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Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla
Comparative Biochemistry and Physiology, Part A 150 (2008) 301–306
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla Asamanja Chattoraj, Mohua Seth, Saumen Kumar Maitra ⁎ Department of Zoology, Visva-Bharati University, Santiniketan-731 235, India
A R T I C L E
I N F O
Article history: Received 10 January 2008 Received in revised form 14 March 2008 Accepted 20 March 2008 Available online 28 March 2008 Keywords: Carp Melatonin MIH Oocytes Serotonin
A B S T R A C T The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 μg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 μM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 μM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24 ± 1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15 ± 1.91% or 14.42 ± 0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58 ± 1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57 ± 0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component – cyclin B and the catalytic component – Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The pineal gland is the major component of the neuroendocrine system which through melatonin (N-acetyl-5-methoxytryptamine) and/or its precursor serotonin (5-hydroxytryptamine) is alleged to be associated with the regulation of reproduction in different vertebrates (Reiter, 1991). Importance of these two indole amines has been demonstrated by the presence of their receptors in the reproductive organs (Woo et al., 2001; Vesela et al., 2003) and by their effects on the functions of the testis and oviduct (Sirotkin and Schaeffer, 1997). However, the existing information on the physiology of these hormones is based mostly on mammalian studies, where influences of these principles on reproduction varied in relation to their dose and duration of treatment as well as their time of application (Donham et al., 1989; Vanêĉek and Klein, 1993).
⁎ Corresponding author. Tel./fax: +91 3463 261268. E-mail address: [email protected] (S.K. Maitra). 1095-6433/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.03.014
The role of pineal gland and its hormones has also been investigated in the seasonal maturation of gonad in fish (VivienRoels et al., 1981; Popek et al., 1992, 1994; Bromage et al., 2001; Bhattacharya et al., 2003, 2007; Bayarri et al., 2004; Maitra et al., 2005), but the results of the study have been diverse. The study on rainbow trout (Popek et al., 1992) suggested that the pineal gland may alter the maturation period and control spawning by acting on the hypothalamo-pituitary-gonadal axis. However, in vitro studies of the oocytes in carp Cyprinus carpio (Popek et al., 1991) and killifish Fundulus heteroclitus (Cerda et al., 1997) could not demonstrate any effect of melatonin on oocyte maturation. On the other hand, serotonin was found to inhibit MIH-induced resumption of meiosis following in vitro study of oocytes in teleost F. heteroclitus (Cerda et al., 1997). The results showing such inhibition in both follicleenclosed and denuded oocytes indicated the presence of oocyteassociated serotonin sensitive sites (Cerda et al., 1995, 1997). In a recent study, it has been shown for the first time that melatonin accelerates the action of MIH when added 4 h prior to MIH in the incubation medium (Chattoraj et al., 2005). However, the relative role
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of serotonin and melatonin on the MIH-induced oocyte maturation in any fish remained unknown. The present study aimed at in vitro demonstration of independent as well as relative influences of serotonin and melatonin on the MIHinduced meiotic resumption in the oocytes of a hitherto unstudied major carp C. catla. Since fish oocytes undergo drastic morphological changes associated with progression of the meiotic cell cycle, in which breakdown of the oocyte nuclear envelope or germinal vesicle breakdown (GVBD) occurring at the prophase/metaphase transition and is usually regarded as a hallmark of the progress of oocyte maturation (Tokumoto et al., 2004), we studied the rate of GVBD in the carp oocytes as the visual marker of meiotic resumption. In most teleosts, 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20 β DHP) serves as the potent MIH (Pang and Ge, 2002; Senthilkumaran et al., 2002), which acts on receptors located on the oocyte membrane and induces the activation of maturation promoting factor (MPF) in the oocyte cytoplasm to initiate final maturation (Nagahama, 1997). Therefore, our study also included the rate of formation of maturation promoting factor (MPF) which is a complex of two proteins, a regulatory component called cyclin B and catalytic component protein kinase known as cyclin-dependent kinase or cdc2 (Yamashita et al., 1992; Chausson et al., 2004). Specific blockers of receptors of melatonin and of serotonin have been appropriately used to provide pharmacological evidence of the specific action of the concerned indolamines on the studied carp oocytes. 2. Materials and methods 2.1. Biological materials Adult C. catla weighing between 600 g and 1000 g (measuring 300 mm to 457 mm) were collected during the spawning phase (July–August) from their natural habitat in India (Lat. 23°39′ Ν., Long. 87°42′ Ε). Soon after catch, only gravid females were quickly transported to the laboratory and were maintained in large aquaria with balanced fish food ad libitum for a week for acclimatization to laboratory conditions before being used in the experiments. 2.2. Chemicals and reagents All the analytical grade chemicals including M199 medium, melatonin, serotonin, MIH, luzindole, metoclopramide HCl, and polyvinylidene difluoride (PVDF) membranes were purchased from Sigma-Aldrich Chemical Co. St. Louis, MO, USA. The primary antibodies of cyclin B and cdc2 were procured from Santa Cruz Biotechnology Inc. California, USA and secondary antibodies were supplied by Bangalore Genei Pvt. Ltd. (Bangalore, India).
Fig. 1. Denuded oocytes collected from the ovary of carp Catla catla during the spawning phase, and freed from somatic follicle cells following cold shock treatment (X 40).
penicillin (100 U/mL) and streptomycin (100 μg/mL). Incubations were carried out separately in M199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 μg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 μM/mL) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 μM/mL) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. The control oocytes were incubated in the medium alone. The selection of dose and time of addition of melatonin in the medium were based on our previous study (Chattoraj et al., 2005). The dose (10 μM/mL) of both luzindole and metoclopramide hydrochloride, used as the specific blockers of the receptors of MEL and SER respectively for demonstration of their pharmacological effects in the medium, has been the same as employed earlier (Cerda et al., 1997; Drazen et al., 2001). In either case, oocytes were incubated further for 4, 8, 12 or 16 h following addition of MIH. Viability of oocytes was about 95% as determined by 0.1% Trypan blue exclusion test. 3.2. Determination of GVBD
3. Methods
At the end of incubation for a given period of time, the oocytes were immersed in a clearing solution (ethanol/ formalin/acetic acid; 6:3:1) for an easy examination of the oocytes (Tokumoto et al., 2004) under a microscope (Olympus, BX51, Japan) with in-built photomicrographic attachments and image analyzing device. The effects of treatment on oocyte maturation were determined by GVBD and the results were expressed in % taking 100 oocytes for each observation.
3.1. Incubation of denuded oocyte
3.3. Preparation of oocyte extracts for study of maturation signals
The large and round shaped yolk-laden Stage-IV oocytes were collected from the ovary of freshly sacrificed gravid females and were manually denuded with the help of forceps under ice cold M199 medium. It is well known that sudden cold shock does not affect the quality of oocytes, rather helps to separate somatic follicle cells from oocytes by forceps (Chattoraj et al., 2005). Accordingly, the same technique has been adopted to avail denuded oocytes. Complete removal of follicle cells was ensured through careful examination under the microscope and only denuded oocytes were selected (Fig. 1). Subsequently, the isolated oocytes were exposed to 95% O2 / 5% CO2 at 27 ± 1 °C before incubating in separate sterilized micro-beakers for different treatments under identical laboratory conditions. Each micro-beaker (5 mL capacity) contained about 50 denuded oocytes which were incubated in 1 mL of Medium M199 supplemented with
Fifty oocytes from each set of incubation at given time points were collected in micro-centrifuge tubes and washed thrice with ice cold extraction buffer (EB: 80 mM sodium β-glycerophosphate, 20 mM HEPES, 15 mM MgCl2, 5 mM EGTA, 0.5 mM PMSF, 1 mM DTT, leupeptin, 10 μg/mL aprotinin pH 7.5) following Hirai et al. (1992) with minor modifications and homogenized in the same buffer at 4 °C. The homogenate was centrifuged at 14,000 g for 15 min. The clear cytosolic supernatant was used for electrophoresis and Western blotting for the detection of cyclin B and p34cdc2 protein. 3.4. Electrophoresis and immunoblotting The methods of electrophoresis and immunoblotting were described earlier (Chattoraj et al., 2005). In brief, for immunodetection
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oocytes following incubation with different agents included in the present investigation (F4,80 = 49.18, p b 0.001; F20,80 = 4.91, p b 0.001).
