Molecular and Cellular Endocrinology 390 (2014) 26–33
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Steroid-induced oocyte maturation in Indian shad Tenualosa ilisha (Hamilton, 1822) is dependent on phosphatidylinositol 3 kinase but not MAP kinase activation Kousik Pramanick a, Sourav Kundu b, Sudipta Paul c, Buddhadev Mallick c, Sujata Roy Moulik c, Puja Pal c, Dilip Mukherjee c,⇑ a
Department of Zoology, Presidency University, 86/1 College Street, Kolkata 73, India Department of Physiology and Biophysics, School of Medicine, Health Sciences Center-A, University of Louisville, KY 40292, USA c Endocrinology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India b
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 28 March 2014 Accepted 1 April 2014 Available online 12 April 2014 Keywords: 17,20b-P Oocyte maturation PI3 kinase MAP kinase cdc2 kinase Tenulosa ilisha
a b s t r a c t Fully grown ﬁsh and amphibian oocytes exposed to a maturation-inducing steroid (MIS) activates multiple signal transduction pathways, leading to formation and activation of maturation-promoting factor (MPF) and induction of germinal vesicle breakdown (GVBD). The present study was to investigate if phosphatidylinositol 3 kinase (PI3 kinase) and mitogen-activated protein kinase (MAP kinase) activation are required for naturally occurring MIS, 17a,20b-dihydroxy-4-pregnen-3-one (17,20b-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that17,20b-P-induced OM was signiﬁcantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20b-P was shown to activate PI3 kinase maximally at 90 min and cdc2 kinase at 16 h of treatment. Relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in 17,20b-P-induced OM was examined. MAP kinase was rapidly phosphorylated and activated (60–120 min) after MIS treatment and this response preceded the activation of cdc2 kinase by several hours. A selective inhibitor of MAP kinase (MEK), PD98059, sufﬁciently blocked the phosphorylation and activation of MAP kinase. Inhibition of MAP kinase activity using PD98059 however, had no effect on MIS-induced cdc2 kinase activation and GVBD. These results demonstrate that activation of the PI3 kinase is required for 17,20b-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20b-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Fully grown oocytes get arrested at prophase of ﬁrst meiosis and in echinoderms and lower vertebrates species-speciﬁc maturation-inducing steroid (MIS) relieves oocytes from this arrest (Masui and Clarke, 1979). Oocytes then undergo a process of meiotic maturation, termed oocyte maturation (OM), characterized by chromosome condensation, germinal vesicle breakdown (GVBD), formation of metaphase spindle and extrusion of ﬁrst polar body to produce an egg that can be fertilized (Masui and Clarke, 1979; Masui, 2001; Contreras et al., 2003). In most ﬁshes, 17a,20b-dihydroxy-4-pregnen-3-one (17,20b-P) is considered to be the potent and effective MIS (for review see Nagahama, 1997). In sciaenid and some other ﬁshes, however, 17a,20b,21-trihydroxy-4⇑ Corresponding author. Fax: +91 33 2582 8282. E-mail addresses: [email protected]
, [email protected]
(D. Mukherjee). http://dx.doi.org/10.1016/j.mce.2014.04.002 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
pregnen-3-one (20b-S) has been identiﬁed as MIS (Trant and Thomas, 1989; Thomas et al., 2002). In ﬁsh and amphibians, OM is initiated by binding of MIS to a novel G protein-coupled membrane progestin receptor (mPRa) on the oocyte surface and activates various signal transduction pathways, ultimately leading to the formation and activation of maturation-promoting factor (MPF) and to GVBD (Lokha et al., 1988; Labbe et al., 1989). Carp MPF, like that of Xenopus (Lokha et al., 1988; Gautier et al., 1988, 1990), consists of two components: one is a homolog of cdc2+ gene product of ﬁssion yeast (Saccharomyces pombe), referred to as p34cdc2 (cdc2), and other is cyclin B. Immature goldﬁsh oocyte contained a 35-kDa cdc2, whereas mature oocytes contained a 34-kDa cdc2 kinase as well as the 35-cdc2 kinase (Hirai et al., 1992; Kajiura et al., 1993). The 34and 35-kDa cdc2 proteins are active and inactive forms respectively (see review Nagahama, 1997). Although the function of MPF in promoting OM is ubiquitous, there are species-speciﬁc differences in the signaling events that leads to MPF activation
