasminogen wild-type mice. Taken together our results
indicate that under the conditions used in this study plasmin is not
required for efficient follicular rupture or for activation of other proteases involved in this process. Alternatively, the role of plasmin may
be effectively compensated for by other mechanisms in the absence of
plasmin. (Endocrinology 140: 5030 –5035, 1999)
correlates with ovulation (7, 8, 11, 14 –18). In addition, other
matrix-degrading proteases, including members of the matrix metalloproteinase family, have been identified in the
ovary, and indirect evidence suggests that these proteases
also play a role in follicular rupture (for review and references, see Refs. 9, 10, 13, and 19 –21).
Mice with deficiencies in different components of the PA
system provide useful model systems to study the role of the
PA system in vivo (22–27). Surprisingly, mice with single
deficiencies for either of the components of the PA system are
born normal in appearance and can produce offspring. Although no firm studies on their fertility have been reported,
the initial characterizations of tPA/uPA double-deficient
(tPA2/2/uPA2/2) mice and plasminogen-deficient (plg2/2)
mice suggest that these mice are less fertile (23, 26).
Successful reproduction involves many biological processes, such as ovulation, fertilization, embryo implantation,
and embryogenesis, in which plasmin has been proposed to
play a role (7, 17, 28 –32). The reduced fertility observed in
mice that cannot generate plasmin (23, 26) could therefore be
due to a defect in one or more of these biological processes.
Alternatively, the reduced fertility of these mice could be
caused by general health problems seen later in life. Our
previous studies on ovulatory mechanisms in young immature PA-deficient mice have revealed that gonadotropin-induced ovulation is normal in mice with a single deficiency
of tPA or uPA and is slightly reduced in tPA2/2/uPA2/2
mice (33). To further assess the physiological relevance of the
PA system on ovulation, we have in an extensive study used
plg2/2 mice to study ovulation efficiency (mean number of
ova released per mouse) during gonadotropin-induced ovulation in young immature mice as well as in physiological
ovulation in adult cycling mice. Our results show that under
5030
OVULATION IN PLASMINOGEN-DEFICIENT MICE
the conditions used in this study plasmin is not required for
efficient follicular wall rupture.
Materials and Methods
Reagents
PMSG and hCG were purchased from Sigma Chemical Co. (St. Louis,
MO). McCoy’s 5A medium (modified without serum) was purchased
from Life Technologies, Inc. (Gaithersburg, MD). Ukidan was obtained
from Serono (Aubonne, Switzerland). S-2251, chromogenic substrate for
plasmin activity, was purchased from Chromogenix (Mölndal, Sweden).
Plates (96-well) were obtained from Nunclon (Nunc A/S, Roskilde,
Denmark), and a Micro Titer-Tek plate reader Multiscan RC was obtained from Labsystems (Stockholm, Sweden). PCR (10-fold concentrated) buffer was purchased from Perkin-Elmer Corp., Roche Molecular
Systems, Inc. (New Jersey, NY), and Taq DNA polymerase (native with
BSA) was obtained from MBI Fermentas (Vilnius, Lithuania). Specific
goat antiserum against murine fibrinogen was purchased from Nordic
Immunology (Tilburg, The Netherlands), an immunohistochemistry kit
was obtained from DAKO Corp. (Copenhagen, Denmark), and Mayer’s
hematoxylin was purchased from Apoteksbolaget (Malmö, Sweden).
Animals
C57BL/6J mice, obtained from Bomholt Gård Breeding and Research
Center Ltd.-Bommice (Ry, Denmark), and plg2/2 mice (26) were kept
on a 12-h light, 12-h dark cycle with the light cycle initiated at 0600 h and
were fed chow and water ad libitum. Experimental protocols were approved by the regional ethical committee of Umeå University (A10/96,
Umeå, Sweden).
Gonadotropin-induced ovulation in 25-day-old mice
Immature 25-day-old female plasminogen wild-type (plg1/1), plasminogen heterozygous (plg1/2), and plg2/2 mice were injected ip with
1.6 IU PMSG to stimulate follicle growth. After 48 h the mice were
injected with 5 IU hCG to induce ovulation. The amounts of gonadotropins used were tested, so that they would result in the release of ova
comparable to physiological ovulation. The animals were killed by cervical dislocation 20 h after injection of hCG, and body weight, ovarian
weight, and number of ova in the oviduct were recorded. Mice that did
not ovulate were excluded from the study.
