Involvement of [Ca.sup.2+] signaling pathway during oocyte maturation of the northern quahog Mercenaria mercenaria.
KEY WORDS: GVBD, northern quahog, Mercenaria mercenaria, hard clam, oocyte maturation, [Ca.sup.2+], signaling pathway
During oogenesis, animal oocytes usually arrest at prophase I of meiosis and develop an enlarged nucleus (germinal vesicle, GV). After staying in the prophase for some time, a fully grown oocyte will be triggered by hormones or other stimuli into maturation during which the oocyte will complete germinal vesicle breakdown (GVBD) and then into metaphase I or II before fertilization (Masui & Clarke 1979). GVBD is usually considered a sign of oocyte maturation that is crucial for subsequent development (Eppig 1982).
In some molluscs, such as Spisula and Barnea, fertilization occurs in prophase I, which allows prophase-arrested oocytes to reinitiate meiotic division (Dube & Guerrier 1982, Dube 1988). Meiosis reinitiation of Spisula oocytes is also triggered by KCl (Allen 1953) or serotonin (5-hydroxytryptamine, 5-HT) (Hirai et al. 1988). In contrast, prophase-arrested oocytes of Ruditapes (Guerrier et al. 1993) and Hiatella (Deguchi & Osanai 1995), undergo meiotic maturation after calcium ionophore, ammonia, of serotonin treatment and secondarily arrest at metaphase I. In the second case, fertilization takes place in metaphase I.
As many immature oocytes rail in fertilization, the mechanism of oocytes maturation has been recently researched, and several signaling pathways have been found important in modulating meiosis resumption, such as cAMP, [Ca.sup.2+], and MAPK pathways (Eckberg 1988, Kishimoto 2003). These pathways finally activate maturation-promoting factor (MPF), which was responsible for the reinitiating meiosis of oocyte (Nebreda & Ferby 2000).
[Ca.sup.2+] is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. During the resumption of meiosis, an increase in intracellular free [Ca.sup.2+] is involved in many animals (Whitaker & Patel 1990). In the nemertean, Cerebratulus lacteus, injecting aqueous extracts from sperm can trigger repetitive [Ca.sup.2+] waves and resumption of meiotic maturation (Stricker 1997). During oocyte maturation in Xenopus laevis oocytes, an increase in free [Ca.sup.2+] is involved in triggering oocyte maturation (Cicirelli & Smith 1987). [Ca.sup.2+] is also required to activate MPF (Maturation-promoting factor) and GVBD during starfish Asterina pectinifera oocyte maturation (Tosuji et al. 2007). In the resumption of meiotic maturation of mammalian oocytes, including mouse, porcine, bovine, and human oocytes, [Ca.sup.2+] signaling pathway also plays an important role (Carroll et al. 1994, Sedmikova et al. 2003, He et al. 1997, Homa et al. 1993, Balakier et al. 2002). However, the mechanisms regulating [Ca.sup.2+] signaling pathway during oocyte maturation in bivalves are less studied.
The northern quahog Mercenaria mercenaria (Linnaeus, 1758) is indigenous to the Atlantic coasts along the North America naturally, from Gulf of St. Lawrence to Gulf of Mexico (Menzel 1970). It was first introduced into China as a candidate for aquaculture in 1997. Its ecological habits, physiology, and artificial breeding had been studied by some researchers in China (Chang et al. 2002, Zhang et al. 2003a, 2003b). An adult female the northern quahog can produce millions of oocytes, which ate 70 ~ 90-[micro]m in diameter with obvious GV. Therefore, the northern quahog is a good experimental animal for studying the mechanisms of oocyte development.
In this paper, the function of [Ca.sup.2+] signaling pathway in serotonin-induced oocyte maturation of the northern quahog is presented. This study not only may help us to understand the mechanism of oocyte maturation, but also can give some guidance to the artificial breeding of the northern quahog.
