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BOR - Papers in Press, published online ahead of print July 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041400
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BIOLOGY OF REPRODUCTION 73, 988–996 (2005)
DOI: 10.1095/biolreprod.105.041400
© 2005 by the Society for the Study of Reproduction, Inc.

Steroid-Induced Oocyte Maturation in Atlantic Croaker (Micropogonias undulatus) Is Dependent on Activation of the Phosphatidylinositol 3-Kinase/Akt Signal Transduction Pathway

Margaret C. Pace 1 , and Peter Thomas 

The University of Texas at Austin Marine Science Institute, Port Aransas, Texas 78373


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.

meiosis, progesterone, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fully grown oocytes from most species are arrested in prophase of meiosis I. In echinoderms and lower vertebrates oocyte meiotic arrest is released upon exposure to a species-specific maturation-inducing hormone [1]. The oocyte then undergoes a process of meiotic maturation, termed oocyte maturation, which is characterized by chromosome condensation, germinal vesicle breakdown (GVBD), and extrusion of the first polar body to produce an egg that can be fertilized [1, 2].

Oocyte maturation in fish and amphibians is one of the best-studied examples of nongenomic steroid hormone signaling [3, 4]. Although steroid hormones traditionally elicit their effects through binding of intracellular nuclear receptors and the alteration of gene transcription, maturation-inducing substances (MISs) bind receptors located on the oocyte plasma membrane to produce rapid changes in intracellular signaling pathways [5, 6]. The steroid membrane receptor mediating oocyte maturation in fish and amphibians has been hypothesized to be either the nuclear progesterone or androgen receptor [7–9], or a novel G-protein-coupled receptor [10–12]. Regardless of its nature, steroid binding to this membrane receptor activates numerous signal transduction pathways, ultimately leading to the formation and activation of maturation-promoting factor (MPF), a complex of Cdk1 and Cyclin B proteins, and GVBD. Although MPF activation is universal to oocyte maturation, the signaling events that lead to MPF activation are quite variable among species [13, 14].

One common signaling event initiated upon hormonal induction of oocyte maturation in lower vertebrates and starfish is a rapid decrease in oocyte 3'-5' cAMP levels by modulation of heterotrimeric G-protein activation, or by heterologous gap junction coupling between the follicle cells and the oocyte, or by both methods [15–19]. It has been hypothesized that decreasing cAMP concentrations in the oocyte is sufficient to promote maturation in Xenopus and mouse oocytes [20, 21], presumably through inhibition of cAMP-dependent protein kinase (Prka) activity, which leads to MPF activation [22].

Another signaling molecule activated during oocyte maturation in many species is phosphatidylinositol 3-kinase (Pik3). Activation of Pik3 is necessary for MIS-mediated oocyte maturation in starfish (1-methyladenine; [23]), striped bass (17,20ß-dihydroxy-4-pregnen-3-one; [24]), and Rana dybowskii (progesterone; [25]). In addition, FSH-induced oocyte maturation in mouse [26] and growth factor-induced oocyte maturation in Xenopus and striped bass [20, 24] require Pik3 activation. Pik3 catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5,)P3) from the plasma membrane lipid phosphtidylinositol-4,5-bisphosphate [27]. Class 1a Pik3s are composed of a regulatory p85 subunit and a catalytic p110{alpha} or p110ß subunit, and are converted to a high activity state by direct interaction with phosphotyrosine residues on activated growth factor receptors. This class of Pik3s likely mediates growth factor-induced oocyte maturation in Xenopus and striped bass [24, 28]. Class 1b Pik3s, including Pik3r3, are composed of a regulatory p101 subunit and a catalytic p110{gamma} subunit, and are converted to a high activity state by binding to free heterotrimeric G-protein ß{gamma} subunits in the plasma membrane [23, 29, 30]. Pik3r3 has been implicated in mediating 1-MA-induced oocyte maturation in starfish [23] and accelerating progesterone-induced oocyte maturation in Xenopus [31].