Temporal onset of follicular wall rupture
To determine the temporal onset of follicular wall rupture, immature
25-day-old female wild-type (C57BL/6J) mice were injected with gonadotropins to induce ovulation as described above. At different time
points after hCG injection (6, 8, 10, 12, 14, 16, and 18 h), groups of four
to seven mice were killed. Their ovaries were removed and analyzed
under a dissecting microscope for the presence of ova in the bursa or
oviduct, indicating that the onset of follicular rupture was initiated.
Ovaries from gonadotropin-primed plg1/1, plg1/2, and plg2/2 mice,
taken at 12 h (8 plg1/1, 20 plg1/2, and 8 plg2/2) and 13 h (3 plg1/1,
7 plg1/2, and 3 plg2/2) after hCG injection were analyzed in the same
manner.
Physiological ovulation in adult mice
The 6-week-old female offspring from plg1/2 breeding pairs were
bred with adult wild-type (C57BL/6J) males for a 5-week period. Every
morning the females were examined for the presence of vaginal plug,
indicating mating the previous night. As mice normally mate when the
female ovulates, the presence of a vaginal plug is an indication that
ovulation has taken place. This method has previously been shown to
be more than 92% successful in the prediction of pregnancy (34). When
a vaginal plug was confirmed, the mouse was killed, and body weight,
ovarian weight, and the number of ova in the oviduct were recorded. In
4 (2 plg1/2 and 2 plg2/2) of the 85 mice in the study, vaginal plugs were
identified, but no ova were found in their oviducts. As it is likely that
these females mated even though they were not in the right stage of their
estrous cycle, they were removed from the study.
5031
Genotyping of the animals
All mice were genotyped by a rapid chromogenic activity assay,
which determines the level of plasminogen in mouse plasma, and by
PCR. In the few cases (,1%) where the results of these two assays did
not concur, Southern blot analysis was used to establish the genotype
(26). To assay the plasminogen level in mouse plasma, urokinase (ukidan) was added, and the amount of plasmin formed was determined.
By comparing the amount of generated plasmin between the different
plasma samples, the genotype of a mouse could be determined. Briefly,
blood was collected from the mouse tail tip in the presence of 0.04 m citric
acid. Plasma was prepared by centrifuging for 10 min at 3000 rpm and
was kept at 220 C until the experiment was performed. Mouse plasma,
diluted 1000 times, was incubated with 65 nm urokinase, 10 mm lysine,
and 80 mm S-2251 in PBS at 37 C in a plate (96-well) with a total volume
of 200 ml/well. Individual sample blanks, to compensate for different
colors of the plasma samples, were identical to the sample, except that
urokinase was excluded. Absorbance was measured at 405 nm every 30
min for 2 h with a Micro Titer-Tek plate reader. The average increase in
absorbance over time was calculated for each mouse. The three different
genotypes could be distinguished on three different levels of average
increase in absorbance over time: high (plg1/1), medium (plg1/2), and
low (plg2/2).
DNA prepared from tail tips was used for PCR reactions. The sequences of the primer pairs used in the reactions were as follows: plg,
59-TCA GCA GGG CAA TGT CAC GG-39 and 59-CTC TCT GTC TGC
CTT CCA TGG-39; and neomycin, 59-ATG ATT GAA CAA GAT GGA
TTG CAC G-39 and 59-TTC GTC CAG ATC ATC CTG ATC GAC-39.
PCR analysis was performed by standard procedure. Briefly, an initial
denaturation at 94 C for 3 min was followed by 25 cycles of denaturation
at 93 C for 30 sec, annealing at 55 C for 30 sec, elongation at 72 C for 45
sec, and finally 5-min elongation at 72 C.
Statistical analysis
The statistical differences between the genotypes were determined by
Student’s t test for two independent samples with unequal variance and
a significance level of P , 0.05 using Excel for Windows version 7.0
(Microsoft Corp.).