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(Schmitt and Nebreda, 2002; Voronina and Wessel, 2004; Pace and Thomas, 2005a,b). It is now known that in lower vertebrates, a decrease in oocyte cAMP levels is sufﬁcient to promote steroid-induced OM, presumably through inhibition of cAMP-dependent protein kinase (Prka) activity (Anderson et al., 1998; Pace and Thomas, 2005a). Steroid-induced OM is also mediated through activation of phosphatidylinositol 3 kinase (PI3 kinase) (Sadler and Ruderman, 1998; Weber and Sullivan, 2001; Ju et al., 2002; Pace and Thomas, 2005b). PI3 kinase activation is also necessary for growth factorinduced OM in Xenopus, striped bass and carp (Anderson et al., 1998; Weber and Sullivan, 2001; Paul et al., 2009). Mitogen-activated protein kinase (MAP kinase) is rapidly activated in response to hormones and growth factors for diverse biological functions (Pearson et al., 2001; Roux and Blenis, 2004). MAP kinase plays an important role in the process of OM in Xenopus (Sagata et al., 1989; Haccard et al., 1995). Involvement of MAP kinase in 2-hydroxyestradiol-17b-induced OM in catﬁsh Heteropneustes fossilis has been reported (Mishra and Joy, 2006). Recently, Khan and Maitra (2013) have shown the participation and activation of cAMP-dependent protein kinase and MAP kinase in MISinduced OM in Anabas testudineus. We also demonstrated the involvement of MAP kinase in insulin- and IGF-I-induced OM in Cyprinus carpio (Paul et al., 2009). Although several earlier studies demonstrated the participation of Mos/MAP kinase in metaphase II arrest (Masui and Clarke, 1979; Josefsberg et al., 2003), involvement of MAP kinase in progesterone-induced MPF activation and GVBD-induction is species-dependent in ﬁsh, amphibian and mammalian model (Yamashita, 1998; Ferrell, 1999; KajiuraKobayashi et al., 2000; Nebreda and Ferby, 2000; Su et al., 2003; Pace and Thomas, 2005b). Tenulosa ilisha, commonly known as Hilsa, is an economically important anadromous food ﬁsh, migrates for breeding from Bay of Bengal to all fresh water inlets (river) of West Bengal, India including the river Hooghly, twice a year; one during early February to April correlated to the general rise in temperature of the water in the estuaries after the closer of winter and another in South-West monsoon (July–September) (Jones and Menon, 1950, 1951). Female ﬁsh produces group-synchronous germ cells (single batch spawner) with biannual-spawning episode, one during April–May and another during August–September (Pramanick et al., 2013). To develop a baseline reproductive biology for wild population, gonadal maturity schedule studies, circulatory sex steroid patterns and their mode of actions are usually applied (Hunter et al., 1992). Very recently, circulatory sex steroid levels including identity of MIS and hormonal regulation of their production by the ovarian tissues of T. ilisha have been reported (Pramanick et al., 2013). But no work has yet been done to know the signal transduction pathways in MIS-induced OM of this ﬁsh. The purpose of the present study was to identify the signal transduction pathways activated in MIS-induced OM in T. ilisha. Our main point was to determine if PI3 kinase and MAP kinase activation are required for cdc2 kinase activation and OM in this ﬁsh.
Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India. Labeled 17a-hydroxy [1,2,6,7-3H] progesterone was procured from Amersham International Plc, UK. Labeled 17,20b-P was prepared from 17a-hydroxy [1,2,6,7-3H] progesterone by sodium borohydrate reduction, followed by chromatographic separation (Kime and Dolben, 1985) as described previously (Sen et al., 2002). MEK inhibitor PD98059, mouse monoclonal antiphospho ERK1/2 antibody P-ERK, mouse monoclonal anti cdc2 p34 antibody and the secondary antibody, goat antimouse IgG2a were purchased from Santa Cruz Biotech, Santa Cruz, CA. Antiphospho p85 subunit of PI3 kinase antibody p85a (B-9) (Santa Cruz Biotech, Santa Cruz, CA) was a gift from Prof. Samir Bhattacharya, VisvaBharati, Santiniketan, India. The phosphospeciﬁc cdc2 monoclonal antibody raised against amino acids 224–230, mapping within a central region of cdc2 of human origin, detects cdc2 P34 of multiple species. PI3 kinase p85a mouse monoclonal antibody raised against amino acids 332–430, mapping within the N-terminus SH2 domain of 85 kDa subunit of PI3 kinase of human origin, detects PI3 kinase p85a of multiple species. Mouse monoclonal P-ERK(P-E-4) antibody recommended for detection of ERK 1 phosphorylate at Tyr-204 and correspondently phosphorylated ERK 2 of multiple species. All other chemicals used were of analytical grade. 