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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
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: tor.ny{at}medchem.umu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 correlates with ovulation (7, 8, 11, 14, 15, 16, 17, 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, 19, 20, 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, 23, 24, 25, 26, 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 (tPA-/-/uPA-/-) mice and plasminogen-deficient (plg-/-) 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, 29, 30, 31, 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 tPA-/-/uPA-/- mice (33). To further assess the physiological relevance of the PA system on ovulation, we have in an extensive study used plg-/- 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 the conditions used in this study plasmin is not required for efficient follicular wall rupture.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
C57BL/6J mice, obtained from Bomholt Gård Breeding and Research
Center Ltd.-Bommice (Ry, Denmark), and plg-/- 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
(plg+/+), plasminogen heterozygous (plg+/-),
and plg-/- 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 plg+/+,
plg+/-, and plg-/- mice, taken at 12 h
(8 plg+/+, 20 plg+/-, and 8
plg-/-) and 13 h (3 plg+/+, 7
plg+/-, and 3 plg-/-) after hCG injection
were analyzed in the same manner.
Physiological ovulation in adult mice
The 6-week-old female offspring from plg+/-
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 plg+/- and 2
plg-/-) 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.
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 -20 C until the experiment was performed. Mouse
plasma, diluted 1000 times, was incubated with 65 nM
urokinase, 10 mM lysine, and 80 µM S-2251 in
PBS at 37 C in a plate (96-well) with a total volume of 200 µl/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 (plg+/+), medium
(plg+/-), and low (plg-/-).
DNA prepared from tail tips was used for PCR reactions. The sequences of the primer pairs used in the reactions were as follows: plg, 5'-TCA GCA GGG CAA TGT CAC GG-3' and 5'-CTC TCT GTC TGC CTT CCA TGG-3'; and neomycin, 5'-ATG ATT GAA CAA GAT GGA TTG CAC G-3' and 5'-TTC GTC CAG ATC ATC CTG ATC GAC-3'.
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.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, no differences in ovulation efficiency
were found between plasminogen wild-type (plg+/+) mice and
plasminogen heterozygous (plg+/-) mice, but there was a
trend toward reduced ovulation efficiency in plg-/- mice.
The difference between plg+/+ control and
plg-/- mice was only 13% and not significant
(P = 0.084). The 25-day-old mice appeared to be healthy
and revealed no macroscopic abnormalities or pathological defects.
However, the plg-/- had both lower body weight (8.9%;
P < 0.001) and ovarian weight (7.7%;
P = 0.030) compared with the plg+/+ 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, plg-/-
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 plg-/- mice to have a slightly reduced, but
not statistically significant, ovulation efficiency compared with
plg+/+ control mice.
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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 plg-/- mice, animals of the three genotypes
(plg+/+, plg+-, and plg-/-) 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
plg-/- mice and plg+/+ 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 plg-/- mice.
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, a total of 81 mice (20
plg+/+, 40 plg+/-, and 21
plg-/-) were included. Vaginal plugs could not be
detected in 2 plg+/+ and 9 plg-/- mice during
the 5-week breeding period. All 9 plg-/- 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
plg+/+, 40 plg+/-, and 12
plg-/-), 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 ± 1.2 weeks (mean ± SD) with no
significant difference in age between mice with different genotypes.
The number of ova for each genotype varied between 6–10
(plg+/+), 3–10 (plg+/-), and 6–13
(plg-/-). As shown in Table 3, the ovulation efficiency
of normally cycling mice was the same regardless of the genotype.
However, the plg-/- mice and plg+/- mice had
5.4% and 5.0% lower body weight, respectively, and 8.5% and 10.2%
lower ovarian weight, respectively, compared with the
plg+/+ mice. The differences in body weight were
statistically significant but not the difference in ovarian weight.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-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, plg-/- mice were used to assess the
physiological importance of plasmin both in an ovulation model, in
which immature mice were stimulated 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 plg-/- mice, but there was a trend (13%;
P = 0.084) toward a slightly reduced ovulation
efficiency in these mice. Compared with the plg+/+ control
mice, the plg-/- mice had both lower body weight (8.9%;
P < 0.001) and lower ovarian weight (7.7%;
P = 0.030). The small reduction observed in ovulation
efficiency of the plg-/- mice may therefore be due to a
delayed maturation of these mice. Surprisingly, ovulation efficiency
was normal in adult cycling plg-/- 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 tissue-remodeling 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
plg-/- mice later in life (24, 26). As shown in Table 1
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there is only a trend (13%; P = 0.084) toward a
reduction in ovulation efficiency in plg-/- mice compared
with plg+/+ mice during gonadotropin-induced ovulation.
However, the plg-/- mice also had 8.9% lower body weight
compared with the plg+/+ mice. This is in agreement with a
previous report in which plg-/- mice were found to have a
lower gain of body weight after weaning (3 weeks of age) compared with
their plg+/- and plg+/+ littermates (26). The
difference in body weight observed between plg-/- mice
and plg+/+ 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
plg-/- mice and plg+/+ control mice of the
same weight.
As there are less heavy mice among the plg-/- mice than among the plg+/+ and plg+/- littermates, it is likely that the lower body weights of the plg-/- 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 plg-/- mice could therefore indicate that these mice develop more slowly than their plg+/+ and plg+/- littermates and therefore may not respond equally well to the hormone treatment. In a previous study (33) we showed that the ovulation efficiency of tPA-/-/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 plg-/-
and control plg+/+ 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 plg-/- mice and plg+/+
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 plg-/- 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 plg+/+ mice and
plg-/- mice. Not even 2 consecutive superovulations with
7 days in between injections revealed any significant difference in
ovulation efficiency between the plg+/+ mice and
plg-/- 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 proteases 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 plg-/- 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.
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Acknowledgments |
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Footnotes |
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Received April 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-antiplasmin. Endocrinology 124:415–421This article has been cited by other articles:
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P. Wahlberg, I. Boden, J. Paulsson, L. R. Lund, K. Liu, and T. Ny Functional Corpora Lutea Are Formed in Matrix Metalloproteinase Inhibitor-Treated Plasminogen-Deficient Mice Endocrinology, March 1, 2007; 148(3): 1226 - 1234. [Abstract] [Full Text] [PDF]
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J. Li, A. Ny, G. Leonardsson, K. S. Nandakumar, R. Holmdahl, and T. Ny The Plasminogen Activator/Plasmin System Is Essential for Development of the Joint Inflammatory Phase of Collagen Type II-Induced Arthritis Am. J. Pathol., March 1, 2005; 166(3): 783 - 792. [Abstract] [Full Text] [PDF]
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Y.-X. Liu, K. Liu, Q. Feng, Z.-Y. Hu, H.-Z. Liu, G.-Q. Fu, Y.-C. Li, R.-J. Zou, and T. Ny Tissue-Type Plasminogen Activator and Its Inhibitor Plasminogen Activator Inhibitor Type 1 Are Coordinately Expressed during Ovulation in the Rhesus Monkey Endocrinology, April 1, 2004; 145(4): 1767 - 1775. [Abstract] [Full Text] [PDF]
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