Results
Gonadotropin-induced ovulation in immature mice
Ovulation efficiency in young 25-day-old mice was studied after stimulation with finite doses of gonadotropins. A
total of 270 mice were included in the study. As shown in
Table 1, no differences in ovulation efficiency were found
between plasminogen wild-type (plg1/1) mice and plasminogen heterozygous (plg1/2) mice, but there was a trend
toward reduced ovulation efficiency in plg2/2 mice. The
difference between plg1/1 control and plg2/2 mice was only
13% and not significant (P 5 0.084). The 25-day-old mice
appeared to be healthy and revealed no macroscopic abnormalities or pathological defects. However, the plg2/2 had
TABLE 1. Gonadotropin-induced ovulation in plg1/1, plg1/2, and
plg2/2 mice
Genotype
plg1/1
plg1/2
plg2/2
No. of mice
used
No. of
ova/mouse
BW
(g)
Ovarian wt
(mg)
53
150
67
8.4 6 3.2
8.3 6 3.3
7.3 6 3.9
13.5 6 1.8
13.5 6 1.9
12.3 6 1.5
6.5 6 1.3
6.3 6 1.5
6.0 6 1.3
Immature 25-day-old female plg1/1, plg1/2, and plg2/2 mice were
injected ip with 1.6 IU PMSG to stimulate follicle growth and 48 h
later with 5 IU hCG to induce ovulation. The animals were killed 20 h
after injection of hCG, and the number of ova in the oviduct, body
weight (grams), and ovarian weight (milligrams) were recorded. The
values represent the mean number of ova per mouse, body weight, and
ovarian weight 6 SD.
5032
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OVULATION IN PLASMINOGEN-DEFICIENT MICE
TABLE 2. Gonadotropin-induced mice divided into subgroups according to body weight
BW (g)
8.0 – 8.9
9.0 –9.9
10.0 –10.9
11.0 –11.9
12.0 –12.9
13.0 –13.9
14.0 –14.9
15.0 –15.9
16.0 –16.9
17.0 –17.9
No. of ova/mouse
plg1/1
No. of ova/mouse
plg1/2
4.0 (1)
5.5 6 3.9 (4)
4.5 6 0.7 (2)
5.9 6 1.6 (11)
7.3 6 3.8 (12)
7.3 6 2.7 (23)
8.9 6 3.4 (34)
8.6 6 3.3 (38)
9.7 6 2.8 (14)
10.2 6 3.7 (10)
11.5 6 2.1 (2)
5.7 6 2.1 (3)
6.2 6 3.5 (6)
8.2 6 1.7 (4)
8.9 6 4.2 (17)
9.2 6 2.2 (12)
8.7 6 1.4 (6)
10.0 6 1.4 (2)
9.5 6 2.1 (2)
No. of ova/mouse
plg2/2
6.7 6 8.1 (3)
5.3 6 1.1 (9)
7.4 6 3.2 (16)
6.6 6 1.5 (20)
7.7 6 3.4 (12)
7.4 6 2.6 (5)
12.0 (1)
28.0 (1)
plg1/1 vs.
plg2/2 (%)
7.0
219
20
13
20
P value
0.81
0.46
0.14
0.38
0.21
220
2195
The 270 mice described in Table 2 were divided into 1-g body weight groups. The values represent the mean number of ova per mouse 6
The numbers in parentheses represent the number of mice in each weight group.
SD.
both lower body weight (8.9%; P , 0.001) and ovarian weight
(7.7%; P 5 0.030) compared with the plg1/1 control mice. To
examine how ovulation efficiency for the different genotypes
correlates to the body weight, we divided the mice according
to body weight into 1-g subgroups. As shown in Table 2, the
number of ova released per mouse increases with the body
weight; however, plg2/2 mice are underrepresented in subgroups containing heavier mice. More than 80% of the mice
in this study had body weights between 10 –15 g. In all but
one of these five weight-matched subgroups there was a
tendency for plg2/2 mice to have a slightly reduced, but not
statistically significant, ovulation efficiency compared with
plg1/1 control mice.
Immunostainings were performed on sections of ovaries
from 10 plg1/1 and 10 plg2/2 mice used in the gonadotropin-induced ovulation experiments. We observed no histological abnormalities and no significant increase in the
number of fibrin depositions in plg2/2 mice compared with
plg1/1 mice (data not shown).
