okadaic-acid and 2-hydroxyestradiol

Mitogen-activated protein kinase (MAPK) was demonstrated in the postvitellogenic follicles (theca-granulosa and oocyte) of catfish by Western blotting using a polyclonal anti-rabbit serum, which recognized both ERK1 and ERK2. Two distinct protein bands resolved in the 46-48 kDa range of 12% SDS-PAGE were immunoblotted. Incubation of the follicles with 5 microM 2-OHE2 elicited GVBD significantly in a duration-dependent manner with a concomitant increase in the expression of MAPK (ERK1 and ERK2). Densitometric analysis of the immunoblots showed significant variations in the intensity of staining. The ERK1 expression increased significantly from 6 h onwards but the changes were less pronounced. On the other hand, ERK2 registered a sharp significant increase after 3h, which paralleled the GVBD response. The MEK inhibitor PD098059 alone did not induce GVBD. Co-incubation of the follicles with 2-OHE2 and PD098059 significantly inhibited the steroid-induced GVBD at all concentrations. Immunoblot analysis showed that PD098059 inhibited MAPK activity significantly compared to the 2-OHE2 group. The addition of okadaic acid (OA) in the incubation medium containing both 2-OHE2 and PD098059 reversed the inhibitory effect of the latter and GVBD was elevated significantly over that of the 2-OHE2 group but significantly lower than that of the 2-OHE2 + OA group. The results suggest an involvement of MAPK in meiotic maturation but the site(s) of action: oocyte, follicular envelope or both needs further investigation.

Topics: 17-alpha-Hydroxyprogesterone; Animals; Catfishes; Estradiol; Female; Flavonoids; Hydroxyprogesterones; Mitogen-Activated Protein Kinases; Okadaic Acid; Oocytes; Phosphoprotein Phosphatases; Progesterone; Sexual Maturation

In vitro effects of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calphostin C (PKC inhibitor) and okadaic acid [OA, a protein phosphatase (PP; PP1 and PP2A) inhibitor] on 2-hydroxyestradiol-17beta (2-OHE(2))-induced oocyte maturation were investigated in the catfish Heteropneustes fossilis. Incubations of postvitellogenic follicles with PMA or OA alone did not induce oocyte maturation. However, co-incubations with 2-OHE(2) and PMA (0.05, 0.5 and 5 microM) or 2-OHE(2) and OA (0.5, 1.0 or 2.0 microM) increased germinal vesicle breakdown (GVBD) significantly over that of 2-OHE(2). Incubation of follicles with calphostin C elicited varied effects on GVBD, low (0.005 and 0.01 microM) and high (5.0 and 10.0 microM) concentrations did not affect GVBD, but medium concentrations (0.05, 0.1, 0.5, 1.0 and 2.5 microM) stimulated it. The medium concentrations elicited a biphasic stimulatory response with peak GVBD at 0.1 microM (54%). Calphostin C (>or=2.5 microM) inhibited the 2-OHE(2)-induced GVBD in a concentration-dependent manner during the 24 h incubation. Pre- or post-treatment with calphostin C inhibited the steroid-induced GVBD only at 6 h. In co-incubation studies, both PMA and OA reversed the inhibitory effect of calphostin C: the former partially and the latter fully. The results of the present study show that PKC appears to modulate the 2-OHE(2)-induced oocyte maturation. The OA-sensitive PP may be involved in the PKC modulation of steroid-induced oocyte maturation.

Topics: Animals; Catfishes; Cell Differentiation; Enzyme Inhibitors; Estradiol; Female; Naphthalenes; Okadaic Acid; Oocytes; Phosphoprotein Phosphatases; Protein Kinase C; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate

In Heteropneustes fossilis, in vitro incubation of postvitellogenic follicles with 2-hydroxyestradiol-17beta (2-OHE2, 5 micromol l(-1)) decreased significantly the total cAMP level, concomitant with germinal vesicle breakdown (GVBD). The incubation of the follicles with cAMP or cAMP-elevating drugs [phosphodiesterase (PDE) inhibitors], such as IBMX (3-isobutyl-1-methyl-xanthine), theophylline and caffeine, inhibited the 2-OHE2-induced GVBD in a concentration-dependent manner. The magnitude of the response varied: both cAMP and IBMX were effective at all concentrations (0.1-2.0 mmol l(-1)), followed by theophylline (0.5-2.0 mmol l(-1)) and caffeine (1-2.0 mmol l(-1)). The protein kinase A (PKA) inhibitor H89 stimulated oocyte maturation in a concentration-dependent manner. However, when co-incubated with 2-OHE2 for 24 h it produced a biphasic effect: low concentrations (0.1 and 1.0 micromol l(-1)) did not alter the 2-OHE2-induced GVBD, but high concentrations (5 and 10 micromol l(-1)) inhibited it. The incubation of the follicles with H89 lowered the inhibitory effect of IBMX on the 2-OHE2-induced GVBD. The incubation of the follicles with okadaic acid (OA), a protein phosphatase 1 and 2A inhibitor did not affect GVBD but when co-incubated with 2-OHE2, it enhanced the GVBD response. OA reversed the inhibitory effect of IBMX. The results suggest that OA may overcome the inhibition of 2-OHE2-induced GVBD by IBMX at a step distal to the cAMP-PKA pathway.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Catfishes; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Estradiol; Female; Isoquinolines; Okadaic Acid; Oocytes; Ovarian Follicle; Phosphodiesterase Inhibitors; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Protein Phosphatase 1; Signal Transduction; Sulfonamides