Activation of Pik3 and the formation of PI(3,4,5)P3 recruits signaling proteins that contain a plekstrin homology domain, such as the serine-threonine kinase Akt (also called Prkb) to the plasma membrane [27]. Activation of Akt is necessary and sufficient to induce oocyte maturation in starfish, Xenopus, and mouse [20, 26, 32]. One potential downstream target for Pik3/Akt is the activation of phosphodiesterases (Pdes), the enzymes that degrade and inactivate cAMP [21]. Activation of oocyte-specific Pde3 by Pik3/Akt was found to mediate insulin-like growth factor (IGF)-induced, but not progesterone-induced, oocyte maturation in Xenopus. In addition, incubation of Xenopus oocytes with the specific Pde3 inhibitor Cilostamide blocks IGF-induced oocyte maturation [20, 21]. Treatment with Cilostamide also completely blocks oocyte maturation in vivo and in vitro in mouse, and Pde3 activity is believed to play an essential role in the resumption of meiosis in mammalian and amphibian oocytes [21].

Activation of mitogen-activated protein kinase (Mapk) is universal during oocyte maturation, although its requirement for GVBD is uncertain [17, 33–35]. In Xenopus oocytes, overexpression of Mos, an Mapk kinase kinase, or constitutively active Mapk1/3, induces GVBD in the absence of progesterone [36, 37]. However, progesterone is able to stimulate GVBD in the presence of various Mapk inhibitors [38]. Activation of MAPK in follicle cells, but not in the oocyte, is necessary for oocyte maturation in mouse [39], and Mapk activation is neither necessary nor sufficient for inducing maturation in goldfish oocytes [34, 40].

The two closely related sciaenid fishes, Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), are well established models of oocyte maturation whose maturation-inducing steroid has been clearly identified as the progestin 17,20beta,21-trihydroxy-4-pregnen-3-one (known throughout as 20ß-S) [41, 42]. Previous studies in spotted seatrout have shown that 20ß-S binds to a receptor located in the oocyte plasma membrane [43], which has recently been identified as a novel seven-transmembrane receptor [44]. 20ß-S binding to this receptor activates a pertussis toxin-sensitive, inhibitory G-protein (Gi), inducing a transient decrease in cAMP [11, 44, 45]. The purpose of the current study was to identify the signal transduction pathways activated downstream of Gi that are necessary for 20ß-S-mediated oocyte maturation in Atlantic croaker. Specifically, we examined whether inhibition of Prka or activation of the Pik3/Akt/Pde or Mapk signal transduction pathways are necessary or sufficient to promote GVBD in Atlantic croaker oocytes. It is hypothesized that activation of signal transduction pathways independent of cAMP, such as the Pik3/Akt pathway, is necessary for 20ß-S-induced oocyte maturation in Atlantic croaker.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

All nonradioactive steroids were obtained from Steraloids (Newport, RI). Dulbecco modified Eagle medium with phenol red (DMEM, nutrient mixture F-12), actinomycin D, penicillin G, hCG, guanosine 5'-diphosphate, guanosine 5'-triphosphate (GTP), phosphoenol pyruvate, pyruvate kinase, and basic laboratory chemicals were purchased from Sigma Aldrich (St. Louis, MO). Inhibitors of Pik3 (Wortmannin and LY294002), Akt (ML-9), Pde3 (Cilostamide), Pde4 (Rolipram), Prka (KT5720 and Rp-cAMP), and Mapk2k1&2 (PD98059 and U0126) were purchased from Biomol (Plymouth, MA). Antibodies for phosphoMapk1&3 (pT202, pY204), total Mapk1&3, phosphoAkt (pSer473), and total Akt proteins were purchased from Cell Signaling Technology (Beverly, MA). Halt protease inhibitor cocktail and Supersignal West Pico Chemiluminescent Substrate System were purchased from Pierce Biotechnology (Westford, IL).

Animal Collection

First-year Atlantic croaker were purchased in September from commercial fishermen in Aransas Pass, TX. Fish were returned to the laboratory and maintained in 16 000-L recirculation tanks on a 10L:14D cycle. Female Atlantic croaker were anesthetized, and an ovarian biopsy sample was obtained by inserting a catheter attached to a syringe via the cloaca into the oviduct. The average diameter of the oocytes was determined under a binocular microscope. Oocytes with an average diameter of 450 to 470 µm were considered fully grown and suitable for experimentation. Fish were humanely killed by severing the spinal cord following procedures approved by the University of Texas at Austin Animal Care and Use Committee. The ovaries were immediately removed, weighed, and either transferred to DMEM pH 7.6 containing 100 mg/L streptomycin and 60 mg/L penicillin for in vitro incubations, or frozen on dry ice and stored at –80°C.

