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First published online June 15, 2006
Journal of Experimental Biology 209, 2567-2575 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02270
2-hydroxyestradiol-17ß-induced oocyte maturation: involvement of cAMP–protein kinase A and okadaic acid-sensitive protein phosphatases, and their interplay in oocyte maturation in the catfish Heteropneustes fossilis
Department of Zoology, Banaras Hindu University, Varanasi-221005, India
* Author for correspondence (e-mail: kpjoy{at}bhu.ac.in)
Accepted 13 April 2006
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Summary |
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Key words: catfish, 2-hydroxyestradiol, GVBD, cAMP, protein kinase A, protein phosphatases, okadaic acid
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionAbbreviationsReferences |
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;
Kagawa, 1994;
Nagahama, 1997;
Yamashita, 1998;
Thomas, 1994;
Thomas, 1999;
Yoshida et al., 2000;
Patino et al., 2001). In
teleosts, chemically different molecules have been demonstrated to elicit MIS
activity and they include 17,20ß-dihydroxy-4-pregnen-3-one
(17,20ß-DP), 17,20ß,21-trihydroxy-4-pregnen-3-one (17ß-S),
corticosteroids, insulin, insulin-like growth factors and activin
(Scott and Canario, 1987;
Nagahama, 1997;
Tyler et al., 1999). Estrogens
are poor inducers of oocyte maturation but a recent report
(Tokumoto et al., 2004) shows
that the synthetic oestrogen diethylstilbestrol induces oocyte maturation in a
manner similar to17,20ß-DP. Recently, we have shown that the hydroxy- and
methoxy-metabolites of estrogens along with their synthesizing enzymes
(estrogen-2-hydroxylase and catechol-O-methyltransferase, respectively) occur
in catfish ovary and that the hydroxyestrogens elicit varying degrees of
maturational activity (Senthilkumaran and
Joy, 2001; Mishra and Joy,
2006a; Mishra and Joy,
2006b). The MIS-like activity of 2-hydroxyestradiol-17ß
(2-OHE2) has been attributed to an indirect action through
follicular steroidogenesis notably by the production of 17,20ß-DP
(Mishra and Joy, 2006c). The
MIS, in turn, initiates a series of phosphorylations and dephosphorylations
catalyzed by protein kinases such as cAMP–protein kinase A (PKA),
protein kinase C (PKC), mitogen-activated protein kinase (MAPK),
cAMP-independent phosphatidylinositide 3-kinase (PI 3-kinase)-serine/threonine
kinase Akt, and protein phosphatases (PP), respectively, leading to the
activation of a cytoplasmic maturation-promoting factor (MPF), which induces
germinal vesicle breakdown (GVBD) and progression of meiosis up to the second
metaphase arrest (Nagahama et al.,
1994; Balamurugan and Haider,
1998; Yamashita,
1998; Pace and Thomas,
2005).
Investigations in starfish, fish, amphibians and mammals support the
contention that the cAMP-PKA pathway is widely held responsible for the
maintenance of the first meiotic arrest and its resumption is attributed to a
decrease in intra-oocyte cAMP level under the influence of a maturation
inducing substance or hormone (MIS or MIH)
(Meijer et al., 1989;
Jalabert et al., 1991;
Chaube and Haider, 1997). The
MIS decreases cAMP by inhibiting adenylyl cyclase
(Yoshikuni and Nagahama, 1994;
Pace and Thomas, 2005) or
increasing phosphodiesterase (PDE) (Chaube
and Haider, 1997) activity. In teleosts, cAMP, adenylyl cyclase
stimulators or PDE inhibitors have been reported to inhibit the MIS-mediated
oocyte maturation (Jalabert et al.,
1991; Haider and Chaube,
1995; Chaube and Haider,
1997; Haider and Baqri,
2000). It has been widely held that a threshold level of cAMP-PKA
activity maintains putative initiator protein(s) in a phosphorylated
(inactive) state inhibiting oocyte maturation and the activation of the
initiator protein (active) dephosphorylation by some unknown mechanisms would
induce maturation-promoting factor (MPF) activity. Recently, the importance of
cAMP-independent signaling pathways in the regulation of oocyte maturation has
been demonstrated (Schmitt and Nebreda,
2002; Pace and Thomas,
2005).