Temporal onset of follicular wall rupture after
gonadotropin-induced ovulation
To determine the temporal onset of follicular wall rupture,
25-day-old wild-type C57BL/6J mice were primed with gonadotropins to induce ovulation. At different time points
after hCG injection the mice were killed and analyzed for the
presence of ova in the ovarian bursa or oviduct. As shown
in Fig. 1A, the first ova were detected at 10 h after hCG
injection in 33% of the mice, and all of the mice had initiated
ovulation at 18 h after hCG. To investigate whether the onset
of follicular wall rupture was delayed in plg2/2 mice, animals of the three genotypes (plg1/1, plg12, and plg2/2)
were killed 12 and 13 h after hCG treatment and analyzed for
the presence of ova. As shown in Fig. 1B, the same number
of the plg2/2 mice and plg1/1 mice had initiated ovulation
at 12 h as well as at 13 h after hCG treatment. In addition,
these mice also had similar ovulation efficiency at these two
time points (data not shown). These data suggest that ovulation is not delayed in plg2/2 mice.
Physiological ovulation in adult mice
In physiological ovulation the development and function
of the ovary are controlled by the endogenous gonadotropins
that are released from the pituitary in response to signals
FIG. 1. Temporal onset of follicular wall rupture. Immature wildtype mice were injected ip with PMSG to stimulate follicle growth and
48 h later with hCG to induce ovulation. A, To monitor the normal
onset of follicular wall rupture, groups of four to seven wild-type mice
were killed at different time points after hCG injection. Ovaries were
removed and examined for ova in the bursa or in the oviduct, indicating the onset of follicular wall rupture. B, Ovaries from plg1/1,
plg1/2, and plg2/2 mice, taken at 12 and 13 h after hCG injection, were
analyzed for the onset of follicular wall rupture as described above.
The ratios above the bars show the number of ovulating mice of the
total number of mice killed at each time point.
from the hypothalamus (4). To study the effect of plasminogen deficiency on physiological ovulation, 6-week-old
plg1/1, plg1/2, and plg2/2 female mice were bred with
wild-type (C57BL/6J) males. Female mice showing vaginal
plug when examined in the morning were killed, and the
numbers of ova in the oviducts were recorded. As shown in
OVULATION IN PLASMINOGEN-DEFICIENT MICE
Table 3, a total of 81 mice (20 plg1/1, 40 plg1/2, and 21
plg2/2) were included. Vaginal plugs could not be detected
in 2 plg1/1 and 9 plg2/2 mice during the 5-week breeding
period. All 9 plg2/2 showed signs of wasting, and 7 of these
mice developed rectal prolapses during the breeding period.
It is likely that the fertility of these mice was impaired due
to general health problems or physical difficulties in mating
due to the presence of rectal prolapses. To avoid effects on
reproduction related to health problems or other reasons,
these mice were not included in the study. For the remaining
70 mice (18 plg1/1, 40 plg1/2, and 12 plg2/2), vaginal plugs
as well as ova in the oviducts were found, indicating that the
reproductive cycle was functional in all three genotypes. On
the average, the vaginal plugs were observed at an age of
7.2 6 1.2 weeks (mean 6 sd) with no significant difference
in age between mice with different genotypes. The number
of ova for each genotype varied between 6 –10 (plg1/1), 3–10
(plg1/2), and 6 –13 (plg2/2). As shown in Table 3, the ovulation efficiency of normally cycling mice was the same regardless of the genotype. However, the plg2/2 mice and
plg1/2 mice had 5.4% and 5.0% lower body weight, respectively, and 8.5% and 10.2% lower ovarian weight, respectively, compared with the plg1/1 mice. The differences in
body weight were statistically significant but not the difference in ovarian weight.
Fibrin depositions were earlier documented in liver, lung,
rectal tissues, and gastric ulcers of 5- to 17-week-old plg2/2
mice as well as in the ovary of a 14-week-old plg2/2 female
(23). We therefore performed immunostaining for fibrin depositions using a fibrinogen/fibrin-specific antibody (24) on
sections of ovaries from four plg1/1 and four plg2/2 mice
with confirmed vaginal plugs and ova in their oviducts.
However, no significant differences in fibrin depositions
were seen between these two genotypes (data not shown).