Fig. 2. Rate of GVBD at different intervals following incubation of the denuded oocytes of Catla catla for 16 h in Medium M 199 with (i) none (control), or (ii) only melatonin (MEL), or (iii) only MIH, or (iv) melatonin added simultaneously with MIH, (v) melatonin added 4 h before [MEL(b)] MIH, or (vi) melatonin with luzindole (L), or (vii) melatonin added simultaneously with MIH and luzindole (L), or (viii) melatonin added 4 h before [MEL(b)] MIH and luzindole (L). Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e N f N g) respectively following one way ANOVA.
of cdc2, the polyvinylidene difluoride (PVDF) membrane was incubated with rabbit anti-PSTAIRE primary antibody (1:1000 dilution). Mouse anti-cyclin B1 antibody was used as the primary antibody (1:1000 dilution) to detect the cyclin B. The respective secondary antibodies (dilution 1:500) conjugated with alkaline phosphatase were used and visualized by treating the membrane with BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium).The densitometric analysis of the Western-blot data was carried out by Image-J software and expressed in line-diagram in arbitrary densitometric units.
4.1.1. The effects of Melatonin and MIH The mean values (±SE) of the rate (%) of GVBD following incubation with only maturation inducing hormone (MIH), or only melatonin (MEL), or MEL with MIH have been diagrammatically presented in Fig. 2. Significant effects were noted both in respect to the treatment (F4,16 = 14.44; p b 0.001) as well as time (F4,16 = 6.00; p b 0.003). Following incubation with MIH (1 μg/mL) alone, significant variations (F = 1561.49; p b 0.001) were noted at different time points. Likewise, significant (F = 524.46; p b 0.001) changes were found in relation to the time of incubation with only MEL (100 pg/mL). However, the rate of GVBD in the oocytes incubated with MIH for 16 h was significantly (F = 960.72; p b 0.001) higher than the oocytes incubated with only MEL at respective time point. An addition of MEL 4 h prior to the application of MIH in the medium resulted in a significant (F = 1149.52; p b 0.001) increase in the rate of GVBD with the progress incubation. More specifically, the rate of meiotic resumption of oocytes noted at 12 h following pre-treatment of MEL and incubated with MIH was significantly (F = 731.46; p b 0.001) higher than that noted in the oocytes incubated with only MIH. 4.1.2. The effects of melatonin, serotonin and MIH Fig. 3 summarizes the results of the study of GVBD% following incubation of oocytes with serotonin (SER) alone, or in combination with MEL and MIH. A two-way ANOVA showed that the effects of SER, MEL and MIH on the rate of GVBD were statistically significant in respect to the treatment (F4,20 = 12.89; p b 0.001) and the time of incubation (F5,20 = 4.18; p b 0.009). Only 14.42 ± 0.43% GVBD were noted following incubation with SER (100 pg/mL) alone for 16 h. The influence of only SER was found significantly (F = 688.63; p b 0.001) inhibitory compared to that noted at 16 h following incubation with MEL and SER, or SER and MIH, or MEL, SER and MIH. Significant variations in GVBD were noted in relation to the time of incubation of oocytes with SER and MEL (F = 508.29, p b 0.001), or SER and MIH (F = 663.66, p b 0.001), or SER, MEL and MIH (F = 607.25, p b 0.001).
3.5. Statistical analysis of experimental data Each experiment was carried out in quadruplicate using fifty oocytes in each set from one fish and the same was followed with three separate donor fish (i.e., n = 3). For example, time kinetics for MIH-induced oocyte maturation was observed in quadruplicate to obtain mean value of %GVBD and densitometric units of cyclin B immunoblot for each fish. Such mean values from three separate donor fish were then used to draw the mean value ± SE for each treatment group at each time point. Data on %GVBD were compared between oocytes cultured for different times and treated in different ways using two-way ANOVAs (analysis of variance). Time and treatment were the grouping factors. Post-hoc comparisons were made between times for each group and between groups for each time using univariate F tests taking p b 0.05 as the statistically significant threshold (Chattoraj et al., 2005). The densitometric data on cyclin B immunoblot for different groups of oocytes were also analyzed in an identical way following the same statistical methods. 4. Results 4.1. The rate of meiotic resumption of oocytes Two-way ANOVA showed significant effects of time and treatment group on GVBD, the visual marker of meiotic resumption of oocytes, in
Fig. 3. Influence of serotonin on the rate of GVBD of the denuded oocytes of Catla catla following the study of the effects of incubation at different intervals following incubation for 16 h in Medium M 199 with (i) none(control), or (ii) only serotonin (SER), or (iii) melatonin (MEL) added simultaneously with serotonin (SER), or (iv) serotonin (SER) added simultaneously with MIH, or (v) serotonin added 4 h before [SER(b)] MIH, or (vi) serotonin added simultaneously with melatonin and MIH. Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e) respectively following one way ANOVA.
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4 h before MIH), M and MIH, (F = 897.74, p b 0.001), or SER (coadministration), M and MIH (F = 1022.88, p b 0.001). 4.1.5. The effects of melatonin, luzindole, serotonin, metoclopramide Incubation of oocytes for 16 h with MEL, SER and M resulted in 77.57 ± 1.44% GVBD, but the rate was significantly (F = 883.81, p b 0.001) decreased to 42.83 ± 1.38% or 7.23 ± 0.43% following treatment with MEL and SER, or MEL, L, SER and M respectively (Figs. 3, 4). A two-way measures ANOVA showed that there were significant effects of MEL, L, SER and M both in respect to the treatment (F4,12 = 3.45; p b 0.04) as well as to the time (F3,12 = 6.46; p b 0.007) of treatment. In consideration to the time groups, significant variations were found in the oocytes which were incubated with MEL, SER and M (F = 686.76, p b 0.001), or MEL, L, SER and M (F = 55.76, p b 0.001).
Fig. 4. Influence of metoclopramide (serotonin receptor blocker) and luzindole (melatonin receptor blocker) on the GVBD of denuded oocytes of Catla catla following the study of the effects of incubation at different intervals following incubation for 16 h in Medium M 199 with (i) none (control), or (ii) metoclopramide (M) with serotonin (SER), or (iii) metoclopramide (M) with melatonin (MEL) and (SER), or (iv) metoclopramide (M) with serotonin (SER) and MIH, or (v) metoclopramide (M) and serotonin added 4 h before [SER(b)] MIH, or (vi) metoclopramide (M), serotonin added simultaneously with melatonin and MIH, or (vii) metoclopramide (M), Luzindole (L), melatonin and serotonin added simultaneously, or (viii) metoclopramide (M), luzindole (L), melatonin, serotonin and MIH added simultaneously, or (ix) luzindole (L), serotonin, melatonin and MIH added simultaneously. Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e N f) respectively following one way ANOVA.