An involvement of okadaic acid (OA)-sensitive PP has been demonstrated
during oocyte maturation in starfish, Xenopus and mammals
(Huchon et al., 1981;
Bornslaeger et al., 1986;
Goris et al., 1989;
Pondaven et al., 1989;
Rime and Ozon, 1990;
Schwartz and Schultz, 1991;
Lu et al., 2001;
Wang et al., 2004). Evidence
for a crosstalk between cAMP-PKA and OA-sensitive PP was presented as well
(Schwartz and Schultz, 1991;
Lu et al., 2001). To our
knowledge, studies on the role of PP in oocyte maturation in fish are not
available.
Heteropneustes fossilis (Bloch) (Order Cypriniformes, Suborder
Siluroidei, Family Saccobranchidae) is a freshwater, air-breathing species
found in ponds, pools, swamps and rivers of India, Sri Lanka, Burma, Laos,
Thailand and Vietnam (see Dutta-Munshi and Hughes, 1992). The catfish is
interesting in that it possesses a pair of long tubular accessory respiratory
organ, which extends through the dorsal muscle of the body on either side of
the vertebral column. The air-breathing adaptation enables it to survive in
waters of low oxygen content and also out of water on wet grounds. The meat is
rich in protein and iron but poor in fat content and, therefore, esteemed for
its invigorating qualities. This species is cultured intensively and is also
ideal for wastewater aquaculture. The catfish has been used as a model for
studying various aspects of reproductive physiology (see
Sundararaj, 1981) and has been
one of the species studied in detail for understanding the hormonal mechanisms
of oocyte maturation.
The objectives of the present study were to demonstrate (1) the involvement of both cAMP-PKA and protein phosphatases in 2-OHE2-induced oocyte maturation and (2) to demonstrate the possible interactions between these two systems in the catfish.
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Materials and methods |
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Animal collection and maintenance
The experiments were performed in accordance with local/national guidelines
for experimentation in animals and all care was taken to prevent cruelty of
any kind.
Gravid female Heteropneustes fossilis Bloch (30–40 g) were obtained from a local fish market in prespawning phase (June) of the reproductive cycle. They were maintained in the laboratory under normal photoperiod (13 h:11 h L:D) and temperature (25±2°C) until used for experiments. The fish were fed goat liver daily ad libitum. A few fish were randomly checked for spontaneous ovulation and sacrificed to determine the maturation of ovary. The fish containing ovaries filled with dark green, postvitellogenic follicles were used in the study.
Preparation of incubation medium and test compounds
The incubation medium was prepared as follows (quantities given are in g):
NaCl 3.74, KCl 0.32, CaCl2 0.16,
NaH2PO4.2H2O 0.10,
MgSO4.7H2O 0.16, glucose 0.40 and Phenol Red 0.008 were
dissolved in 1 l of triple distilled water. The pH was adjusted to 7.5 with 1
mol l–1 sodium bicarbonate and autoclaved. Penicillin
(2x105 i.u.) and streptomycin sulphate (200 mg) were added
and filtered. The medium was stored at 4°C and prepared fresh every
week.
Stock solutions of cAMP, IBMX and caffeine were prepared by dissolving known amounts in the incubation medium. Theophylline, OA and 2-OHE2 were dissolved in the medium containing 50 µl ethanol. H89 was dissolved in acidic incubation medium (10 µl, 5 mmol l–1 HCl). The stock solutions were prepared on the day of the experiment and kept at 0°C. Just before the incubation, they were diluted with the incubation medium to give different working concentrations.
Collection, selection and incubation of ovarian follicles
All instruments and glassware were sterilized. The acclimatized, gravid
female H. fossilis were sacrificed by decapitation and ovaries were
transferred to a Petri dish containing fresh cooled incubation medium. Rounded
dark green postvitellogenic follicles were separated from each other with the
help of a fine brush and watchmaker's forceps. The follicles were incubated in
embryo cups containing 3 ml of the incubation medium or the medium containing
various test compounds, at 25±2°C. As controls, follicles were
incubated in plain medium (control) or the medium containing vehicle (vehicle
control). At the end of the incubations, the follicles were cleared in
ethanol:acetic acid:formalin to determine germinal vesicle (GV) breakdown
(GVBD) as an index of oocyte maturation. Translucent follicles without GVs and
opaque follicles containing GVs were counted separately. The percentage of
GVBD was determined from the total number of the follicles incubated.