Discussion
Numerous studies have suggested that plasmin generated
from plasminogen is involved in the proteolytic process essential for ovulation. 1) Ovulation is preceded by a transient
and cell-specific expression of PA that causes proteolytic
activity localized to the surface of the ovary just before ovulation (11, 15, 16, 18, 35). 2) Intrabursal administration of tPA
antibodies and a2-antiplasmin was associated with suppression of gonadotropin induced ovulation in rats (36). 3) Addition of bacterial streptokinase to in vitro perfused rabbit
ovaries was shown to induce ovulation in the absence of
gonadotropins (37). In the present study, plg2/2 mice were
used to assess the physiological importance of plasmin both
in an ovulation model, in which immature mice were stim-
5033
ulated to ovulate by injections with defined amounts of gonadotropin, and during physiological ovulation in adult
mice with normal reproductive cycles. Our study shows that
during gonadotropin-induced ovulation the onset of follicular wall rupture was not delayed in plg2/2 mice, but there
was a trend (13%; P 5 0.084) toward a slightly reduced
ovulation efficiency in these mice. Compared with the
plg1/1 control mice, the plg2/2 mice had both lower body
weight (8.9%; P , 0.001) and lower ovarian weight (7.7%; P 5
0.030). The small reduction observed in ovulation efficiency
of the plg2/2 mice may therefore be due to a delayed maturation of these mice. Surprisingly, ovulation efficiency was
normal in adult cycling plg2/2 mice. Under the conditions
used in this study plasmin is therefore not required for efficient follicular wall rupture. A physiological role for plasmin in ovulation may merely be manifested under conditions
other than those used in this study. Alternatively, plasmin
may be compensated for by other mechanisms in its absence
or may merely play its role in other proteolytic or tissueremodeling processes in the ovary.
Gonadotropin-induced ovulation is a well characterized
model in which sexually immature animals are treated with
gonadotropins to induce ovulation (34). This model has several advantages and has been widely used in previous investigations where the effect of different agents on ovulation
has been studied. As immature mice have not started their
estrous cycles, ovulation can be triggered by injecting defined doses of gonadotropins. Fluctuations in endogenous
hormone levels can thereby be avoided, and possible influences that plasminogen deficiency might have on the signal
pathways that regulate ovulation are minimized. By using
young mice we can also study ovulation before the onset of
the pathological conditions that have been documented for
plg2/2 mice later in life (24, 26). As shown in Table 1, there
is only a trend (13%; P 5 0.084) toward a reduction in ovulation efficiency in plg2/2 mice compared with plg1/1 mice
during gonadotropin-induced ovulation. However, the
plg2/2 mice also had 8.9% lower body weight compared
with the plg1/1 mice. This is in agreement with a previous
report in which plg2/2 mice were found to have a lower gain
of body weight after weaning (3 weeks of age) compared
with their plg1/2 and plg1/1 littermates (26). The difference
in body weight observed between plg2/2 mice and plg1/1
mice complicates analysis of the data. As shown in Table 2,
the difference in ovulation efficiency between lighter and
heavier mice of the same genotype is much larger than that
between plg2/2 mice and plg1/1 control mice of the same
weight.
As there are less heavy mice among the plg2/2 mice than
TABLE 3. Physiological ovulation in adult plg1/1, plg1/2, and plg2/2 mice
Genotype
plg1/1
plg1/2
plg2/2
No. of mice
used
No. of mice
with no plugs
No. of
ovulating mice
No. of
ova/mouse
BW
(g)
Ovarian wt
(mg)
20
40
21
2
0
9
18 (90)
40 (100)
12 (57)
8.3 6 1.1
8.2 6 1.3
8.2 6 1.8
20.2 6 1.3
19.2 6 1.8
19.1 6 1.3
5.9 6 1.2
5.3 6 0.9
5.4 6 1.4
plg1/1, plg1/2, and plg2/2 female mice, aged 6 weeks, were bred with adult wild-type (C57BL/6J) males for a 5-week period. Every morning
the females were examined for the presence of a vaginal plug. When a vaginal plug was detected, the mouse was killed, and the number of ova
in the oviduct, body weight (grams), and ovarian weight (milligrams) were recorded. The values represent the mean number of ova per mouse,
body weight, and ovarian weight 6 SD; percentages are given in parentheses.