2.2. Animal Adult T. ilisha were captured from the river Hooghly between Naihati (Dist. North 24 Parganas) and Triveni (Dist. Nadia), West Bengal, India, 60–80 km upstream from the inlet of this river to Bay of Bengal during the month of July to August 2009 to 2010 in the early morning depending on the tidal times. During these months, ovary of this ﬁsh mostly contained post-vitellogenic follicles (diameter 0.4–0.5 mm). In such follicles oocytes were found to initiates coalescence of lipid droplets around a centrally located germinal vesicle. Details of ﬁsh capture, collection of ovaries and their incubation in Idler’s medium in aseptic conditions have been described previously (Pramanick et al., 2013). All these were done on the ﬁsherman boat. Ovarian tissues in oxygenated medium were then quickly transported on crushed ice to the laboratory and processed for isolation and collection of post-vitellogenic follicles following the procedure used in our laboratory (Paul et al., 2009, 2010a; Pramanick et al., 2013). Growth stages of oocytes were determined according to Chonder (1999). This was done by taking ovarian follicles in glass beakers that contained clearing solution of acetic acid:ethanol:formalin mixture (1:6:3 v/v) for 12 h and percentages of oocytes at different developmental stages were determined as described early (Pramanick et al., 2013). Oocytes were denuded and defolliculated by enzymatic treatment with collagenase (0.1%) following the procedure routinely used in our laboratory (Paul et al., 2010b). Viability of intact follicles and denuded oocytes was checked by trypan blue dye exclusion technique. 2.3. Incubation of intact follicles and denuded oocytes
2. Materials and methods 2.1. Chemicals Human chorionic gonadotropin (HCG) was a gift from National Hormone and Pituitary Programme, Torence, California. 17b-estradiol, 17,20b-P, PI3 kinase inhibitor, Wortmannin, collagenase typeI and nitrobluetetrazolium/5-bromo-4-chloro-3-indoylphosphate were purchased from Sigma Chemical, St. Louis, MO. Another PI3 kinase inhibitor, LY294002 (RBI, Natick, NY) was a gift from Dr. Sib Sankar Roy, Molecular Endocrinology Laboratory, Indian
Approximately, 100 intact follicles (60 mg) and 50 fully denuded oocytes were transferred into individual wells of a 24well culture plate (Tarsons, India) for 2 h containing 1 ml ice-cold Idler’s medium supplemented with streptomycin (100 lg/ml) and penicillin (100 IU/ml) adjusted to pH 7.4. This 2 h pre-incubation time was required to waive the surgical shock. Medium from each well of intact follicles was replaced with fresh medium containing 17,20b-P and inhibitors as mentioned in results and incubated for different time intervals up to 16 h in a metabolic shaker bath at 23 ± 1 °C under air. Prior to the addition of 17,20b-P and inhibitors, denuded oocytes were incubated with
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E2 (50 nM) for 2 h. This is because enzymatic or manual removal of follicle layers in ﬁsh oocytes caused marked increase in spontaneous OM, which could be partially reversed by the treatment of E2 (Pang and Thomas, 2010). Dose of E2 and incubation time were optimized for T. ilisha oocytes. Denuded oocytes were then incubated with 17,20b-P for 8 h, because in such oocytes spontaneous maturation increased during longer incubation period, thereby obscuring the treatment effects. Inhibitors in both the cases were added 1 h prior to the addition of test compounds. Steroids were dissolved in ethanol and inhibitors in DMSO and added to each well in 1 ll aliquots. An equal volume of solvent (1 ll) was added to the vehicle control treatment wells. At the end of incubation, medium from each well of intact follicle was aspirated and kept at -20 °C for extraction and assay of steroid. A portion of intact follicles and denuded oocytes were processed for immunoblotting and the remaining were ﬁxed in a clearing solution for 12 h as mentioned previously and OM was examined by scoring GVBD under the microscope.
ANOVA within and across different effectors. Individual comparisons between treatments were made by adopting Bonferroni’s multiple comparison tests using SPSS (Chicago, IL, USA). The level of signiﬁcance chosen was p < 0.05.