Effects of 2-OHE2 on oocyte maturation
About 30–40 follicles in triplicate from each fish (N=3)
were incubated in the medium containing 5 µmol l–1
2-OHE2 (Mishra and Joy,
2006b) for 0, 3, 6, 12, 24 and 30 h. As controls, the follicles
were incubated in the incubation medium alone or in the medium containing the
same volume of vehicle. At the end of each time interval, the follicles were
transferred to fresh medium to complete a total of 30 h incubation (except the
30 h group). The follicles were cleared and scored for GVBD.
Effect of 2-OHE2 incubation on total follicular cAMP level
About 70–80 follicles in duplicate from 5 fish each were incubated
for different time intervals (0, 3, 6, 12, 24 and 30 h) in the medium
containing 5 µmol l–1 2-OHE2. The incubations
continued up to 30 h in plain medium for all groups except the 30 h group. For
the controls, the follicles were incubated in the vehicle medium for similar
durations. The follicles were harvested in groups; they were homogenized in
200 µl trichloroacetic acid (TCA, 5%), centrifuged at 1500
g at 4°C for 10 min and the supernatant was collected. TCA
was extracted from the supernatant with water-saturated ether (5 volumes, two
times), the ether layer was discarded and the supernatant was collected. The
residual ether was removed with a N2 stream. Extracted samples were
kept at –20°C for cAMP estimation.
Cyclic AMP concentration was measured using an EIA kit, following the
method of Pradelles et al. (Pradelles et
al., 1989). On the day of the analysis, the samples were diluted
with assay buffer (provided with the kit). Samples (50 µl) were reacted
with 50 µl each of cAMP–acetycholine esterase (AchE) tracer and cAMP
antibody in mouse monoclonal anti-rabbit IgG precoated wells, followed by 18 h
incubation at 4°C. After the incubations, the solutions were decanted and
the wells rinsed five times with the wash buffer. 200 µl Ellman's Reagent
(provided with the kit) was added to the wells and incubated in dark for
90–120 min for optimum color development (0.3–0.8 a.u., after
blank subtraction). The developed plate was read at 405 nm in a Multiskan EX
(Thermo Labsystems, Milford, CA, USA) ELISA reader. The concentration of total
cAMP was calculated as pmol mg–1 oocyte. The minimum
detection limit with the kit was 4 pmol ml–1. The intra- and
inter-assay coefficients of variation were 4.5% and 7.4%, respectively.
Effects of cAMP and cAMP-elevating drugs on 2-OHE2-induced-oocyte maturation
Concentration-response study
About 30–40 follicles in triplicate (N=3 fish) were
incubated with different concentrations of cAMP, IBMX, theophylline or
caffeine (0.1, 0.5, 1 and 2 mmol l–1) alone or in combination
with 5 µmol l–1 2-OHE2 for 24 h. Control groups
were maintained concurrently. After the completion of the incubations, the
follicles were cleared and counted for the percentage of GVBD.
Pre-incubation study
About 30–40 follicles in triplicate (N=3 fish) were
incubated with 1 mmol l–1 each of cAMP, IBMX, theophylline
and caffeine for 2, 4 or 6 h. After the respective intervals, the follicles
were rinsed in fresh incubation medium and transferred to the medium
containing 5 µmol l–1 2-OHE2 for 6 h (this
duration elicited 
50% GVBD). Subsequently, the follicles were briefly
rinsed in fresh incubation medium and maintained in the plain medium for up to
30 h (total incubation time). Control incubations were also conducted side by
side. At the end of the incubations, the follicles were cleared and scored for
the percentage of GVBD.
Post-incubation study
About 30–40 follicles in triplicate (N=3 fish) were
incubated with 5 µmol l–1 2-OHE2 for 6 h. The
follicles were then rinsed with fresh medium and transferred into the medium
containing 1 mmol l–1 each of cAMP, IBMX, theophylline or
caffeine for different time intervals (2, 4 or 6 h). After the respective
intervals, the follicles were placed in plain incubation medium for the
completion of 30 hincubation period. Control incubations were set up
simultaneously. At the end of the incubations, the follicles were cleared and
scored for GVBD.