4.1.3. The effects of melatonin, luzindole (antagonist of melatonin receptors) and MIH The mean values (± SE) of GVBD% in the oocytes following incubation with MEL and luzindole (L) with or without MIH have been shown in Fig. 2. A two-way ANOVA revealed that the effects of L, MEL and MIH were statistically significant in respect to the treatment (F4,28 = 16.72; p b 0.001) as well as to the time (F7,28 = 5.98; p b 0.001). The oocytes exhibited 16.53 ± 0.51%, or 38.49 ± 1.04%, or 39.3 ± 1.58% GVBD when incubated for 16 h with MEL (co-administration) and L, or MEL, L and MIH, or MEL (added 4 h before MIH), L and MIH respectively. Significant variations were noted in relation to the time of incubation with MEL and MIH (F = 2116.60, p b 0.001), or MEL and L (F = 430.93, p b 0.001), or MEL (co-administration), L and MIH (F = 308.63, p b 0.001), or MEL (added 4 h before MIH), L and MIH. (F = 433.22, p b 0.001). An inhibitory influence of L in each experiment was found at both 12 h (F = 731.46; p b 0.001) and 16 h (F = 960.72; p b 0.001) of incubation. 4.1.4. The effects of serotonin, metoclopramide (antagonist of serotonin receptors) and MIH The mean values (±SE) of the rate (%) of GVBD in different groups of oocytes incubated with SER and metoclopramide (M), or SER (added 4 h before MIH), MIH and M, or SER (co-administration), MIH and M have been presented in Fig. 4. A two-way ANOVA showed that the effects of SER, M and MIH were statistically significant in both treatment (F4,12 = 8.44; p b 0.001) and time groups (F3,12 = 3.49; p b 0.04). At the end of 16 h of incubation with SER (added 4 h before MIH), M and MIH, the rate of GVBD was 89.93 ± 2.03%, while 94.25 ± 2.15% oocytes underwent GVBD following incubation with SER (co-administration), MIH and M. The rate of GVBD was significantly higher in the oocytes which were incubated for 16 h with SER (added 4 h before MIH), MIH and M (F = 122.14; p b 0.001), or SER (co-administration), M and MIH (F = 163.05; p b 0.001) compared to those which were incubated in the corresponding media but without M (Figs. 3, 4). Significant variations were noted in relation to the duration of incubation with SER (added
4.1.6. The effects of melatonin, luzindole, serotonin, metoclopramide and MIH The mean values (±SE) of GVBD% in the oocytes which were incubated with MEL, L, SER, M and MIH or, MEL, L, SER and MIH, or MEL, SER, M and MIH have been presented as bar diagram in Fig. 4. It has been revealed from two-way ANOVA that the effects of MEL, L, SER, M and MIH were statistically significant in respect to the treatment (F4,16 = 9.77; p b 0.001) and the duration (F4,16 = 5.29; p b 0.006) of incubation (Figs. 3, 4). 95.91 ± 0.85% oocytes underwent GVBD following incubation with MEL, SER, M and MIH, while the value was reduced to 64.42 ± 0.3% or 39.29 ± 1.67% following incubation with MEL, L, SER and MIH, or with MEL, L, SER, M and MIH (Fig. 4). The rate of GVBD in the oocytes incubated with MEL, SER, M and MIH was significantly (F = 426.02, p b 0.001) higher than induced by MEL, SER and MIH, or MEL, L, SER and MIH, or MEL, L, SER, M and MIH (Figs. 3, 4). A significant effect of the duration of incubation was noted in the oocytes which were incubated with MEL, SER, M and MIH (F = 956.81, p b 0.001), or MEL, L, SER and MIH (F = 853.73, p b 0.001) or, MEL, L, SER, M and MIH (F = 209.90, p b 0.001).
Fig. 5. (A) SDS PAGE of the carp oocyte extract showing a 50 kDa protein as cyclin B and a 34 kDa protein as cdc2. (B) The immunoblot data of cyclin B in the extracts of oocytes at different time points following incubation in a M199 medium showing a higher expression of maturation promoting factor in presence of melatonin added 4 h before the addition of MIH, and a weak expression of the same proteins in the absence of melatonin or presence of serotonin. (C) Densitometric analysis of the Immunoblot of cyclin B. Each densitometric data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the graph indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c) respectively following one way ANOVA.
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Fig. 6. Immunoblot of p34cdc2 demonstrates time dependent changes in the protein in the oocytes which were incubated with only serotonin or only MIH for 16 h or preincubated with melatonin for 4 h before addition of MIH and incubation for 16 h thereafter. The increase in the expression of the 34 kDa protein is notable at early hours of incubation with melatonin, while no expression of this catalytic protein is found in presence of serotonin.
4.2. Cyclin B—cdc2 activity in the incubated oocytes The oocyte extracts used in SDS PAGE (Figs. 5A) and immunoblotting with anti-cyclin B antibody or anti-PSTAIRE (anti-Cdk1 or cdc2) antibody demonstrated the presence of 50 kDa protein as cyclin B and 34 kDa protein as cdc2 respectively. Maximum cyclin B was found at 16 h following incubation with only MIH, but at 12 h in the oocytes which were treated with MEL 4 h before incubation with MIH (Fig. 5B). A two-way measures of the densitometric data on the immunoblot of cyclin B revealed that there were significant effects of MEL, SER and MIH in both treatment (F4,8 = 6.89; p b 0.01) and time (F2,8 = 72.49; p b 0.001) groups (Fig. 5C). In consideration to the duration of incubation, significant variations were found in groups treated with SER (F = 12323.88, p b 0.001), or MIH (F = 249.43, p b 0.001), or MEL and MIH (F = 336.80, p b 0.001). The densitometric units of cyclin B in the SER treated oocytes were significantly (F = 1997.9, p b 0.001) lower than those found following incubation with MIH alone, or MEL (4 h before) and MIH for 12 h. However, there were two bands which showed differential expressions with time. There was a gradual decrease in 35 kDa band protein and concomitant increase in 34 kDa protein band (cdc2) with the progress of time (Fig. 6). The profiles of 35 kDa band and 34 kDa band proteins were mostly identical in the oocytes incubated only with MIH for 16 h or treated with MEL 4 h before incubation with MIH for 12 h. On the contrary, a reduction in the formation of the cdc2 was noted in all the oocytes which were incubated with SER (Fig. 6). The results of the study of both cyclin B and cdc2 in the oocytes incubated with the blockers of receptors of MEL or SER were complimentary to the findings on the rate of GVBD in the concerned oocytes. 5. Discussion The importance of both serotonin and melatonin in the regulation of reproductive processes has been emphasized with the mammalian studies (Vesela et al., 2003). Presence of serotonin has also been reported in various non-neural tissues in different vertebrates and invertebrates, including the gonads of many invertebrates in which it regulates spawning and oocyte meiotic maturation (Amireault and
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Dubé, 2005; Dubé and Amireault, 2007). But to our utter surprise, prior to this study, no information was available on the influence of serotonin on the actions of melatonin on the same oocytes in any vertebrate species. Our present study demonstrated for the first time that serotonin not only inhibits the independent actions of MIH, but also the actions of melatonin on the MIH-induced oocytes maturation in carp. We found that stimulatory actions of melatonin alone or of melatonin and MIH on the rate of GVBD were significantly lost as a result of incubation of oocytes with serotonin and melatonin or serotonin, melatonin and MIH respectively (Figs. 2 and 3). Pharmacological evidence of the actions of concerned indole amines on the studied features of oocytes gained further support from the results of the study using luzindole and metoclopramide in the incubation medium as the specific blocker of receptors for melatonin (Browning et al., 2000; Drazen et al., 2001) and serotonin (Cerda et al., 1997) respectively. While luzindole was found to reduce the stimulatory actions of melatonin, metoclopramide suppressed the inhibitory actions of serotonin on the studied features of carp oocytes (Fig. 4). In each case, both electrophoretic and immunoblot studies (Figs. 5 and 6) revealed that the rate of GVBD was associated with the rate of MPF formation in the concerned oocytes. The results of the present study showing a significantly (F = 731.46 p b 0.001) higher rate of GVBD and an accelerated rate of formation of MPF (maturation promoting factor) in the carp denuded oocytes following pre-treatment of MEL and incubation with MIH compared to those incubated with only MIH for 12 h are complimentary to our previous findings (Chattoraj et al., 2005) and underline the importance of melatonin in the regulation of meiotic resumption in carp oocytes. However, it is notable that only 14.42 ± 0.43% oocytes underwent GVBD following incubation with serotonin alone for 16 h. Although compared to the independent actions of melatonin (77.15 ± 1.91% GVBD) and MIH (93.24 ± 1.57% GVBD), the stimulatory actions (14.42 ± 0.43% GVBD) of serotonin