3. Results 3.1. HCG-stimulated 17,20b-P production and oocyte maturation Fig. 1A and B shows that HCG at its increasing concentrations stimulated 17,20b-P production and GVBD induction in vitro in post-vitellogenic ovarian follicles of T. ilisha in a dose-dependent manner after 16 h incubation. Maximum effective dose of HCG
2.4. Immunoblotting At the end of each incubation, oocytes were washed with fresh medium, homogenized in 500 ll ice-cold lysis buffer containing 25 mM glycerophosphate (pH 7.4), 10 mM sodium pyrophosphate, 1 mM sodium molybdate, 1 mM sodium orthovanadate, 10 mM sodium ﬂuoride, 0.2% (v/v) triton X 100, 1 mM EGTA, 1 mM magnesiumchloride, 100 mM potassium chloride, 20 mM imidazole–HCl (pH 6.8), and inhibitors of leupeptin, aprotinin, PMSF and trypsin. All protease inhibitors were added at the dose of 1 lg/ml. The homogenate was centrifuged at 12,000 g for 5 min at 4 °C, supernatant was sonicated for 5 s on ice and protein content was determined (Lowry et al., 1951). For immunoblotting, 20 lg of total protein were electrophoresed through a 10% SDS PAGE and transferred to polyvinylideneﬂuoride (PVDF) membrane (Fermentas, Life Sciences). Membranes were blocked for 1 h in 5% blocking solution (Tris buffered saline with 0.1% Tween-20 and 5% non fat milk) followed by incubation with primary antibodies for overnight at 4 °C. Mouse monoclonal anti PI3K p85a antibody, mouse monoclonal anti phospho ERK 1/2 antibody P-ERK (E4) and mouse monoclonal P34 cdc2 antibody (all are from Santa Cruz) were used at 1:2000 dilutions. Bound primary antibodies were visualized using corresponding secondary antibodies at 1:2000 dilutions, which were tagged with alkaline phosphatase and were developed with nitrobluetetrazolium/5-bromo 4-chloro-3-indoylphosphate. 2.5. Extraction and assay of steroids The method for extraction and assay of 17,20b-P from incubation medium was similar to the previously described procedure (Sen et al., 2002; Mukherjee et al., 2006; Pramanick et al., 2013). The antiserum of 17,20b-P was highly speciﬁc and cross reacted with 17,20b-P; 5b-pregnen-3a,17b-triol; 5a,3a,17a,20b-P; progesterone; 17a-hydroxyprogesterone; testosterone and 17b-estradiol at 100%, 42%, 4%, 0.001%, 0.01%, 0.25%, and 0.01% respectively. Intra- and inter-assay coefﬁcient of variation was 9% and 12% respectively. 2.6. Statistical analysis Data obtained from three replicate incubations of intact follicles and denuded oocytes from a single donor ﬁsh showed a similar tendency and therefore a mean of all the three data was considered as one experiment. All data were expressed as mean ± S.E.M. of three such experiments taking intact follicles or denuded oocytes from three donor ﬁsh. After the test for normality and homogeneity, the signiﬁcance of treatment effects was determined by one-way
Fig. 1. HCG-induced 17,20b-P production (A), oocyte maturation (%GVBD) (B) and17,20b-P-induced GVBD (C) in intact post-vitellogenic follicles of T. ilisha after 16 h incubation. Effects of 17b-estradiol (E2, 50 nM) in blocking spontaneous GVBD in fully denuded oocytes and 17,20b-P treatment for 8 h to override this inhibitory estrogen action (D). Approximately 100 intact follicles and 50 denuded oocytes per experiment were counted and scored for GVBD. Bars represent the mean ± SEM of three such experiments taking follicles from three donor ﬁsh. Different letters denote signiﬁcant differences from each other (p < 0.05). Asterisk denotes values signiﬁcantly different from Veh (p < 0.05; one way ANOVA and Bonferroni test). Veh., vehicle control.