Effect of H89 on 2-OHE2-induced oocyte maturation
Concentration-response study
About 30–40 follicles in triplicate (N=3 fish) were
incubated with different concentrations of H89 (0.1, 1, 5 and 10 µmol
l–1) alone or in combination with 5 µmol
l–1 2-OHE2 for 24 h. Control groups were
maintained concurrently. After the incubations, the follicles were cleared and
scored for GVBD.
Effect of H89 on IBMX inhibition of 2-OHE2-induced oocyte maturation
About 30–40 follicles in triplicate (N=3 fish) were
incubated with 5 µM 2-OHE2 and 1 mmol l–1 IBMX.
After 1 h, the medium was replaced by the medium containing 2-OHE2,
IBMX and 10 µmol l–1 H89 for 7 h. Then, the follicles were
briefly rinsed and maintained in plain incubation medium to complete the 30 h
incubation. Control series consisted of incubation of the follicles with
2-OHE2 (5 µmol l–1, 8 h), IBMX (1 mmol
l–1, 8 h), H89 (10 µmol l–1, 7 h),
2-OHE2 and IBMX (8 h), 2-OHE2 (8 h) and H89 (7 h) and
plain incubation medium with or without the vehicle. At the end of the
incubations, the follicles were cleared and scored for the percentage of
GVBD.
Effect of okadaic acid on 2-OHE2-induced oocyte maturation
Concentration-response study
About 30–40 follicles in triplicate (N=3 fish) were
incubated with different concentrations of OA (0.5, 1 and 2 µmol
l–1) alone or in combination with 5 µmol
l–1 2-OHE2 for 24 h. Control groups were
maintained concurrently. After the incubations, the follicles were cleared and
scored for GVBD.
Effect of okadaic acid (OA) on IBMX inhibition of 2-OHE2-induced oocyte maturation
About 30–40 follicles in triplicate (N=3 fish) were
co-incubated with 5 µmol l–1 2-OHE2 and 1 mmol
l–1 IBMX. After 1 h, the medium was replaced by fresh medium
containing 2-OHE2, IBMX and 1 µmol l–1 OA for 7
h. Then, the follicles were transferred to plain incubation medium to complete
the 30 h incubation. Control series consisted of incubation of the follicles
with 2-OHE2 (5 µmol l–1, 8 h), IBMX (1 mmol
l–1, 8 h), OA (1 µmol l–1, 7 h),
2-OHE2 and IBMX (8 h), 2-OHE2 (8 h) and OA (7 h) and
plain incubation medium with or without the vehicle. At the end of the
incubations, the follicles were cleared and scored for the percentage of
GVBD.
Statistical analysis
Data were expressed as means ± s.e.m. and were analyzed by one-way
analysis of variance (ANOVA), followed by Newman–Keuls' test
(P<0.05) for multiple group comparisons.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionAbbreviationsReferences |
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Effect of 2-OHE2 on GVBD and total follicular cAMP level
A significant GVBD was registered in the 2-OHE2 groups, which
increased from 3 h onwards (Fig.
1; F=680.10; P<0.001, one-way ANOVA). The
response was significantly high at all intervals as compared to the control
group (P<0.05, Newman–Keuls' test).
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The incubation of the follicles with 2-OHE2 decreased cAMP significantly in a duration-dependent manner (Fig. 1; F=137.32; P<0.001, one-way ANOVA). The decrease was significant at all intervals compared with the control groups (P<0.05, Newman–Keuls' test). The control values (plain medium and vehicle groups) were not significantly different.
Effect of cAMP and cAMP-elevating drugs on 2-OHE2-induced oocyte maturation
Cyclic AMP, IBMX, theophylline and caffeine all produced a significant
suppression of 2-OHE2-induced GVBD
(Fig. 2; cAMP,
F=294.91; IBMX, F=364.14; theophylline, F=214.89;
caffeine, F=267.67; P<0.001 one-way ANOVA) whereas when
applied without 2-OHE2 they did not produce any significant effect
compared to the vehicle control. The inhibition due to cAMP and IBMX was
concentration-dependent (P<0.05, Newman–Keuls' test). Only
higher concentrations of theophylline (0.5, 1.0 or 2.0 mmol
l–1) and caffeine (1.0 or 2.0 mmol l–1)
inhibited GVBD significantly (P<0.05, Newman–Keuls' test).