Oocyte Membrane Preparation

Oocyte membranes were prepared as previously described [46]. Briefly, ovarian tissue fragments were finely minced in ice-cold homogenization buffer containing 25 mM Hepes pH 7.6, 10 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 1 mM EDTA, and the fragments were repeatedly expelled through an 18- and then a 22-gauge needle. Oocytes were washed through a 400-µm Nitex mesh to remove follicle cells and immature oocytes, and homogenized in 2 ml of homogenization buffer with five passes through a glass tissue grinder. The homogenate was centrifuged twice at 500 x g for 5 min, and the pellet discarded. The supernatant was centrifuged at 20 000 x g for 20 min, and the resulting supernatant discarded. The pellet was resuspended in 1.5 ml of homogenization buffer and further separated over a 1.2 M sucrose pad (1 volume pellet resuspension:1 volume sucrose buffer) by centrifugation at 6500 x g for 45 min. The middle-layer plasma membrane fraction was collected, washed, and the final membrane pellet was resuspended to a concentration of 1 mg of total protein per milliliter of buffer containing 75 mM Tris-HCl, 5 mM MgCl2, 2 mM EDTA, and 1x Halt protease inhibitor cocktail.

Measurement of Adenylyl Cyclase Activity

Defolliculated Atlantic croaker oocyte membranes (oocyte membranes) were thawed on ice. Ten micrograms of oocyte membranes were incubated on ice with assay buffer containing 0.1 mM ATP, 5 nM GTP, 0.25 mM phosphoenolpyruvate, 10 µg pyruvate kinase, 1 mM isobutylmethylxanthine, and either 290 nM 20ß-S, 290 nM cortisol, or vehicle (0.01% ethanol) in a final volume of 100 µl. Incubations were initiated by the addition of the oocyte membranes to the assay buffer. Cyclic AMP production was measured at 0-, 1-, 5-, 10-, 15-, and 30-min intervals. Incubations were terminated by boiling the samples for 5 min. Samples were centrifuged for 15 min at 12 000 x g and the supernatant collected. The cAMP concentration of the supernatants was measured using a cAMP enzyme immunoassay kit according to the manufacturer's instructions (Biomedical Technologies Inc., Stoughton, MA).

GVBD Bioassay

Ovarian tissue was collected from reproductively mature female seatrout as described above and placed in DMEM supplemented with antibiotics. Ovarian fragments containing approximately 100 follicles were incubated in 24-well culture plates in 1 ml DMEM containing 15 U hCG at 24°C for 9 h (priming). After priming, follicles were washed once with 1 ml DMEM and then transferred to fresh DMEM containing 290 nM 20ß-S, unless otherwise noted with or without inhibitor, and incubated for an additional 12 h at 24°C. After 12 h the oocytes were scored for completion of GVBD, the first easily identifiable event in oocyte maturation [41]. Follicles were then collected, defolliculated as described above, and frozen at –80°C. Thirty minutes before collection follicles were treated with 10 µM Calyculin A, a phosphatase inhibitor. Concentrations of inhibitors were as follows: Wortmannin, 0.1 nM, 1 nM, and 10 nM; LY294002, 2.5 µM, 5 µM, and 25 µM; ML-9, 3 µM, 6 µM, 12 µM, and 25 µM; Cilostamide, 100 nM, 1 µM, and 10 µM; Rolipram, 10 nM, 100 nM, and 1 µM; KT-5720, 1 µM; Rp-cAMP, 5 mM; PD98059, 500 nM, 2.5 µM, 25 µM, and 50 µM; U0126, 10 nM, 100 nM, 1 µM, and 10 µM. Control incubations for each bioassay included oocytes incubated without hCG or 20ß-S (negative GVBD control), hCG-treated (primed) oocytes incubated without 20ß-S (overpriming control), primed oocytes incubated with either 0.01% ethanol or 0.01% to 0.1% dimethylsulfoxide and 290 nM 20ß-S (vehicle control), and unprimed oocytes incubated with 290 nM 20ß-S (in vivo priming control). Inhibitors were diluted according to the manufacturer's instructions and were added 30 min before the addition of steroid.