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was shown to be 100 ng/ml and minimum tried concentration was 25 ng/ml for these two events. Effectiveness of 17,20b-P in inducing OM in intact follicles and fully denuded oocytes was examined. For this, intact follicles (Fig. 1C) were incubated without or with increasing concentrations of 17,20b-P for 16 h and denuded oocytes (Fig. 1D), after a pre-incubation with E2 for 2 h, were incubated without (vehicle control) or with increasing concentrations of steroid for 8 h. Fig 1C shows that 17,20b-P was effective in inducing OM in intact follicles in a dose-dependent manner. Fig. 1D shows that denuded oocytes underwent spontaneous OM by 8 h which was partially reversed by E2 treatment and addition of increasing doses of 17,20b-P to the incubations gradually and signiﬁcantly override this inhibitory estrogen action. Maximum effective dose of 17,20b-P in inducing GVBD both in intact follicles and denuded oocytes was 100 ng/ml. 3.2. 17,20b-P-stimulated cdc2 kinase activation and oocyte maturation To investigate the functional relationship between MPF activation and GVBD induction, intact follicles and denuded oocytes were incubated with 17,20b-P (100 ng/ml) for different time intervals as described previously (Fig. 1C and D). A portion of intact follicles and denuded oocytes were then examined for GVBD and remaining portion was processed for Western blot analysis using antibody speciﬁc for 34 kDa cdc2 (P-34 cdc2). Fig. 2A shows that intact follicles and denuded oocytes both (0 h control) contained a 35 kDa cdc2 (inactive) whereas in 17,20b-P-treated follicles/oocytes, inactive 35 kDa cdc2 was activated through chemical modiﬁcation with an electrophoretic mobility shift to 34 kDa cdc2. This electophoretic mobility shift by the induction of 17,20b-P in intact follicles was started from 4 h and maximum was shown at 16 h, whereas in denuded oocytes, it was from 2 h with a maximum at 8 h. Presence of only 35 kDa inactive cdc2 kinase in denuded oocyte (0 h control), pre-treated with E2 indicates reversal of spontaneous OM. The lower panel shows the signiﬁcant induction of GVBD after 17,20b-P treatment both in intact follicles and denuded oocytes compared to 0 h control (Fig. 2 B).
3.3. Effects of inhibitors of PI3 kinase on 17,20b-P-induced oocyte maturation To examine whether PI3 kinase activation was necessary in 17,20b-P-induced OM, intact follicles were incubated for 16 h and denuded oocytes for 8 h either with Wortmannin or LY294002, two different PI3 kinase inhibitors at their increasing concentrations in absence or presence of 17,20b-P (100 ng/ml). Denuded oocytes were incubated with E2 (50 nM) for 2 h prior to the addition of PI3 kinase inhibitors and 17,20b-P. It appears from Fig. 3A and B that Wortmannin and LY 294002 both attenuated 17,20b-P-induced OM in intact follicles and denuded oocytes gradually and signiﬁcantly in a dose-dependent manner. The lowest concentration of Wortmannin that strongly inhibited the OM was 10 lM as compared to LY 294002, which was 50 lM. These data suggest that PI3 kinase activation was involved in MIS-induced OM in T. ilisha.
3.4. Effects of 17,20b-P on PI3 kinase activation and effects of PI3 kinase inhibitors This experiment was conducted only with denuded oocytes. For this, oocytes were initially treated with E2 (50 nM) for 2 h and then exposed to 17,20b-P (100 ng/ml) for different time intervals from 0 to 180 min in presence or absence of PI3 kinase inhibitors. After incubation, oocyte lysates were immunoblotted using antiphospho p85 PI3 kinase antibody, speciﬁcally recognize the activated form of the protein. Fig. 4A shows that 85 kDa subunit of endogenous PI3 kinase in T. ilisha oocytes could be detected by immunoblotting (0 min control) and become activated by 17,20b-P from 60 min onwards with a maximum at 90 min. The stimulatory effect of 17,20b-P on phosphorylated p85 PI3 kinase was not attributed to increased p85 PI3 kinase protein levels, as total protein was unaffected by the treatments. Oocytes treated with 17,20b-P (100 ng/ ml) for 90 min in the presence of maximally effective dose of either Wortmannin (10 lM) or LY294002 (50 lM) efﬁciently blocked p85 PI3 kinase phosphorylation (Fig. 4B).
Fig. 2. Effects of 17,20b-P on time-dependent cdc2 activation (A) and GVBD induction (B) in intact follicles and denuded oocytes of T. ilisha. Denuded oocytes were initially incubated with E2 (50 nM) for 2 h and then intact follicles and denuded oocytes both were incubated separately with 17,20b-P (100 ng/ml) for different time intervals. At the end of incubation, they were examined for GVBD and also processed for Western blot analysis using antibody speciﬁc for 34 KDa cdc2 (P-34-cdc2). Approximately 100 intact follicles and 50 denuded oocytes per experiment were counted and scored for GVBD. Bars represent the mean ± SEM of three such experiments taking follicles from three donor ﬁsh. Mobilities of molecular mass standard are given in kDa on the left. Immunoblot analyses were performed at least three times with nearly identical results. The expression of GAPDH was used as loading control. Different letters denote signiﬁcant differences from control (0) and different time hours (p < 0.05, one way ANOVA and Bonferroni test).