The magnitude of the inhibition was in the order,
cAMP>IBMX>theophylline> caffeine.
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Effect of H89 on 2-OHE2-induced GVBD
Concentration effect
The incubation of the follicles with H89 alone produced an overall
significant increase in the percentage of GVBD
(Fig. 3A; F=237.21;
P<0.001, one-way ANOVA) as compared to the control groups. The
percentage of GVBD increased significantly in a concentration-dependent manner
(P<0.05, Newman–Keuls' test). The co-incubation of the
follicles with H89 and 2-OHE2 produced varied effects
(Fig. 3A; F=775.52;
P<0.001, one-way ANOVA). The low concentrations of the drug (0.1
and 1 µmol l–1) did not affect the GVBD in response to
2-OHE2 but higher concentrations (5 and 10 µmol
l–1) inhibited the response of significantly
(P<0.05, Newman–Keuls' test).
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Effect of okadaic acid on 2-OHE2-induced oocyte maturation
Concentration-response study
The incubation of the follicles with OA alone did not alter the GVBD
response but when co-incubated with 2-OHE2, GVBD was significantly
higher than with 2-OHE2 alone
(Fig. 4A; F=824.13;
P<0.001, one-way ANOVA; P<0.05, Newman–Keuls'
test).
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Effect of OA on the IBMX inhibition of 2-OHE2-induced GVBD
The incubation of the follicles with 2-OHE2, IBMX and OA for 7 h
reversed the inhibitory effect of IBMX on the 2-OHE2-induced GVBD
response, elevating it to that of the 2-OHE2 group
(Fig. 4B; F=454.54;
P<0.001, one-way ANOVA; P<0.05, Newman–Keuls'
test).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionAbbreviationsReferences |
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Total oocyte cAMP levels decreased significantly and persistently
concomitant with the increase in GVBD. The time-course study showed that the
effect was evident at 3 h (the first sampling interval) and was maximal at 30
h (the end sampling time). The inhibition varied from 20.18% at 3 h to 62.29%
at 30 h. The changes were similar to those reported in catfish (Clarias
batrachus) oocytes after 17,20ß-DP treatment
(Haider and Chaube, 1995;
Haider and Baqri, 2000). These
workers correlated the changes in cAMP level with the morphological stages of
the oocytes; it was high in centrally located GV oocytes, decreased in
relation to GV migration and lower still in oocytes that had undergone GVBD. A
10–21% decrease in cAMP level was sufficient to induce GVBD in
Xenopus (Huchon et al.,
1981; Cicirelli and Smith,
1985).
A negative correlation of cAMP level with the GVBD response was further
obtained from the results of the cAMP supplementation experiment. In the
presence of cAMP, the 2-OHE2-induced GVBD was inhibited in a
concentration-dependent manner in the co-incubation (24 h), pre-stimulation
(2, 4 and 6 h) and post-stimulation (4 and 6 h) studies. In the catfish, 0.1
mmol l–1 cAMP inhibited GVBD, whereas in yellow perch,
concentrations of 1.0 and 0.5 mmol l–1 were not inhibitory
(DeManno and Goetz, 1987a).
Further, the PDE inhibitors IBMX, theophylline and caffeine also inhibited the
2-OHE2-induced GVBD. The response of the PDE inhibitors appears to
vary with species, inhibitor type and concentration and duration
(DeManno and Goetz, 1987a;
DeManno and Goetz, 1987b;
Chaube and Haider, 1997;
Haider and Baqri, 2000;
Pace and Thomas, 2005). In the
present study, the inhibitory response was evident at >0.1 mmol
l–1 for IBMX, >0.5 mmol l–1 for
theophylline and at >1.0 mmol l–1 for caffeine in the
co-incubation studies. At 2.0 mmol l–1, both IBMX and
theophylline inhibited 90% of GVBD, whereas caffeine blocked only about
65%. In C. batrachus >1.0 mmol l–1 theophylline
or IBMX completely blocked the 17,20ß-DP-induced GVBD
(Chaube and Haider, 1997;
Haider and Baqri, 2000). In
H. fossilis, a prior 4 h incubation of the follicles with IBMX or
theophylline inhibited the GVBD response to 2-OHE2. In the
post-stimulation experiment, only a 6 h treatment of IBMX or theophylline
could inhibit the GVBD response. The post-treatment with cAMP or
cAMP-elevating drugs produced a low inhibition of GVBD unlike the
pre-treatment. This may be due to the prior exposure of the follicles to a
high level of cAMP, which strongly countered the steroid's effect, resulting
in higher inhibition than in the post-treatment groups. In the post-treatment
groups, the follicles were first exposed to 2-OHE2, which lowered
the endogenous cAMP level initiating the GVBD response normally. After the
stimulation, maintaining the follicles at elevated cAMP level (post-treatment)
might have suppressed only the subsequent maturational activity.