Immunoblotting

Defolliculated oocytes were homogenized in 200 µl of buffer containing 50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3PO4, 1 mM NaF, and 1x Halt protease inhibitor cocktail with five passes through a glass tissue grinder. Samples were incubated with slight agitation for 45 min at 4°C. The homogenate was centrifuged twice at 15 000 x g for 5 min and the pellet discarded. The supernatant was sonicated for 5 sec on ice and the final protein concentration was determined using the Bradford protein assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA). For immunoblotting, 5 µg of total protein were electrophoresed through a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in 5% dry milk dissolved in Tris-buffered saline containing 0.1% Tween (TBS-T) and then incubated with antiphosphoMapk1/3 antibody (1:500), anti-Mapk1/3 antibody (1:500), antiphosphoAkt antibody (1:500), or anti-Akt antibody (1:500) diluted in TBS-T overnight at 4°C. Membranes were washed three times in TBS-T and incubated for an additional 1 h at room temperature with horseradish peroxidase-labeled anti-rabbit secondary antibody (1:8000). Membranes were developed using the Supersignal West Pico Chemiluminescent Substrate System according to the manufacturer's instructions and exposed to film for 5 sec to 5 min to visualize immunoreactive bands. The positive control for the anti-Akt antibodies was Calyculin A-treated Jurkat cell extracts (human T lymphocytes) and the positive control for the anti-Mapk1/3 antibodies was 10% serum-treated MDA-MB-231 cell extracts (human breast cancer cell line).

Statistical Analyses

Values are reported as means ± SEM and were analyzed using GraphPad Prism software version 3.02 (GraphPad Prism Software, San Diego, CA; http://www.graphpad.com). Results were analyzed by one-way random analysis of variance (RM-ANOVA) with the Dunnett multiple comparison test, and significance is reported as P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of 20ß-S Treatment on cAMP Production

To test whether treatment with 20ß-S caused a measurable decrease in cAMP production in Atlantic croaker oocytes, isolated oocyte membranes were treated with vehicle (0.01% ethanol), 290 nM cortisol, or 20ß-S, and cAMP production was measured over 30 min. Addition of 290 nM 20ß-S significantly reduced adenylyl cyclase activity and cAMP production by approximately 50% within 1 min and for up to 5 min, as compared to control membranes with no steroid added (Fig. 1). The effect of 20ß-S on adenylyl cyclase activity was specific to the MIS. Addition of 290 nM cortisol, a C21 steroid that does not bind the 20ß-S receptor or promote GVBD, did not reduce adenylyl cyclase activity.



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  		FIG. 1. Effect of 20ß-S treatment on oocyte adenylyl cyclase activity. Cyclic AMP production was measured in unstimulated oocyte membranes (Control) or oocyte membranes stimulated with 290 nM 20ß-S or cortisol. Data are reported as mean pmol cAMP produced per milligram of protein per minute ± SEM (n = 4). *P < 0.05, **P < 0.01 compared to control membranes by one-way RM-ANOVA with the Dunnett multiple comparisons test

Effect of Inhibitors of Prka, Pde3, and Pde4 on GVBD

To test whether the 20ß-S-induced decrease in cAMP concentration was sufficient to initiate GVBD, the effects of pharmacologic inhibitors of Prka, oocyte-specific Pde3, and follicle-specific Pde4 on GVBD were investigated. Incubation of primed Atlantic croaker follicles with either of two cell-permeable inhibitors of Prka, KT5720, or Rp-cAMP did not induce maturation in the absence of steroid (data not shown), nor did they enhance the efficacy of the steroid to promote GVBD (Fig. 2A) or accelerate the timing of GVBD (Fig. 2B). In contrast, inhibition of Pde3 by Cilostamide (100 nM to 10 µM) significantly decreased GVBD in Atlantic croaker follicles by approximately 55% compared to controls (Fig. 3A). However, Cilostamide concentrations as high as 50 µM were not able to further reduce the percentage of GVBD (data not shown). Inhibition of Pde4 using 1 µM Rolipram significantly reduced GVBD by 72% (Fig. 3B); however, inhibitor concentrations up to 25 µM did not further reduce the percentage of GVBD (data not shown).