on the meiotic resumption of carp oocytes are negligible, a significant reduction in the rate (4.33 ± 0.35%) of GVBD in the oocytes incubated with both serotonin and the antagonist of its receptors metoclopramide clearly indicates that serotonin per se may have a stimulatory influence on the studied features of carp oocytes. These results lend support to the findings on medaka (Oryzias latipes) in which serotonin was found to induce oocyte maturation in vitro by stimulating the production of MIH and 17β-estradiol from granulose (follicular) cells (Iwamatsu et al., 1992, 1993). However, the concentrations of none of the alleged steroids have been measured in the present study and, therefore, the endocrine mechanism of such actions of serotonin in carp oocytes should remain speculative. One of the major objectives of the current investigation was to demonstrate the influences, if any, of serotonin on the actions of melatonin and/or MIH on the rate of meiosis resumption in concerned carp oocytes. It was notable that incubation of oocytes with serotonin and MIH resulted in significant decrease in the rate of GVBD compared to the independent effects of MIH (Figs. 2 and 3). But the study showing that the effects were reversible in presence of metoclopramide (Fig. 4) suggests an inhibitory role of serotonin on the actions MIH on meiosis resumption in carp oocytes. A direct inhibitory action of serotonin on the MIH-activated meiosis resumption in the oocyte was also noted in another teleost F. heteroclitus in which serotonin reversibly inhibited both gonadotropin and MIH-induced oocyte meiosis resumption without any effect on the steroidogenic pathways (Cerda et al., 1995). Likewise, serotonin was detected within the ovarian follicles of amphibians where it exerted a reversible antagonistic effect on progesterone-induced oocyte maturation through putative receptors located on the follicle cells, on the oocyte membrane, and in the ooplasm (Buznikov et al., 1993; Nikitina et al., 1993). The quantitative assay of various serotonergic agonists and antagonists on the oocytes of F. heteroclitus suggested the role of specific serotonin membrane receptor sites in the mediation of the
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inhibitory effects of serotonin on oocyte meiosis resumption. In the same teleost species, serotonin was found to have no effect on the steroidogenic pathways in the granulose cells associated with the oocyte, and thereby indicated that serotonin inhibited the steroidinduced oocyte maturation by a direct action on the oocyte (Cerda et al., 1995). It was also demonstrated that serotonin was more effective at inhibiting MIH-stimulated GVBD in denuded oocytes than in the follicle-enclosed oocytes of F. heteroclitus which taken together with the polar nature of serotonin suggested that the serotonin receptor, mediating the inhibitory action on the steroid-induced meiotic maturation, is located at the surface of prophase-arrested oocytes (Cerda et al., 1997). The conjecture of a receptor mediated action of serotonin on the process of meiotic maturation also gained support from the present study where a significant decrease in the rate of GVBD and associated formation of MPF (revealed from electrophoresis and immunoblot studies; Figs. 5 and 6) was shown by the oocytes following incubation with serotonin, its receptor blocker, and MIH (Fig. 4). A vast evidence is available to show that in fish oocytes MIH promotes the formation of a dimeric protein kinase, known as MPF, which in turn triggers meiotic maturational events like germinal vesicle breakdown (GVBD) through dispersion of nuclear lamina, chromosome condensation and formation of metaphase spindles in the oocytes of fish (Nagahama, 1997). MPF is a complex of two proteins, a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, cdc2 (Lohka et al., 1988; Yamashita et al., 1992; Chausson et al., 2004). We confirmed the presence of a 50 kDa cyclin B and a conserved p34cdc2 protein (Fig. 5A) in the oocyte extract of carp using mouse anti-cyclin monoclonal and anti-PSTAIRE antibodies (Chattoraj et al., 2005). Notably, expression of 50 kDa cyclin B (Fig. 5B), supported by densitometric analysis of the immunoblot data (Fig. 5C), in the cytosolic extracts of oocytes are higher in the melatonin pre-treated than that in only MIH-treated oocytes, but significantly lower in the oocytes which were incubated with serotonin. These observations, therefore, provides evidence that both melatonin and MIH are stimulatory to formation of a 34 kDa protein band and serotonin is inhibitory to their actions (Fig. 6). Collectively, these observations may be considered as persuasive evidence of a hitherto unknown aspect of serotonin melatonin MIH interactions on the process of meiotic maturation in teleosts in general and carp in particular. However, it remains unknown whether respective receptor mediated actions of serotonin and melatonin involve the receptors of MIH on the concerned oocytes, and requires further study. In conclusion, serotonin is antagonistic to the actions of melatonin and MIH on the process of meiotic maturation in carp. Acknowledgment The authors thankfully acknowledge financial assistance from the Council of Scientific and Industrial Research, Govt. of India, New Delhi (37(1220)/05/EMR-II). References Amireault, P., Dubé, F., 2005. Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos. Biol. Reprod. 73, 358–365. Bayarri, M.J., Rodriguez, L., Zanuy, S., Madrid, J.A., Sanchez-Vazquez, F.J., Kagawa, H., Okuzawa, K., Carrillo, M., 2004. Effect of photoperiod manipulation on the daily rhythms of melatonin and reproductive hormones in caged European sea bass (Dicentrarchus labrax). Gen. Comp. Endocrinol. 136, 7–81. Bhattacharya, S., Dey, R., Basu, A., Maitra, S.K., Banerji, T.K., 2003. The structure of the pineal complex in a common Indian teleost, Catla catla: evidence for pinealinduced inhibition of testicular function within an annual reproductive cycle. Endocr. Res. 29, 141–156. Bhattacharya, S., Chattoraj, A., Maitra, S.K., 2007. Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light and continuous darkness. Chronobiol. Int. 24, 629–650.
Bromage, N.R., Porter, M.J.R., Randall, C.F., 2001. The environmental regulation of maturation in farmed finfish with special reference to the role of photoperiod and melatonin. Aquaculture 197, 63–98. Browning, C., Beresford, I., Fraser, N., Giles, H., 2000. Pharmacological characterization of human recombinant melatonin MT(1) and MT(2) receptors. Br. J. Pharmacol. 129, 877–886. Buznikov, G.A., Nikitina, L.A., Galanov, A.Y., Malchenko, L.A., Trubnikova, O.B., 1993. The control of oocyte maturation in the starfish and amphibians by serotonin and its antagonists. Int. J. Dev. Biol. 37, 363–364. Cerda, J., Petrino, T.R., Lin, Y.W.P., Wallace, R.A., 1995. Inhibition of Fundulus heteroclitus oocyte maturation by serotonin (5-hydroxytryptamine). J. Exp. Zool. 273, 224–233. Cerda, J., Zanuy, S., Carrillo, M., 1997. Evidence for dietary effects on plasma level of sexual steroids during spermatogenesis in the sea bass. Aquat. Int. 5, 473–477. Chattoraj, A., Bhattacharya, S., Basu, D., Bhattacharya, S., Bhattacharya, S., Maitra, S.K., 2005. Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in Carps. Gen. Comp. Endocrinol. 140, 145–155. Chausson, F., Paterson, L.A., Betteley, K.A., Hannah, L., Meijer, L., Bentley, M.G., 2004. CDK1/cyclin B regulation during oocyte maturation in two closely related lugworm species, Arenicola marina and Arenicola defodiens. Dev. Growth Differ. 46, 71–82. Donham, R.S., Horton, T.H., Rollag, M.D., Stetson, M.H., 1989. Age, photoperiodic responses, and pineal function in meadow voles, Microtus pennsylvanicus. J. Pineal Res. 7, 243–252. Drazen, D.L., Bilu, D., Bilbo, S.D., Nelson, R.J., 2001. Melatonin enhancement of splenocyte proliferation is attenuated by luzindole, a melatonin receptor antagonist. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1476–R1482. Dubé, F., Amireault, P, 2007. Local serotonergic signaling in mammalian follicles, oocytes and early embryos. Life Sci. 81, 1627–1637. Hirai, T., Yamashita, M., Yoshikuni, M., Lou, Y.H., Nagahama, Y., 1992. Cyclin B in fish oocytes: its cDNA and amino acid sequence, appearance during maturation, and induction of p34cdc2 activation. Mol. Reprod. Dev. 33, 131–140. Iwamatsu, T., Toya, Y., Ouchi, H., Aoyama, T., Yoneima, J., Kondo, T., Imai, K., Hattori, H., Ikegami, S., Onda, M., 1992. Characterization of a low molecular weight factor in chicken serum with oocyte maturation-inducing activity. Biomed. Res. 13, 429–437. Iwamatsu, T., Sakai, Y.T.N., Terada, Y., Nagata, R., Nagahama, Y., 1993. Effect of 5hydroxytryptamine on steroidogenesis and oocyte maturation in pre-ovulatory follicles of the medaka Oryzias latipes. Dev. Growth Differ. 35, 625–630. Lohka, M.J., Hayes, M.K., Maller, J.L., 1988. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. U. S. A. 85, 3009–3013. Maitra, S.K., Chattoraj, A., Bhattacharyya, S., 2005. Implication of melatonin in oocyte maturation in Indian major carp Catla catla. Fish. Physiol. Biochem. 31, 201–207. Nagahama, Y., 1997. 17a, 20 b-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 62, 190–196. Nikitina, L.A., Trubnikova, O.B., Buznikov, G.A., 1993. Effects of neurotransmitters and their antagonists on oocyte maturation: the effect of serotonin antagonists on in vitro oocyte maturation in amphibians. Ontogenez 24, 29–38. Pang, Y., Ge, W., 2002. Gonadotropin and activin enhanced maturational competence of oocytes in the zebra fish (Danio rerio). Biol. Reprod. 66, 259–265. Popek, W., Bieniarz, K., Epler, P., 1991. Role of pineal gland in the sexual cycle in common carp. In: Surowiak, J.M., Lewandowski, H. (Eds.), Chronobiology and Chronomedicine. Verlag Peter Lang, Frankfurt, pp. 99–102. Popek, W., Bieniarz, K., Epler, P., 1992. Participation of the pineal gland in sexual maturation of female rainbow trout (Onchrhynchus mykiss Walbaum). J. Pineal Res. 13, 97–100. Popek, W., Breton, B., Sokolowska-Mikolajczyk, M., Epler, P., 1994. The effects of bicuculline (a GABA receptor antagonist) on LHRH-A and pimozide stimulated gonadotropin (GtH2) release in female carp (Cyprinus carpio L.) in normal and in polluted environments. Arch. Pol. Fish 5, 59–75. Reiter, R.J., 1991. The pineal gland: reproductive interactions. In: Pang, P.K.T., Schreibman, M.P. (Eds.), Vertebrate Endocrinology: Fundamental and Biomedical Implications, vol. 4. Academic Press, San Diego, pp. 269–310 (Part B). Senthilkumaran, B., Sudhakumari, C., Chang, X., Kobayashi, T., Oba, Y., Guan, G., Yoshiura, Y., Yoshikuni, M., Nagahama, Y., 2002. Ovarian carbonyl reductase-like 20b hydroxysteroid dehydrogenase shows distinct surge in mRNA expression during natural and gonadotropin-induced meiotic maturation in Nile tilapia. Biol. Reprod. 67, 1080–1086. Sirotkin, A.V., Schaeffer, H.J., 1997. Direct regulation of mammalian reproductive organs by serotonin and melatonin. J. Endocrinol. 154, 1–5. Tokumoto, T., Tokumoto, M., Horiguchi, R., Ishikawa, K., Nagahama, Y., 2004. Diethylstilbestrol induces fish oocyte maturation. Proc. Natl. Acad. Sci. U. S. A. 101, 3686–3690. Vanêĉek, J., Klein, D.C., 1993. A sub-population of neonatal gonadotrophin – releasing hormone– sensitive pituitary cells is responsive to melatonin. Endocrinology 133, 360–367. Vesela, J., Rehak, P., Mihalik, J., Czikkova, S., Pokorny, J., Koppel, J., 2003. Expression of serotonin receptors in mouse oocytes and preimplantation embryos. Physiol. Res. 52, 223–228. Vivien-Roels, B., Pevet, P., Dubois, M.P., Arendt, J., Brown, G.M., 1981. Immunohistochemical evidence for the presence of melatonin in the pineal gland, the retina and the Harderian gland. Cell Tissue Res. 217, 105–115. Woo, M.M.M., Tai, C.J., Kang, S.K., 2001. Direct action of melatonin in human granulosaluteal cells. J. Clin. Endocrinol. Metab. 86, 4789–4797. Yamashita, M., Fukuda, S., Yoshikuni, M., Bulet, P., Hirai, T., Yamaguchi, A., Lou, Y.-H., Zhao, Z., Nagahama, Y., 1992. Purification and characterization of maturation -promoting factor in fish. Dev. Biol. 148, 8–15.
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla Asamanja Chattoraj, Mohua Seth, Saumen Kumar Maitra ⁎ Department of Zoology, Visva-Bharati University, Santiniketan-731 235, India
A R T I C L E
I N F O
Article history: Received 10 January 2008 Received in revised form 14 March 2008 Accepted 20 March 2008 Available online 28 March 2008 Keywords: Carp Melatonin MIH Oocytes Serotonin
A B S T R A C T The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 μg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 μM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 μM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24 ± 1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15 ± 1.91% or 14.42 ± 0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58 ± 1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57 ± 0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component – cyclin B and the catalytic component – Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The pineal gland is the major component of the neuroendocrine system which through melatonin (N-acetyl-5-methoxytryptamine) and/or its precursor serotonin (5-hydroxytryptamine) is alleged to be associated with the regulation of reproduction in different vertebrates (Reiter, 1991). Importance of these two indole amines has been demonstrated by the presence of their receptors in the reproductive organs (Woo et al., 2001; Vesela et al., 2003) and by their effects on the functions of the testis and oviduct (Sirotkin and Schaeffer, 1997). However, the existing information on the physiology of these hormones is based mostly on mammalian studies, where influences of these principles on reproduction varied in relation to their dose and duration of treatment as well as their time of application (Donham et al., 1989; Vanêĉek and Klein, 1993).
⁎ Corresponding author. Tel./fax: +91 3463 261268. E-mail address: [email protected] (S.K. Maitra). 1095-6433/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.03.014
The role of pineal gland and its hormones has also been investigated in the seasonal maturation of gonad in fish (VivienRoels et al., 1981; Popek et al., 1992, 1994; Bromage et al., 2001; Bhattacharya et al., 2003, 2007; Bayarri et al., 2004; Maitra et al., 2005), but the results of the study have been diverse. The study on rainbow trout (Popek et al., 1992) suggested that the pineal gland may alter the maturation period and control spawning by acting on the hypothalamo-pituitary-gonadal axis. However, in vitro studies of the oocytes in carp Cyprinus carpio (Popek et al., 1991) and killifish Fundulus heteroclitus (Cerda et al., 1997) could not demonstrate any effect of melatonin on oocyte maturation. On the other hand, serotonin was found to inhibit MIH-induced resumption of meiosis following in vitro study of oocytes in teleost F. heteroclitus (Cerda et al., 1997). The results showing such inhibition in both follicleenclosed and denuded oocytes indicated the presence of oocyteassociated serotonin sensitive sites (Cerda et al., 1995, 1997). In a recent study, it has been shown for the first time that melatonin accelerates the action of MIH when added 4 h prior to MIH in the incubation medium (Chattoraj et al., 2005). However, the relative role
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of serotonin and melatonin on the MIH-induced oocyte maturation in any fish remained unknown. The present study aimed at in vitro demonstration of independent as well as relative influences of serotonin and melatonin on the MIHinduced meiotic resumption in the oocytes of a hitherto unstudied major carp C. catla. Since fish oocytes undergo drastic morphological changes associated with progression of the meiotic cell cycle, in which breakdown of the oocyte nuclear envelope or germinal vesicle breakdown (GVBD) occurring at the prophase/metaphase transition and is usually regarded as a hallmark of the progress of oocyte maturation (Tokumoto et al., 2004), we studied the rate of GVBD in the carp oocytes as the visual marker of meiotic resumption. In most teleosts, 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20 β DHP) serves as the potent MIH (Pang and Ge, 2002; Senthilkumaran et al., 2002), which acts on receptors located on the oocyte membrane and induces the activation of maturation promoting factor (MPF) in the oocyte cytoplasm to initiate final maturation (Nagahama, 1997). Therefore, our study also included the rate of formation of maturation promoting factor (MPF) which is a complex of two proteins, a regulatory component called cyclin B and catalytic component protein kinase known as cyclin-dependent kinase or cdc2 (Yamashita et al., 1992; Chausson et al., 2004). Specific blockers of receptors of melatonin and of serotonin have been appropriately used to provide pharmacological evidence of the specific action of the concerned indolamines on the studied carp oocytes. 2. Materials and methods 2.1. Biological materials Adult C. catla weighing between 600 g and 1000 g (measuring 300 mm to 457 mm) were collected during the spawning phase (July–August) from their natural habitat in India (Lat. 23°39′ Ν., Long. 87°42′ Ε). Soon after catch, only gravid females were quickly transported to the laboratory and were maintained in large aquaria with balanced fish food ad libitum for a week for acclimatization to laboratory conditions before being used in the experiments. 2.2. Chemicals and reagents All the analytical grade chemicals including M199 medium, melatonin, serotonin, MIH, luzindole, metoclopramide HCl, and polyvinylidene difluoride (PVDF) membranes were purchased from Sigma-Aldrich Chemical Co. St. Louis, MO, USA. The primary antibodies of cyclin B and cdc2 were procured from Santa Cruz Biotechnology Inc. California, USA and secondary antibodies were supplied by Bangalore Genei Pvt. Ltd. (Bangalore, India).