5034
OVULATION IN PLASMINOGEN-DEFICIENT MICE
among the plg1/1 and plg1/2 littermates, it is likely that the
lower body weights of the plg2/2 mice have contributed to
their slightly reduced ovulation efficiency in this model.
Studies have shown that there is a strong correlation between
a critical body weight and the amount of adipose tissue to the
onset of puberty (38). Leptin, which is synthesized in adipose
tissue, is thought to cause maturation of reproductive tissues
and to be a trigger of puberty (39, 40). The lower body and
ovarian weight observed for plg2/2 mice could therefore
indicate that these mice develop more slowly than their
plg1/1 and plg1/2 littermates and therefore may not respond equally well to the hormone treatment. In a previous
study (33) we showed that the ovulation efficiency of tPA2/
2/2
2/uPA
mice during gonadotropin-induced ovulation is
reduced. It is possible that the reduced ovulation efficiency
in that study was due to delayed maturation of the reproduction tissues of those mice.
Although the ovulation efficiency is similar in plg2/2 and
control plg1/1 mice, the pathways leading to ovulation may
vary. To investigate whether the lack of plasmin could cause
a delay in the initiation of ovulation, we studied the temporal
onset of follicular wall rupture. As shown in Fig. 1A, for the
wild-type (C57BL/6J) mice, the first ovulated ova were
found in the bursa or oviduct 10 h after hCG injection, and
most of the mice had started to ovulate at 12–16 h after hCG
injection. As shown in Fig. 1B, the same percentage of plg2/2
mice and plg1/1 mice had initiated ovulation at 12–13 h after
hCG, suggesting that that ovulation is not delayed due to
plasminogen deficiency. Studies in other systems have
shown that gene-deficient mice exhibiting no obvious phenotype can be provoked to reveal a phenotype after experimental challenges (23, 41). In an attempt to challenge the
ovulatory process to enhance a possible phenotype in plg2/2
mice, we increased the dose of gonadotropins to 5 IU PMSG
and 10 IU hCG to induce superovulation. With this amount
of gonadotropins, the number of ova released varied between 15–54. Our preliminary results reveal no difference in
ovulation efficiency between plg1/1 mice and plg2/2 mice.
Not even 2 consecutive superovulations with 7 days in between injections revealed any significant difference in ovulation efficiency between the plg1/1 mice and plg2/2 mice
(data not shown).
Ovulation is a complex physiological process regulated at
many levels. This includes a coordinated action of the two
pituitary gonadotropins on the ovary as well as the action of
other intraovarian factors (4). To study the effect of plasminogen deficiency on the ability to ovulate, we used two
different methods to induce ovulation. Physiological ovulation is dependent on regulatory mechanisms controlled by
endogenous hormone levels (5). In the physiological ovulation model, we have studied the whole, complex, ovulatory
process where the surge of gonadotropins from the pituitary
leads to the release of mature ova from the ovary. As shown
in Table 3, the ovulation efficiency was the same in all three
genotypes, and the difference in body weight and ovarian
weight between the different genotypes did not seem to
affect ovulation efficiency.
In conclusion, the data presented here provide genetic
evidence that plasmin is not required to sustain normal ovulation efficiency in mice or for the activation of other pro-
Endo • 1999
Vol 140 • No 11
teases involved in degradation of the follicular wall. However, a substantial body of indirect evidence obtained from
studies in several species favors a role for plasmin in ovulation (7, 8, 11, 15, 16, 35, 42). In this regard, it should be noted
that our results do not exclude the possibility that plasmin
plays a role in ovulation that could be manifested under
conditions other than those used in this study. It is also
possible that plasmin plays its role in other proteolytic or
tissue-remodeling processes in the ovary or that plasmin
might play a less important role in the mouse than in other
species, such as rats and rabbits. There might also be back-up
systems that compensate for the impaired protease function
in plg2/2 mice. These might involve the signaling molecules
that induce ovulation and/or other to date unknown mechanisms, including up-regulation of still unidentified ovarian
proteases.
Acknowledgments
We thank Dr. Aaron Hsueh at Stanford University School of Medicine
for conclusive discussions, and James Snell for critically reading this
manuscript.
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