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  		FIG. 2. Effects of Prka inhibition on the efficacy of 20ß-S to promote or accelerate GVBD in Atlantic croaker follicles. A) Primed Atlantic croaker follicles were incubated with either media only (Control), 1 µM KT5720, or 5 mM Rp-cAMP in the presence of 145, 73, or 35 nM 20ß-S for 12 h and scored for GVBD. Bars are mean percent GVBD + SEM (n = 4). B) Primed Atlantic croaker follicles were incubated with media (control), 1 µM KT5720, or 5 mM Rp-cAMP in the presence of 290 nM 20ß-S and the time course for GVBD recorded. Symbols represent the mean percent GVBD per hour ± SEM (n = 4)



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  		FIG. 3. Effects of Pde inhibition on GVBD in Atlantic croaker follicles. Primed Atlantic croaker follicles were incubated with either vehicle (Control), 100 nM to 10 µM Cilostamide (A) or 10 nM to 1 µM Rolipram (B) in the presence of 290 nM 20ß-S for 12 h and scored for GVBD. Bars represent the mean percent GVBD ± SEM (n = 4). **P < 0.01, ***P < 0.001 compared to control follicles by one-way RM-ANOVA with the Dunnett multiple comparisons test

Effect of Inhibitors of Pik3 on GVBD

To examine whether Pik3 activation was necessary for steroid-mediated GVBD, primed Atlantic croaker follicles were incubated with either Wortmannin or LY294002, two mechanistically different Pik3 inhibitors. Both inhibitors significantly reduced GVBD and relatively low concentrations of Wortmannin (10 nM) or LY294002 (25 µM) completely blocked GVBD (Fig. 4A). Additionally, the efficacy of LY294002 inhibition on GVBD was dependent on the concentration of 20ß-S. Increased inhibition of GVBD by 1 µM LY294002 was observed in oocytes stimulated with 35 nM 20ß-S, a physiologically relevant concentration of steroid, as compared to oocytes incubated with 145 nM 20ß-S (64% versus 23%, respectively; Fig. 4B). Due to the large variability in the percentage of GVBD in control fish from these experiments, a representative fish is shown. Inhibition of Pik3 using either 10 nM Wortmannin or 25 µM LY294002 also blocked the phosphorylation and activation of Akt and Mapk1/3 (Fig. 4C).



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  		FIG. 4. Effect of Pik3 inhibition on GVBD in Atlantic croaker oocytes. A) Primed Atlantic croaker follicles were incubated with media only (control), 0.1–10 nM Wortmannin, or 2.5–25 µM LY294002 in the presence of 290 nM 20ß-S for 12 h and scored for GVBD. Bars represent the mean percent GVBD ± SEM (n = 4). ***P < 0.001 compared to control membranes by one-way RM-ANOVA with the Dunnett multiple comparisons test. B) Primed Atlantic croaker follicles were incubated with media only (Control), or 1 µM or 2.5 µM LY294002 in the presence of 145, 73, or 35 nM 20ß-S. Bars are percent GVBD from a representative fish (n = 3). C) Defolliculated oocyte lysates (5 µg) of ovarian follicles incubated with 290 nM 20ß-S and 0.1 to 10 nM Wortmannin or 2.5 to 25 µM LY294002 for 4 h were analyzed by SDS-PAGE and immunoblotting using antiphosphoAkt (top panel), anti-Akt (second panel), antiphosphMapk1/3 (third panel), or anti-Mapk1/3 antibodies (fourth panel). Positive controls (+) for Akt are Calyculin A-treated Jurkat cell extracts and positive controls for Mapk1/3 are serum-treated MDA-MB-231 cell extracts. Data are representative of three independent experiments

Effect of 20ß-S Treatment on Akt Activation and Effect of Inhibitors of Akt on GVBD

A potential next step in the steroid-initiated Pik3 signaling pathway is activation of the serine/threonine kinase Akt. Immunoblot analyses of Atlantic croaker oocyte lysates using an antiphosphoAkt antibody that specifically recognizes the activated form of the protein showed that Akt was activated within 1 h of 20ß-S treatment (Fig. 5A). Incubation of primed Atlantic croaker follicles with 3 µM ML-9, an inhibitor of Akt, significantly reduced GVBD by 34% compared to control follicles, and 25 µM ML-9 completely blocked GVBD (Fig. 5B). These concentrations are below those reported to inhibit Prka (IC50 30 µM) or S6 kinase activity (IC50 50 µM) by ML-9, suggesting the inhibitor effect was specific for Akt [47]. GVBD was completely inhibited by 6 µM ML-9 in follicles treated with 35 nM 20ß-S (Fig. 5C). Finally, immunoblot analysis using antiphosphoAkt antibody confirmed the effectiveness of ML-9 to block Akt activation by 20ß-S in Atlantic croaker oocytes (Fig. 5D).