Fig. 1. Denuded oocytes collected from the ovary of carp Catla catla during the spawning phase, and freed from somatic follicle cells following cold shock treatment (X 40).
penicillin (100 U/mL) and streptomycin (100 μg/mL). Incubations were carried out separately in M199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 μg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 μM/mL) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 μM/mL) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. The control oocytes were incubated in the medium alone. The selection of dose and time of addition of melatonin in the medium were based on our previous study (Chattoraj et al., 2005). The dose (10 μM/mL) of both luzindole and metoclopramide hydrochloride, used as the specific blockers of the receptors of MEL and SER respectively for demonstration of their pharmacological effects in the medium, has been the same as employed earlier (Cerda et al., 1997; Drazen et al., 2001). In either case, oocytes were incubated further for 4, 8, 12 or 16 h following addition of MIH. Viability of oocytes was about 95% as determined by 0.1% Trypan blue exclusion test. 3.2. Determination of GVBD
3. Methods
At the end of incubation for a given period of time, the oocytes were immersed in a clearing solution (ethanol/ formalin/acetic acid; 6:3:1) for an easy examination of the oocytes (Tokumoto et al., 2004) under a microscope (Olympus, BX51, Japan) with in-built photomicrographic attachments and image analyzing device. The effects of treatment on oocyte maturation were determined by GVBD and the results were expressed in % taking 100 oocytes for each observation.
3.1. Incubation of denuded oocyte
3.3. Preparation of oocyte extracts for study of maturation signals
The large and round shaped yolk-laden Stage-IV oocytes were collected from the ovary of freshly sacrificed gravid females and were manually denuded with the help of forceps under ice cold M199 medium. It is well known that sudden cold shock does not affect the quality of oocytes, rather helps to separate somatic follicle cells from oocytes by forceps (Chattoraj et al., 2005). Accordingly, the same technique has been adopted to avail denuded oocytes. Complete removal of follicle cells was ensured through careful examination under the microscope and only denuded oocytes were selected (Fig. 1). Subsequently, the isolated oocytes were exposed to 95% O2 / 5% CO2 at 27 ± 1 °C before incubating in separate sterilized micro-beakers for different treatments under identical laboratory conditions. Each micro-beaker (5 mL capacity) contained about 50 denuded oocytes which were incubated in 1 mL of Medium M199 supplemented with
Fifty oocytes from each set of incubation at given time points were collected in micro-centrifuge tubes and washed thrice with ice cold extraction buffer (EB: 80 mM sodium β-glycerophosphate, 20 mM HEPES, 15 mM MgCl2, 5 mM EGTA, 0.5 mM PMSF, 1 mM DTT, leupeptin, 10 μg/mL aprotinin pH 7.5) following Hirai et al. (1992) with minor modifications and homogenized in the same buffer at 4 °C. The homogenate was centrifuged at 14,000 g for 15 min. The clear cytosolic supernatant was used for electrophoresis and Western blotting for the detection of cyclin B and p34cdc2 protein. 3.4. Electrophoresis and immunoblotting The methods of electrophoresis and immunoblotting were described earlier (Chattoraj et al., 2005). In brief, for immunodetection
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oocytes following incubation with different agents included in the present investigation (F4,80 = 49.18, p b 0.001; F20,80 = 4.91, p b 0.001).
Fig. 2. Rate of GVBD at different intervals following incubation of the denuded oocytes of Catla catla for 16 h in Medium M 199 with (i) none (control), or (ii) only melatonin (MEL), or (iii) only MIH, or (iv) melatonin added simultaneously with MIH, (v) melatonin added 4 h before [MEL(b)] MIH, or (vi) melatonin with luzindole (L), or (vii) melatonin added simultaneously with MIH and luzindole (L), or (viii) melatonin added 4 h before [MEL(b)] MIH and luzindole (L). Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e N f N g) respectively following one way ANOVA.
of cdc2, the polyvinylidene difluoride (PVDF) membrane was incubated with rabbit anti-PSTAIRE primary antibody (1:1000 dilution). Mouse anti-cyclin B1 antibody was used as the primary antibody (1:1000 dilution) to detect the cyclin B. The respective secondary antibodies (dilution 1:500) conjugated with alkaline phosphatase were used and visualized by treating the membrane with BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium).The densitometric analysis of the Western-blot data was carried out by Image-J software and expressed in line-diagram in arbitrary densitometric units.
4.1.1. The effects of Melatonin and MIH The mean values (±SE) of the rate (%) of GVBD following incubation with only maturation inducing hormone (MIH), or only melatonin (MEL), or MEL with MIH have been diagrammatically presented in Fig. 2. Significant effects were noted both in respect to the treatment (F4,16 = 14.44; p b 0.001) as well as time (F4,16 = 6.00; p b 0.003). Following incubation with MIH (1 μg/mL) alone, significant variations (F = 1561.49; p b 0.001) were noted at different time points. Likewise, significant (F = 524.46; p b 0.001) changes were found in relation to the time of incubation with only MEL (100 pg/mL). However, the rate of GVBD in the oocytes incubated with MIH for 16 h was significantly (F = 960.72; p b 0.001) higher than the oocytes incubated with only MEL at respective time point. An addition of MEL 4 h prior to the application of MIH in the medium resulted in a significant (F = 1149.52; p b 0.001) increase in the rate of GVBD with the progress incubation. More specifically, the rate of meiotic resumption of oocytes noted at 12 h following pre-treatment of MEL and incubated with MIH was significantly (F = 731.46; p b 0.001) higher than that noted in the oocytes incubated with only MIH. 4.1.2. The effects of melatonin, serotonin and MIH Fig. 3 summarizes the results of the study of GVBD% following incubation of oocytes with serotonin (SER) alone, or in combination with MEL and MIH. A two-way ANOVA showed that the effects of SER, MEL and MIH on the rate of GVBD were statistically significant in respect to the treatment (F4,20 = 12.89; p b 0.001) and the time of incubation (F5,20 = 4.18; p b 0.009). Only 14.42 ± 0.43% GVBD were noted following incubation with SER (100 pg/mL) alone for 16 h. The influence of only SER was found significantly (F = 688.63; p b 0.001) inhibitory compared to that noted at 16 h following incubation with MEL and SER, or SER and MIH, or MEL, SER and MIH. Significant variations in GVBD were noted in relation to the time of incubation of oocytes with SER and MEL (F = 508.29, p b 0.001), or SER and MIH (F = 663.66, p b 0.001), or SER, MEL and MIH (F = 607.25, p b 0.001).
3.5. Statistical analysis of experimental data Each experiment was carried out in quadruplicate using fifty oocytes in each set from one fish and the same was followed with three separate donor fish (i.e., n = 3). For example, time kinetics for MIH-induced oocyte maturation was observed in quadruplicate to obtain mean value of %GVBD and densitometric units of cyclin B immunoblot for each fish. Such mean values from three separate donor fish were then used to draw the mean value ± SE for each treatment group at each time point. Data on %GVBD were compared between oocytes cultured for different times and treated in different ways using two-way ANOVAs (analysis of variance). Time and treatment were the grouping factors. Post-hoc comparisons were made between times for each group and between groups for each time using univariate F tests taking p b 0.05 as the statistically significant threshold (Chattoraj et al., 2005). The densitometric data on cyclin B immunoblot for different groups of oocytes were also analyzed in an identical way following the same statistical methods. 4. Results 4.1. The rate of meiotic resumption of oocytes Two-way ANOVA showed significant effects of time and treatment group on GVBD, the visual marker of meiotic resumption of oocytes, in
Fig. 3. Influence of serotonin on the rate of GVBD of the denuded oocytes of Catla catla following the study of the effects of incubation at different intervals following incubation for 16 h in Medium M 199 with (i) none(control), or (ii) only serotonin (SER), or (iii) melatonin (MEL) added simultaneously with serotonin (SER), or (iv) serotonin (SER) added simultaneously with MIH, or (v) serotonin added 4 h before [SER(b)] MIH, or (vi) serotonin added simultaneously with melatonin and MIH. Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e) respectively following one way ANOVA.