A comparison of the inhibitory effect of cAMP on the one hand and IBMX and
theophylline on the other showed that cAMP acted faster (2 h) and relatively
at a lower concentration (0.1 mmol l–1) to inhibit the GVBD
response. The slower rate of response to IBMX and theophylline may be due to
their indirect action through PDE inhibition. The inhibitory effect of
caffeine on the GVBD response was relatively low and slow, unlike IBMX or
theophylline. This may be due to the fact that the potency of caffeine to
inhibit PDE activity is low, with IC50 values in mmol
l–1 range (Vadziuk and
Kosterin, 2003), compared with IBMX and theophylline, which have
lower IC50 values (in µmol l–1 range)
(Lee et al., 2002;
Yuasa et al., 2005).
It has been reported that the MIS decreases cAMP by inhibiting adenylate
cyclase or cAMP-dependent PDE activity
(Finidori-Lepicard et al.,
1981; Chaube and Haider,
1997; Haider and Baqri,
2000). The adenylyl cyclase activator forskolin, which stimulates
cAMP, has been demonstrated to inhibit MIS-induced maturation in fish (see
Jalabert et al., 1991;
Haider and Chaube, 1995).
Likewise, the PDE inhibitors, such as IBMX and theophylline that elevate cAMP,
have been shown to inhibit the MIS-induced maturation
(DeManno and Goetz, 1987a;
DeManno and Goetz, 1987b;
Chaube and Haider, 1997;
Haider and Baqri, 2000).
Haider and Baqri (Haider and Baqri,
2000) showed a correlation between PDE activity, cAMP level and
GVBD response. Hydroxyestrogens have been shown to decrease cAMP level in
other tissue systems (Paul and Skolnick,
1977). By contrast, we have shown that the steroid-induced GVBD is
associated with the production of ovarian MIS such as 17,20ß-DP
(Mishra and Joy, 2006a),
which, in turn, might have inhibited the follicular cAMP level. The
significant and persistent decrease in the cAMP level during the maturation of
the catfish oocytes might be due to the combined action of all these
mechanisms.
The available evidence indicates that cAMP maintains meiotic arrest or
inhibits the MIS-induced GVBD response by stimulating PKA activity
(Huchon et al., 1981;
Bornslaeger et al., 1986;
Jalabert et al., 1991;
Rime et al., 1992;
Matten et al., 1994). The
involvement of PKA in oocyte maturation has been demonstrated in Fundulus
heteroclitus (Cerda et al.,
1997) and C. batrachus
(Haider and Baqri, 2002). In
both species, the PKA inhibitor H-8 or H89 stimulated GVBD, bypassing the MIS.
In F. heteroclitus, the response was similar to that of
17,20ß-DP but with a time lag of several hours
(Cerda et al., 1997). In C.
batrachus, the response was significantly low compared to the MIS effect
but the time lag of the response was shorter than that reported in F.
heteroclitus (Haider and Baqri,
2002). These workers have demonstrated a negative correlation
between H89, 17,20ß-DP-induced GVBD response and PKA activity. In H.
fossilis, H89 stimulated GVBD in a concentration-dependent manner (66.5%
at 10 µmol l–1) but was less effective than
2-OHE2 (80.4% at 5 µmol l–1). However, in
Atlantic croaker, PKA inhibitors (Rp-cAMP and KT5720), did not stimulate GVBD
in the absence of the MIS (20ß-S) nor did they enhance the efficacy of
the MIS (Pace and Thomas,
2005).