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Fig. 4. p85-Associated PI3 kinase activation and effects of PI3 kinase inhibitors in denuded oocytes. Denuded oocytes were initially exposed to E2 (50 nM) for 2 h and then incubated with 17,20b-P (100 ng/ml) for 0 to 180 min (A) or with 17,20b-P in presence of PI3 kinase inhibitors either Wortmannin (Wn) or LY294002 (Ly) for 90 min (B). p85-associated phosphorylated PI3 kinase (PPI-3) and total PI3 kinase (T-PI3) are shown. Mobilities of molecular mass standard are given in kDa on the left. Immunoblot analyses were performed at least three times with nearly identical results. The expression of GAPDH was used as loading control. (+) denotes presence and ( ) denotes absence.
Fig. 3. Percent GVBD of intact follicles (A) after 16 h and denuded oocytes (B) after 8 h incubation without or with 17,20b-P in presence of graded doses of PI3 kinase inhibitors, either Wortmannin (Wn) or LY294002 (Ly). Denuded oocytes were treated with E2 (50 nM) for 2 h and then both intact follicles and denuded oocytes, after 1 h pre-incubation with PI3 kinase inhibitors, were incubated with 17,20b-P (100 ng/ml). Approximately 100 intact follicles and 50 denuded oocytes per experiment were counted and scored for GVBD. Bars represent the mean ± SEM of three such experiments taking follicles from three donor ﬁsh. Asterisks denote values signiﬁcantly different (p < 0.05) from those treated with hormone. Different letters denote signiﬁcant differences from those treated with hormone (17,20b-P for intact and E2 + 17,20b-P for denuded oocytes) and inhibitors (p < 0.05, one way ANOVA and Bonferroni test).
3.5. Effect of MEK1/2 inhibitor on 17,20b-P-induced oocyte maturation To examine whether MAP kinase activated signaling was necessary for 17,20b-P-induced OM of this ﬁsh, denuded oocytes were initially treated with E2 (50 nM) for 2 h and then incubated for 1 h with increasing doses of MEK inhibitor PD98059 (0.1, 1.0 and 5.0 lM) followed by incubation with 17,20b-P (100 ng/ml) for 8 h and GVBD was examined. Fig. 5A shows that PD98059 at its increasing concentrations had no effect on GVBD even at its highest concentration tested (5.0 lM) indicating that MAP kinase activation was not necessary for MIS-induced OM in this ﬁsh. 3.6. Effects of 17,20b-P on ERK1/2phosphorylation and effects of inhibitor of MEK1/2 Because the phosphorylated state of ERK1/2 correlates closely with its kinase activity, the activity of ERK1/2 was indirectly determined using a phosphospeciﬁc ERK1/2 antibody, P-ERK. For this, denuded oocytes were initially incubated with E2 (50 nM) for 2 h and then incubated with 17,20b-P (100 ng/ml) for different time intervals from 0 to180 min. Immunoblot analyses of the oocyte
lysate demonstrate increased levels of phosphorylated ERK1/2 in response to 17,20b-P and the strongest response occurred between 90 and 120 min (Fig. 5B). The stimulatory effects of 17,20b-P on phosphorylated ERK1/2 was not attributed to increased ERK protein levels, as total ERK1/2 protein was unaffected by incubation time. Oocytes treated with E2 (50 nM) plus 17,20b-P (100 ng/ml) for 120 min in presence of PD98059 (5.0 lM) sufﬁciently blocked ERK1/2 activation (Fig. 5C). 3.7. Effect of inhibitors of PI3 kinase on 17,20b-P-stimulated ERK1/2 phosphorylation To examine a link between activation of PI3 kinase and ERK1/2 phosporylation during oocyte maturation, denuded oocytes were initially incubated with E2 (50 nM) for 2 h and then with either Wortmannin (10 lM) or LY294002 (50 lM) for 1 h followed by incubation with 17,20b-P (100 ng/ml) for 120 min. Oocyte lysates were immunoblotted using antiphospho ERK1/2 and anti ERK1/2. Immunoblot analyses of the lysate demonstrate that both Wortmannin and LY294002 blocked the 17,20b-P-induced phosphorylation of ERK1/2 after 120 min (Fig. 6A). 3.8. Effects of PI3 kinase inhibitors on cdc2 kinase activity To examine whether PI3 kinase activation was necessary for 17,20b-P-induced cdc2 activation, denuded oocytes were initially incubated for 2 h with E2 (50 nM) followed by incubation with 17,20b-P (100 ng/ml) for 8 h in presence or absence of either Wortmannin (10 lM) or LY294002 (50 lM) and cdc2 kinase activity was examined. Immunoblot analyses of the oocyte lysate show that 17,20b-P-induced activation of cdc2 kinase was strongly inhibited by both the inhibitors (Fig. 6B). 3.9. Effects of MEK kinase inhibitor on cdc2 kinase activity To investigate the functional relationship between MAP kinase and cdc2 kinase activation in 17,20b-P-induced OM,