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4 h before MIH), M and MIH, (F = 897.74, p b 0.001), or SER (coadministration), M and MIH (F = 1022.88, p b 0.001). 4.1.5. The effects of melatonin, luzindole, serotonin, metoclopramide Incubation of oocytes for 16 h with MEL, SER and M resulted in 77.57 ± 1.44% GVBD, but the rate was significantly (F = 883.81, p b 0.001) decreased to 42.83 ± 1.38% or 7.23 ± 0.43% following treatment with MEL and SER, or MEL, L, SER and M respectively (Figs. 3, 4). A two-way measures ANOVA showed that there were significant effects of MEL, L, SER and M both in respect to the treatment (F4,12 = 3.45; p b 0.04) as well as to the time (F3,12 = 6.46; p b 0.007) of treatment. In consideration to the time groups, significant variations were found in the oocytes which were incubated with MEL, SER and M (F = 686.76, p b 0.001), or MEL, L, SER and M (F = 55.76, p b 0.001).
Fig. 4. Influence of metoclopramide (serotonin receptor blocker) and luzindole (melatonin receptor blocker) on the GVBD of denuded oocytes of Catla catla following the study of the effects of incubation at different intervals following incubation for 16 h in Medium M 199 with (i) none (control), or (ii) metoclopramide (M) with serotonin (SER), or (iii) metoclopramide (M) with melatonin (MEL) and (SER), or (iv) metoclopramide (M) with serotonin (SER) and MIH, or (v) metoclopramide (M) and serotonin added 4 h before [SER(b)] MIH, or (vi) metoclopramide (M), serotonin added simultaneously with melatonin and MIH, or (vii) metoclopramide (M), Luzindole (L), melatonin and serotonin added simultaneously, or (viii) metoclopramide (M), luzindole (L), melatonin, serotonin and MIH added simultaneously, or (ix) luzindole (L), serotonin, melatonin and MIH added simultaneously. Each data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the bars indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c N d N e N f) respectively following one way ANOVA.
4.1.3. The effects of melatonin, luzindole (antagonist of melatonin receptors) and MIH The mean values (± SE) of GVBD% in the oocytes following incubation with MEL and luzindole (L) with or without MIH have been shown in Fig. 2. A two-way ANOVA revealed that the effects of L, MEL and MIH were statistically significant in respect to the treatment (F4,28 = 16.72; p b 0.001) as well as to the time (F7,28 = 5.98; p b 0.001). The oocytes exhibited 16.53 ± 0.51%, or 38.49 ± 1.04%, or 39.3 ± 1.58% GVBD when incubated for 16 h with MEL (co-administration) and L, or MEL, L and MIH, or MEL (added 4 h before MIH), L and MIH respectively. Significant variations were noted in relation to the time of incubation with MEL and MIH (F = 2116.60, p b 0.001), or MEL and L (F = 430.93, p b 0.001), or MEL (co-administration), L and MIH (F = 308.63, p b 0.001), or MEL (added 4 h before MIH), L and MIH. (F = 433.22, p b 0.001). An inhibitory influence of L in each experiment was found at both 12 h (F = 731.46; p b 0.001) and 16 h (F = 960.72; p b 0.001) of incubation. 4.1.4. The effects of serotonin, metoclopramide (antagonist of serotonin receptors) and MIH The mean values (±SE) of the rate (%) of GVBD in different groups of oocytes incubated with SER and metoclopramide (M), or SER (added 4 h before MIH), MIH and M, or SER (co-administration), MIH and M have been presented in Fig. 4. A two-way ANOVA showed that the effects of SER, M and MIH were statistically significant in both treatment (F4,12 = 8.44; p b 0.001) and time groups (F3,12 = 3.49; p b 0.04). At the end of 16 h of incubation with SER (added 4 h before MIH), M and MIH, the rate of GVBD was 89.93 ± 2.03%, while 94.25 ± 2.15% oocytes underwent GVBD following incubation with SER (co-administration), MIH and M. The rate of GVBD was significantly higher in the oocytes which were incubated for 16 h with SER (added 4 h before MIH), MIH and M (F = 122.14; p b 0.001), or SER (co-administration), M and MIH (F = 163.05; p b 0.001) compared to those which were incubated in the corresponding media but without M (Figs. 3, 4). Significant variations were noted in relation to the duration of incubation with SER (added
4.1.6. The effects of melatonin, luzindole, serotonin, metoclopramide and MIH The mean values (±SE) of GVBD% in the oocytes which were incubated with MEL, L, SER, M and MIH or, MEL, L, SER and MIH, or MEL, SER, M and MIH have been presented as bar diagram in Fig. 4. It has been revealed from two-way ANOVA that the effects of MEL, L, SER, M and MIH were statistically significant in respect to the treatment (F4,16 = 9.77; p b 0.001) and the duration (F4,16 = 5.29; p b 0.006) of incubation (Figs. 3, 4). 95.91 ± 0.85% oocytes underwent GVBD following incubation with MEL, SER, M and MIH, while the value was reduced to 64.42 ± 0.3% or 39.29 ± 1.67% following incubation with MEL, L, SER and MIH, or with MEL, L, SER, M and MIH (Fig. 4). The rate of GVBD in the oocytes incubated with MEL, SER, M and MIH was significantly (F = 426.02, p b 0.001) higher than induced by MEL, SER and MIH, or MEL, L, SER and MIH, or MEL, L, SER, M and MIH (Figs. 3, 4). A significant effect of the duration of incubation was noted in the oocytes which were incubated with MEL, SER, M and MIH (F = 956.81, p b 0.001), or MEL, L, SER and MIH (F = 853.73, p b 0.001) or, MEL, L, SER, M and MIH (F = 209.90, p b 0.001).
Fig. 5. (A) SDS PAGE of the carp oocyte extract showing a 50 kDa protein as cyclin B and a 34 kDa protein as cdc2. (B) The immunoblot data of cyclin B in the extracts of oocytes at different time points following incubation in a M199 medium showing a higher expression of maturation promoting factor in presence of melatonin added 4 h before the addition of MIH, and a weak expression of the same proteins in the absence of melatonin or presence of serotonin. (C) Densitometric analysis of the Immunoblot of cyclin B. Each densitometric data represents mean ± SE of three independent observations. Different alphabets in capital scripts and separate alphabets in small scripts on the graph indicate significant (p b 0.001) difference between the time points within the specific treatment group (A N B N C N D N E) and between the treatment groups at a specific time point (a N b N c) respectively following one way ANOVA.
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Fig. 6. Immunoblot of p34cdc2 demonstrates time dependent changes in the protein in the oocytes which were incubated with only serotonin or only MIH for 16 h or preincubated with melatonin for 4 h before addition of MIH and incubation for 16 h thereafter. The increase in the expression of the 34 kDa protein is notable at early hours of incubation with melatonin, while no expression of this catalytic protein is found in presence of serotonin.