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  		FIG. 5. Effect of Akt inhibition on GVBD in Atlantic croaker oocytes. A) Primed Atlantic croaker follicles were incubated with 290 nM 20ß-S and samples were collected at 0, 1, 2, 4, 8, and 12 h. Defolliculated oocyte lysates (5 µg) were analyzed by SDS-PAGE and immunoblotting using antiphosphoAkt (top panel) and anti-Akt (bottom panel) antibodies. Positive control samples (+) are Calyculin A-treated Jurkat cell extracts. B) Primed Atlantic croaker follicles were incubated with vehicle (Con) or 3, 6, 12, or 25 µM ML-9 and 290 nM 20ß-S for 12 h and scored for GVBD. Bars are mean percent GVBD + SEM (n = 4). C) Primed Atlantic croaker follicles were incubated with vehicle (Control), or 6 or 12 µM ML-9 in the presence of 145, 73, or 35 nM 20ß-S. Bars are mean percent GVBD ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control follicles by one-way RM-ANOVA with the Dunnett multiple comparisons test. D) Defolliculated oocyte lysates (5 µg) of ovarian follicles incubated with 290 nM 20ß-S and 6, 12, or 25 µM ML-9 for 4 h were analyzed by SDS-PAGE and immunoblotting using antiphospoAkt (top panel) and anti-Akt (bottom panel) antibodies

Effect of 20ß-S Treatment on Mapk1/3 Activation and Effect of Inhibitors of Mapk1/3 on GVBD

We examined the effect of two inhibitors of Mapk2k1/ 2, the kinase that directly activates Mapk1/3, on Atlantic croaker oocyte maturation. Immunoblot analysis of oocyte lysates using an antiphosphoMapk1/3 antibody that is specific for the activated form of the protein detected an increase in Mapk1/3 activation in 20ß-S-treated follicles within 4 h of steroid addition (Fig. 6A). Treatment of primed follicles with either of two different Mapk1/3 inhibitors, 50 µM PD98059 or 10 µM U0126, had no effect on GVBD (Fig. 6B). These concentrations are well above the reported IC50 for either inhibitor, 2 µM and 72 nM, respectively. Higher concentrations of either inhibitor killed the follicles (data not shown). Immunoblot analysis confirmed the effectiveness of the inhibitors to block Mapk1/ 3 activity in 20ß-S-treated oocytes (Fig. 6C).



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  		FIG. 6. Effect of Mapk inhibition on GVBD in Atlantic croaker oocytes. A) Primed Atlantic croaker follicles were incubated with 290 nM 20ß-S and samples were collected at 0, 1, 2, 4, 8, and 12 h. Defolliculated oocyte lysates were analyzed by SDS-PAGE and immunoblotting using antiphosphoMapk1/3 (top panel) and anti-Mapk1/3 (bottom panel) antibodies. Positive control samples (+) are serum-treated MDA-MB-213 cell extracts. B) Primed Atlantic croaker follicles were incubated with vehicle (Control), 500 nM to 25 µM PD98059 or 10 nM to 10 µM U0126, and 290 nM 20ß-S for 12 h and scored for GVBD. Bars are mean percent GVBD ± SEM (n = 4). C) Defolliculated oocyte lysates (5 µg) of ovarian follicles incubated with 290 nM 20ß-S and 500 nM to 25 µM PD98059 or 100 nM to 10 µM U0126 for 12 h were analyzed by SDS-PAGE and immunoblotting using antiphosphMapk1/3 (top panel) or anti-Mapk1/3 (bottom panel) antibodies. Data are representative of three independent experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that whereas 20ß-S treatment of oocyte membranes significantly decreases cAMP production, activation of cAMP-independent signal transduction pathways, specifically the Pik3/Akt pathway, is necessary for oocyte maturation in Atlantic croaker. This finding is in agreement with similar studies in other species, including starfish, striped bass, and Rana dybowskii, that found Pik3 activation, which is independent of cAMP and Prka, is necessary for MIS-mediated oocyte maturation [24, 25, 48].