An interesting observation of the present study was that H89 produced
concentration-dependent differential effects on the 2-OHE2-induced
GVBD when co-incubated for 24 h: high concentrations (5 and 10 µmol
l–1) were inhibitory, and low concentrations (0.1 and 1
µM) were ineffective. The underlying mechanism of this dual effect is not
clear. The H89 treatment also partially reversed the inhibitory effect of IBMX
on the 2-OHE2-induced GVBD. The differential effects can be
attributed to the compartmentalization of PKA isoforms or functions. The PKA
isoforms exert opposite effects in mouse oocyte maturation; PKA I within the
oocytes suppresses GVBD, whereas PKA II within the somatic cell stimulates
maturation (Downs and Hunzicker-Dunn,
1995; Rodriguez et al.,
2002). The inhibition of 2-OHE2-induced GVBD at higher
concentrations of H89 and partial release of the inhibitory effect of IBMX on
the steroid-induced GVBD might be due to differential response of the PKA
isoforms or due to the action of the drug on other kinases
(Chijiwa et al., 1990;
Leemhuis et al., 2002). These
kinases may interact with cAMP-PKA pathway
(Liu and Simon, 1996). Further
detailed studies are required to define the differential responses of H89 on
the GVBD response in the catfish.
The role of PKA activity in oocyte maturation needs to be re-examined in
the context of recent studies. In Xenopus, catalytic activity of PKA
is not necessary to block meiotic maturation induced by progesterone or Mos,
although it is important for the inhibition of Cdc25C-induced maturation
(Schmitt and Nebreda, 2002).
In Atlantic croaker, inhibition of the cAMP-independent signal pathway by PI
3-kinase/Akt blocked 20ß-S-induced GVBD. The involvement of the PI
3-kinase/Akt pathway in inhibition of GVBD has also been reported in striped
bass (Weber and Sullivan,
2001). These studies are to be extended to the catfish; however,
we have shown a positive involvement of other signaling pathways such as PKC
and MAPK in oocyte maturation (Mishra and
Joy, 2006d; Mishra and Joy,
2006e). It appears that the relative importance of various signal
transduction pathways and their interactions during oocyte maturation may show
species differences.
Okadaic acid-sensitive PP (PP1 and PP2A) has been demonstrated to play an
important role in oocyte maturation
(Huchon et al., 1981;
Schwartz and Schultz, 1991;
Sun et al., 2002). OA
stimulates oocyte maturation in starfish, Xenopus and mammals
(Goris et al., 1989;
Rime and Ozon, 1990;
Gavin et al., 1991;
Schwartz and Schultz, 1991;
Sun et al., 2002) and has been
attributed to MPF (Goris et al.,
1989) or MAPK (Zernika-Goetz et al., 1997;
Sun et al., 2002) activation.
In the catfish, the incubation of follicles with various concentrations of OA
alone did not induce GVBD but together with 2-OHE2 it did
(Mishra and Joy, 2006d). Thus,
the effect of OA on meiotic resumption is dependent on the presence of the MIS
in the catfish unlike in Xenopus, starfish or mammals in which the
action is MIS (hormone) independent (Goris
et al., 1989; Pondaven et al.,
1989; Rime and Ozon,
1990). OA has stimulated MAPK activation by suppressing PP1 and
PP2A activity, leading to a higher percentage of GVBD.
In the present study, OA reversed fully the inhibitory effect of IBMX on
the 2-OHE2-induced GVBD. Similar observations were also reported in
mouse (Rime and Ozon, 1990;
Gavin et al., 1991;
Schwartz and Schultz, 1991)
and rat (Lu et al., 2001). In
Xenopus, in vivo activation of the MPF by OA does not involve PKA
(Rime et al., 1992). Rime et
al. have suggested that the OA-induced inhibition of PP1 and/or PP2A acts at a
step distal to the site of action of cAMP-PKA, activating both MPF and MAPK
(see also Sun et al., 2002;
Fan et al., 2002). In the
catfish, a similar mechanism may operate to overcome the inhibitory effect of
IBMX on the 2-OHE2-induced GVBD. Further studies are required to
identify the specific site(s) in the cascade of MPF and/or MAPK
activation.
In conclusion, the results of the present study show that cAMP-PKA inhibits the 2-OHE2-induced GVBD in the catfish. Okadaic acid per se did not support oocyte maturation, as it does in other species, but it potentiated the effect of 2-OHE2 on GVBD. OA could overcome the inhibitory effect of IBMX on GVBD, implying an action distal to the cAMP–PKA cascade.
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