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Fig. 5. Percent GVBD of denuded oocytes incubated with 17,20b-P in presence of a MEK inhibitor PD98059 (PD) (A). Time-dependent activation of ERK1/2 in oocytes incubated with 17,20b-P in absence (B) or presence of PD (C). Denuded oocytes were exposed to E2 (50 nM) for 2 h and then incubated for 1 h in presence of graded doses of PD followed by incubation with 17,20b-P (100 ng/ml) for 8 h. Immunoblot analyses of phosphorylated ERK (P-ERK1/2) and total ERK (T-ERK1/2) induced by 17,20b-P (100 ng/ml) for 0–180 min (B) or with MEK inhibitor PD for 120 min (C) are shown. Approximately 50 denuded oocytes per experiments were counted and scored for GVBD. Bars represent the mean ± SEM of three such experiments taking follicles from three donor ﬁsh. Asterisks denote values signiﬁcantly different from those treated with E2 or E2 + PD (p < 0.05). Mobilities of molecular mass standards are given in kDa on the left. Immunoblot analyses were performed at least three times with nearly identical results. The expression of GAPDH was used as loading control. (+) Denotes presence and ( ) denotes absence.
denuded oocytes were initially incubated with E2 as previously described and then with a MEK inhibitor PD98059 for 1 h followed by 8 h incubation with 17,20b-P and cdc2 kinase activity was examined. Fig. 6C shows that PD98059 at two doses tested (1.0 and 5.0 lM) had no effect on 17,20b-P-induced cdc2 kinase activation.
4. Discussion The present study demonstrates that 17,20b-P produced in follicle cells by the induction of gonadotropin is able to induce OM in T. ilisha. This ﬁnding corroborates earlier study on GVBD induction in same species by 17,20b-P (Pramanick et al., 2013). We demonstrated that 17,20b-P-induced OM in this ﬁsh is mediated through activation of PI3 kinase pathway. This ﬁnding is in agreement with similar studies in other species, including starﬁsh, striped bass, Atlantic croaker and Rana dybowskii that found PI3 kinase activation is necessary for MIS-induced OM (Sadler and Ruderman, 1998; Weber and Sullivan, 2001; Ju et al., 2002; Pace and Thomas, 2005a). In contrast, reports are also available that activation of PI3 kinase is not necessary for progesterone-induced OM in Xenopus (Liu et al., 1995; Mood et al., 2004).
The ﬁndings that incubation of T. ilisha oocytes with two mechanistically different PI3 kinase inhibitors, either Wortmannin or LY294002, sufﬁciently blocked PI3 kinase activation within 90 min followed by inhibition of GVBD induction support the requirement and involvement of PI3 kinase in 17,20b-P-induced OM in T. ilisha. The effectiveness of both the inhibitors to block GVBD in this ﬁsh was dependent on the concentrations of inhibitors, presumably due to full occupancy of MIS receptor with inhibitors. We also observed that effectiveness of these inhibitors to block GVBD was dependent on the concentrations of MIS (data not shown), presumably due to reduction in the number of occupied MIS receptors. Inhibition started at a very low concentration of MIS (20 ng/ml) and attained maximum at 100 ng/ml. Similar ﬁndings have been observed in MIS-induced OM in starﬁsh, striped bass, Rana dybowski and Atlantic croaker (Sadler and Ruderman, 1998; Weber and Sullivan, 2005; Ju et al., 2002; Pace and Thomas, 2005a) and also in growth factor-induced oocyte maturation in many species including Xenopus (Chuang et al., 1993; Fabian et al., 1993; Liu et al., 1995; Browayes-Poly et al., 2000), striped bass (Weber and Sullivan, 2001) and C. carpio (Paul et al., 2009). Result of the present study demonstrates that 17,20b-P-induced activation of PI3 kinase leads to GVBD induction and that takes place several hours before cdc2 becomes activated. The evidence for such cdc2 activation comes from Western blot analysis using cdc2 p34 antibody. We observed that 17,20b-P induces cdc2 activation by converting inactive cdc2 p35 to high activity stage cdc2 p34 and maximum