The lack of GVBD observed in Atlantic croaker oocytes incubated with Prka inhibitors contrasts with observations in catfish and mouse, in which pharmacologic inhibition of Prka activity induces oocyte maturation in the absence of steroid [49, 50]. It is possible that physiological differences between Atlantic croaker oocytes, compared to catfish and mouse oocytes, prevented the Prka inhibitors from entering the oocyte in sufficient concentrations to effectively block the kinase activity. However, these findings, in conjunction with results from the Pik3/Akt inhibition experiments, support the hypothesis that activation of cAMP-independent signaling pathways is necessary for oocyte maturation in Atlantic croaker.

Previous studies have identified PDE3 as the predominant PDE isoform in mammalian oocytes and PDE4 as the predominant PDE isoform in mammalian follicle cells [51, 52]. The current finding that inhibition of either Pde3 or Pde4 significantly reduced the percentage of GVBD in Atlantic croaker oocytes suggests that high cAMP levels in the follicle cells and oocyte assist in maintaining meiotic arrest. However, failure of the inhibitors to completely block GVBD may be an indication that increased Pde3/4 activity alone is not sufficient to promote oocyte maturation in the absence of MIS. In contrast, inhibition of PDE3 blocked spontaneous and hCG-stimulated GVBD in >90% of rat and mouse oocytes, and completely blocked IGF-induced GVBD in Xenopus [21, 53]. The effect of PDE4 inhibition on oocyte maturation in mammals is less clear, with some authors reporting delayed meiotic resumption in the presence of the inhibitor [54], whereas others found no effect [55]. One explanation for the significant reduction in GVBD observed with Pde4 inhibition in Atlantic croaker follicles is that, in contrast to other species, heterologous gap junctions between the follicle cells and oocyte are maintained, and even increase, during the onset of meiotic maturation [56]. The significant effect of follicle-specific Pde4 inhibition on GVBD in Atlantic croaker oocytes suggests that decreasing cAMP levels in the follicle cells, perhaps by reducing transfer of cAMP from the follicle cells to the oocyte through gap junctions, assists in releasing the oocyte from meiotic arrest.

The finding that incubation of Atlantic croaker follicles with either of two mechanistically different Pik3 inhibitors, Wortmannin or LY294002, completely blocked GVBD further supports the hypothesis that activation of cAMP-independent signaling pathways is necessary for oocyte maturation. Further, the effectiveness of Pik3 inhibition by LY29400 to block GVBD in Atlantic croaker was dependent on the concentration of 20ß-S, presumably due to a reduction in the number of occupied 20ß-S receptors and Pik3 activation at low steroid concentrations, 35 nM versus 145 nM. Similar findings have been observed in starfish, striped bass, and Rana dybowskii [23–25]. In contrast, both LY294002 and Wortmannin failed to inhibit progesterone-stimulated GVBD in Xenopus oocytes [57, 58]. Activated forms of class 1a Pik3s, which are recruited to the plasma membrane by activated growth factor receptors, can induce oocyte maturation in Xenopus in the absence of progesterone [59] and microinjection of the class 1a Pik3 p85 regulatory subunit blocks progesterone-stimulated GVBD [60]. However, these findings likely represent the growth factor-mediated, and not progesterone-mediated, signal transduction pathway leading to oocyte maturation.

Previous studies with Xenopus and starfish oocytes have shown that Akt activation is necessary and sufficient to induce oocyte maturation, although Akt acts on different downstream effectors; Pde3 in Xenopus and Myt 1 in starfish [20, 32, 45]. The increased effectiveness of Akt inhibition to block GVBD in Atlantic croaker follicles incubated with a physiologically relevant concentration of 20ß-S (35 nM), presumably through a reduction in 20ß-S receptor binding and Akt activation, provides evidence that the effects of ML-9 are specific for 20ß-S-induced activation of Akt and not the result of a nonspecific or toxic effect on the oocyte. That inhibition of Akt but not Pde3 blocks GVBD suggests that Akt is activating multiple downstream effectors, one of which is Pde3 (Fig. 7A). These results, in addition to those described above, indicate that Akt is an essential component of the 20ß-S/Gß{gamma}/Pik3 signal transduction pathway mediating meiotic maturation in Atlantic croaker oocytes (Fig. 7A).