4.2. Cyclin B—cdc2 activity in the incubated oocytes The oocyte extracts used in SDS PAGE (Figs. 5A) and immunoblotting with anti-cyclin B antibody or anti-PSTAIRE (anti-Cdk1 or cdc2) antibody demonstrated the presence of 50 kDa protein as cyclin B and 34 kDa protein as cdc2 respectively. Maximum cyclin B was found at 16 h following incubation with only MIH, but at 12 h in the oocytes which were treated with MEL 4 h before incubation with MIH (Fig. 5B). A two-way measures of the densitometric data on the immunoblot of cyclin B revealed that there were significant effects of MEL, SER and MIH in both treatment (F4,8 = 6.89; p b 0.01) and time (F2,8 = 72.49; p b 0.001) groups (Fig. 5C). In consideration to the duration of incubation, significant variations were found in groups treated with SER (F = 12323.88, p b 0.001), or MIH (F = 249.43, p b 0.001), or MEL and MIH (F = 336.80, p b 0.001). The densitometric units of cyclin B in the SER treated oocytes were significantly (F = 1997.9, p b 0.001) lower than those found following incubation with MIH alone, or MEL (4 h before) and MIH for 12 h. However, there were two bands which showed differential expressions with time. There was a gradual decrease in 35 kDa band protein and concomitant increase in 34 kDa protein band (cdc2) with the progress of time (Fig. 6). The profiles of 35 kDa band and 34 kDa band proteins were mostly identical in the oocytes incubated only with MIH for 16 h or treated with MEL 4 h before incubation with MIH for 12 h. On the contrary, a reduction in the formation of the cdc2 was noted in all the oocytes which were incubated with SER (Fig. 6). The results of the study of both cyclin B and cdc2 in the oocytes incubated with the blockers of receptors of MEL or SER were complimentary to the findings on the rate of GVBD in the concerned oocytes. 5. Discussion The importance of both serotonin and melatonin in the regulation of reproductive processes has been emphasized with the mammalian studies (Vesela et al., 2003). Presence of serotonin has also been reported in various non-neural tissues in different vertebrates and invertebrates, including the gonads of many invertebrates in which it regulates spawning and oocyte meiotic maturation (Amireault and
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Dubé, 2005; Dubé and Amireault, 2007). But to our utter surprise, prior to this study, no information was available on the influence of serotonin on the actions of melatonin on the same oocytes in any vertebrate species. Our present study demonstrated for the first time that serotonin not only inhibits the independent actions of MIH, but also the actions of melatonin on the MIH-induced oocytes maturation in carp. We found that stimulatory actions of melatonin alone or of melatonin and MIH on the rate of GVBD were significantly lost as a result of incubation of oocytes with serotonin and melatonin or serotonin, melatonin and MIH respectively (Figs. 2 and 3). Pharmacological evidence of the actions of concerned indole amines on the studied features of oocytes gained further support from the results of the study using luzindole and metoclopramide in the incubation medium as the specific blocker of receptors for melatonin (Browning et al., 2000; Drazen et al., 2001) and serotonin (Cerda et al., 1997) respectively. While luzindole was found to reduce the stimulatory actions of melatonin, metoclopramide suppressed the inhibitory actions of serotonin on the studied features of carp oocytes (Fig. 4). In each case, both electrophoretic and immunoblot studies (Figs. 5 and 6) revealed that the rate of GVBD was associated with the rate of MPF formation in the concerned oocytes. The results of the present study showing a significantly (F = 731.46 p b 0.001) higher rate of GVBD and an accelerated rate of formation of MPF (maturation promoting factor) in the carp denuded oocytes following pre-treatment of MEL and incubation with MIH compared to those incubated with only MIH for 12 h are complimentary to our previous findings (Chattoraj et al., 2005) and underline the importance of melatonin in the regulation of meiotic resumption in carp oocytes. However, it is notable that only 14.42 ± 0.43% oocytes underwent GVBD following incubation with serotonin alone for 16 h. Although compared to the independent actions of melatonin (77.15 ± 1.91% GVBD) and MIH (93.24 ± 1.57% GVBD), the stimulatory actions (14.42 ± 0.43% GVBD) of serotonin on the meiotic resumption of carp oocytes are negligible, a significant reduction in the rate (4.33 ± 0.35%) of GVBD in the oocytes incubated with both serotonin and the antagonist of its receptors metoclopramide clearly indicates that serotonin per se may have a stimulatory influence on the studied features of carp oocytes. These results lend support to the findings on medaka (Oryzias latipes) in which serotonin was found to induce oocyte maturation in vitro by stimulating the production of MIH and 17β-estradiol from granulose (follicular) cells (Iwamatsu et al., 1992, 1993). However, the concentrations of none of the alleged steroids have been measured in the present study and, therefore, the endocrine mechanism of such actions of serotonin in carp oocytes should remain speculative. One of the major objectives of the current investigation was to demonstrate the influences, if any, of serotonin on the actions of melatonin and/or MIH on the rate of meiosis resumption in concerned carp oocytes. It was notable that incubation of oocytes with serotonin and MIH resulted in significant decrease in the rate of GVBD compared to the independent effects of MIH (Figs. 2 and 3). But the study showing that the effects were reversible in presence of metoclopramide (Fig. 4) suggests an inhibitory role of serotonin on the actions MIH on meiosis resumption in carp oocytes. A direct inhibitory action of serotonin on the MIH-activated meiosis resumption in the oocyte was also noted in another teleost F. heteroclitus in which serotonin reversibly inhibited both gonadotropin and MIH-induced oocyte meiosis resumption without any effect on the steroidogenic pathways (Cerda et al., 1995). Likewise, serotonin was detected within the ovarian follicles of amphibians where it exerted a reversible antagonistic effect on progesterone-induced oocyte maturation through putative receptors located on the follicle cells, on the oocyte membrane, and in the ooplasm (Buznikov et al., 1993; Nikitina et al., 1993). The quantitative assay of various serotonergic agonists and antagonists on the oocytes of F. heteroclitus suggested the role of specific serotonin membrane receptor sites in the mediation of the
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inhibitory effects of serotonin on oocyte meiosis resumption. In the same teleost species, serotonin was found to have no effect on the steroidogenic pathways in the granulose cells associated with the oocyte, and thereby indicated that serotonin inhibited the steroidinduced oocyte maturation by a direct action on the oocyte (Cerda et al., 1995). It was also demonstrated that serotonin was more effective at inhibiting MIH-stimulated GVBD in denuded oocytes than in the follicle-enclosed oocytes of F. heteroclitus which taken together with the polar nature of serotonin suggested that the serotonin receptor, mediating the inhibitory action on the steroid-induced meiotic maturation, is located at the surface of prophase-arrested oocytes (Cerda et al., 1997). The conjecture of a receptor mediated action of serotonin on the process of meiotic maturation also gained support from the present study where a significant decrease in the rate of GVBD and associated formation of MPF (revealed from electrophoresis and immunoblot studies; Figs. 5 and 6) was shown by the oocytes following incubation with serotonin, its receptor blocker, and MIH (Fig. 4). A vast evidence is available to show that in fish oocytes MIH promotes the formation of a dimeric protein kinase, known as MPF, which in turn triggers meiotic maturational events like germinal vesicle breakdown (GVBD) through dispersion of nuclear lamina, chromosome condensation and formation of metaphase spindles in the oocytes of fish (Nagahama, 1997). MPF is a complex of two proteins, a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, cdc2 (Lohka et al., 1988; Yamashita et al., 1992; Chausson et al., 2004). We confirmed the presence of a 50 kDa cyclin B and a conserved p34cdc2 protein (Fig. 5A) in the oocyte extract of carp using mouse anti-cyclin monoclonal and anti-PSTAIRE antibodies (Chattoraj et al., 2005). Notably, expression of 50 kDa cyclin B (Fig. 5B), supported by densitometric analysis of the immunoblot data (Fig. 5C), in the cytosolic extracts of oocytes are higher in the melatonin pre-treated than that in only MIH-treated oocytes, but significantly lower in the oocytes which were incubated with serotonin. These observations, therefore, provides evidence that both melatonin and MIH are stimulatory to formation of a 34 kDa protein band and serotonin is inhibitory to their actions (Fig. 6). Collectively, these observations may be considered as persuasive evidence of a hitherto unknown aspect of serotonin melatonin MIH interactions on the process of meiotic maturation in teleosts in general and carp in particular. However, it remains unknown whether respective receptor mediated actions of serotonin and melatonin involve the receptors of MIH on the concerned oocytes, and requires further study. In conclusion, serotonin is antagonistic to the actions of melatonin and MIH on the process of meiotic maturation in carp. Acknowledgment The authors thankfully acknowledge financial assistance from the Council of Scientific and Industrial Research, Govt. of India, New Delhi (37(1220)/05/EMR-II). References Amireault, P., Dubé, F., 2005. Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos. Biol. Reprod. 73, 358–365. Bayarri, M.J., Rodriguez, L., Zanuy, S., Madrid, J.A., Sanchez-Vazquez, F.J., Kagawa, H., Okuzawa, K., Carrillo, M., 2004. Effect of photoperiod manipulation on the daily rhythms of melatonin and reproductive hormones in caged European sea bass (Dicentrarchus labrax). Gen. Comp. Endocrinol. 136, 7–81. Bhattacharya, S., Dey, R., Basu, A., Maitra, S.K., Banerji, T.K., 2003. The structure of the pineal complex in a common Indian teleost, Catla catla: evidence for pinealinduced inhibition of testicular function within an annual reproductive cycle. Endocr. Res. 29, 141–156. Bhattacharya, S., Chattoraj, A., Maitra, S.K., 2007. Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light and continuous darkness. Chronobiol. Int. 24, 629–650.
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