activation was recorded at 16 h in intact follicles and 8 h in denuded oocytes. We further observed that both the inhibitors of PI3 kinase inhibited17,20b-P-induced cdc2 activation leading to OM after 16 h and 8 h incubation in intact follicles and denuded oocytes respectively. This ﬁnding indicates that activation of PI3 kinase is essential for the 17,20b-P-induced cdc2 activation in T. ilisha for ﬁnal OM. This ﬁnding is in agreement with similar study in IGF-I and insulin-induced OM in carp (Paul et al., 2009). Molecular mechanism of MPF activation in ﬁsh oocytes has been well documented in goldﬁsh. Immature goldﬁsh oocytes contain monomeric 35-kDa cdc2, and cyclin B is absent. MIS induces the de novo synthesis of cyclin B. Binding of cyclin B to 35-kDa cdc2 allows MO15 (the catalytic subunit of a protein kinase that activate cdc2 kinase through phosphorylation of Thr161) to phosphorylate cdc2 on Thr161, which activates cdc2 and induces an electrophoretic mobility shift from 35 to 34 kDa. Thereafter, Ser79 and/or Ser94 of cyclin B is phosphorylated by the active cdc2, yielding MPF (see review Nagahama, 1997). Earlier studies reported the role of MAP kinase on progesteroneinduced OM in Xenopus (Sagata et al., 1989). A crucial step in this process is the expression Mos, which activates MAP kinase. Later studies in starﬁsh, mouse, Xenopus, and goldﬁsh, using pharmacologic inhibitors, mos morpholino antisense oligonucleotides, Mos gene knockouts, and several other techniques showed that MAP kinase activation is not necessary for OM, but rather acting as a cytostatic factor to suppress DNA replication between meiosis I and meiosis II (Ferrell, 1999; Nebreda and Ferby, 2000; Yoshida et al., 2000; Voronina and Wessel, 2004). Activation of MAP kinase in follicle cells, but not in oocytes, is necessary for OM in mouse (Su et al., 2003) and MAP kinase activation is neither necessary nor sufﬁcient for inducing OM in goldﬁsh oocytes (Yamashita, 1998; Kajiura-Kobayashi et al., 2000). In Atlantic croacker also MAP kinase activation is not necessary in steroid-induced OM (Pace and Thomas, 2005a). Failure of MAP kinase inhibition using a speciﬁc MEK inhibitor, PD98059 to block MIS-induced cdc2 activation and GVBD induction in T. ilisha in the present study is in agreement with these studies and conﬁrms that MAP kinase activation is not necessary for steroid-induced OM in this ﬁsh. In contrast, some recent studies on catﬁsh, H. fossilis and perch, A. testudineus
K. Pramanick et al. / Molecular and Cellular Endocrinology 390 (2014) 26–33
Fig. 6. Effects of PI3 kinase inhibitors on 17,20b-P-induced ERK1/2 phosphorylation (A) and cdc2 activation (B) and MEK inhibitor PD98059 (PD) on 17,20b-P-induced cdc2 activation (C). Denuded oocytes after pre-incubation for 2 h with E2 were incubated for 120 min with 17,20b-P (100 ng/ml) in presence of Wortmannin (Wn) or LY294002 (Ly) for immunoblot analyses of ERK1/2 phosphorylation (A) or incubated 8 h for analyses of cdc2 activation with Wn or Ly (B) or with PD (C). Mobilities of molecular mass standard are given in kDa on the left. Immunoblot analyses were performed at least three times with nearly identical results. The expression of GAPDH was used as loading control. (+) Denotes presence and ( ) denotes absence.
suggested the involvement of MAP kinase in steroid-induced OM (Mishra and Joy, 2006; Khan and Maitra, 2013). 4.1. Conclusion In summary, the present study suggests that PI3 kinase activation is required for steroid-induced cdc2 activation and OM in T. ilisha. This study further indicates that although MAP kinase activated after steroid treatment but is not necessary for cdc2 activation and GVBD induction in this ﬁsh. Funding This work is supported by grants in aid from Council of Scientiﬁc and Industrial Research (CSIR) [Ref. No. 3F-1/CSIR/Z001/651/ 2005/S-768 (3)] to KousikPramanick as CSIR-NET fellow. The authors acknowledge DST-PARS Kalyani University for providing partial ﬁnancial support. There is no conﬂict of interest that would prejudice the impartiality of the research.
Acknowledgement The authors are thankful to Prof. Samir Bhattacharya, VisvaBharati, Santiniketan, India for his constant inspiration.
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