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  		FIG. 7. Proposed model of the signal transduction pathways activated in the oocyte in response to 20ß-S treatment of primed Atlantic croaker follicles. A) Binding of 20ß-S to its G-protein-coupled receptor located in the oocyte plasma membrane activates an inhibitory G-protein. The G-protein {alpha} or ß{gamma} subunits (or both) decrease adenylyl cyclase activity, lowering cytosolic cAMP concentrations and reducing Prka activation. The Gß{gamma} subunits also recruit Pik3 to the plasma membrane. Pik3 catalyzes the formation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) from the plasma membrane lipid phosphtidylinositol-4,5-bisphosphate (PIP2). Plekstrin homology domain-containing proteins such as the serine/threonine kinase Akt are then recruited to the plasma membrane by binding to the newly formed PIP3. Activation of Akt activates or inactivates (or both) a number of downstream effectors, including Pde3 and Myt1. Activation of Pde further reduces cAMP concentrations and Prka activation. These events culminate in the activation of maturation-promoting factor (MPF) and GVBD. B) Treatment of primed Atlantic croaker follicles with 20ß-S also initiates translation of the Mapk kinase kinase, mos. Mos protein phosphorylates and activates Map2k1/2, which in turn, phosphorylates and activates Mapk1/3. Alternatively, Gß{gamma}, Pik3 or both may recruit the small G-protein Ras to the plasma membrane and activate Mapk1/3 through the Raf/Map2k1/2 pathway [63]. Activated Mapk1/3 is not necessary for GVBD but may act as a cyctostatic factor (CSF) preventing DNA replication during the second meiotic division

Early studies on the role of Mapk activation on GVBD suggested that expression of Mos, which activates Mapk, was required for progesterone-induced meiotic maturation in Xenopus oocytes [36]. Later studies in starfish, mouse, Xenopus, and goldfish using a variety of techniques including pharmacologic inhibitors, mos morpholino antisense oligonucleotides, and Mos gene knockouts, showed that Mapk activation is not necessary for GVBD, but rather is acting as a cytostatic factor to suppress DNA replication between the two meiotic divisions [14, 35, 61, 62]. Failure of Mapk inhibition to block GVBD in Atlantic croaker oocytes is in agreement with these studies and confirms that Mapk1/3 activation is not necessary for GVBD. Inhibition of Pik3 blocks Mapk1/3 activation, suggesting that Pik3 activates multiple downstream signal transduction pathways, not all of which are necessary for GVBD (Fig. 7B).

Previous studies on oocyte maturation in sciaenid fishes have shown that the 20ß-S oocyte membrane receptor is coupled to an inhibitory G-protein and pertussis toxin microinjection experiments have established that activation of this G-protein is necessary for GVBD [11, 44, 45]. It has also been shown that 20ß-S membrane receptor concentrations increase during hCG induction of oocyte maturation in vitro and also in vivo in fish undergoing oocyte maturation in the laboratory or on their spawning grounds [46]. There is recent evidence of a role in sciaenid oocyte maturation for the novel membrane progestin receptor, mPR{alpha} [12]. Similar to the changes observed with 20ß-S membrane receptor-binding activity, levels of oocyte mPR{alpha} mRNA and protein increase in response to gonadotropin treatment (priming) in in vitro GVBD bioassays [12]. Additionally, recent unpublished heterologous expression studies (Thomas and Pang) in MDA-MB-231 cells show that mPR{alpha} specifically binds progestins to directly activate a pertussis toxin sensitive G-protein and decrease cAMP production, mirroring the effects of 20ß-S in the oocyte. The current study characterizes the signal transduction events initiated downstream of G-protein activation that are necessary for 20ß-S-induced GVBD in a sciaenid fish. The present results, together with previous findings demonstrating Gi activation in oocytes treated with 20ß-S, provide detailed descriptions of the signal transduction pathways activated in sciaenid fish oocytes by the MIS, which are summarized in the models in Figure 7. Identification of the naturally occurring MIS (20ß-S) in sciaenid fishes and the extensive characterization of its receptor and the signal transduction pathways activated upon steroid binding to the receptor make sciaenids an excellent biological model for the study of nongenomic actions of maturation-inducing steroid to initiate oocyte maturation.


   FOOTNOTES
 
1 Correspondence: Margaret C. Pace, The University of Texas at Austin Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373. FAX: 361 749 6777; mpace{at}utmsi.utexas.edu Back

Received: 1 March 2005.

First decision: 8 April 2005.

Accepted: 13 July 2005.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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