MATERIALS AND METHODS
Artificial seawaters (ASW) were prepared in MBL formulae (Cavanaugh 1975) and buffered with 10-mM Tris-HCl, pH 8.2. Serotonin, A23187 (calcium ionophore), verapamil (calcium channel blocker), triftuoperazine (calmodulin antagonist) were purchased from Sigma company.
Handling of Oocytes
The northern quahog were collected from a farming zone in Laizhou Bay (Shandong, China) (67.47 [+ or -] 8.18 mm; 99.95 [+ or -] 12.12 g; n = 40) and brought into reproductive condition in a mollusc hatchery. Six female clams were used in each trial. Oocytes were obtained and handled according to Allen (1953). Oocytes suspension was filtered through nylon mesh sieve with ASW and the oocytes were then washed 3-4 times to remove the tissue debris and other impurities.
Artificial Activation and Quantification of Oocyte Maturation
The oocytes were induced in a 24-well cell culture cluster (Corning, USA). 50 [micro]L settled oocytes (about 20,000 oocytes) were added to each well containing 850 [micro]L ASW. Then 100 [micro]L various stocking solutions were added to each well to achieve the final concentration (3 replicates per treatment). The entire experiment was carried out at room temperature (20[degrees]C) with various doses of chemical agents over a 60 min or 90 min period. Oocytes were examined under a microscopy with photographs taken by a Nikon Coolpix 4500 camera. GVBD rates were calculated according to the photographs. About 30~50 oocytes were scored for each examination.
Values are presented as mean [+ or -] SD. Two-way analysis of variance (two-way ANOVA) and one-way ANOVA were performed for all the data analysis with the SPSS 13.0 statistical software. When a significant effect (P < 0.05) was found, a multiple comparison (Tukey) test was conducted to compare the significant difference among treatments. Percentage data (above 5%) were transformed (arcsine of the square root) before ANOVA, but presented in figures as nontransformed percentage.
5-HT-Induced Oocyte Maturation
The effect of 5-HT on prophase-arrested oocytes was tested at concentrations from 0.01 [micro]M to 100 [micro]M. GVBD first occurred 15 min later (Fig. 1), and GVBD rate increased as time increased. About 70% of the oocytes showed GVBD after 60 min incubation, whereas in ASW group the rate was only <10%. The highest GVBD rate was induced by 10 [micro]M, with a percentage of 72.57% at 90 min. GVBD rates enhanced as time increased, and 5-HT induced oocyte maturation in a time-dependent manner (two-way ANOVA, P < 0.001). However, there was no difference between 40 min and 50 min, so was 60 min and 90 min (Tukey test, P > 0.05). 5-HT (0.01 ~ 100 [micro]M) could promote the maturation in a dose-dependent manner, but only GVBD rate of 0.001 [micro]M was significantly lower than those of other concentrations (Tukey test, P < 0.05). (Fig. 2)
[FIGURE 1 OMITTED]
Effect of [Ca.sup.2+] on Oocyte Maturation
ASW was the control group. As illustrated in Figure 3, High concentration of [Ca.sup.2+] (50 ~ 200 mM) could promote oocyte maturation significantly at 60 min and 90 min (two-way ANOVA, P < 0.001). Nevertheless, it was less effective than 5-HT. For example, 200 mM [Ca.sup.2+] had the best effect in stimulating oocyte maturation after 90 min, but its GVBD rate could only reach to 31.53%. In addition, [Ca.sup.2+] needed more time to induce maturation than that of 5-HT. [Ca.sup.2+] (50 ~ 200 mM) stimulated the maturation of oocytes in a dose-dependent manner significantly at 60 min (one-way ANOVA, P < 0.001), but not significantly at 90 min (one-way ANOVA, P > 0.05).
Effect of A23187 on Oocyte Maturation
ASW was the control group. A23187 (1 ~ 10 [micro]M) could stimulate the maturation remarkably (Tukey test, P < 0.001), whereas the relative lower concentration group (0.01 ~ 0.1 [micro]M) had no remarkable effect on oocyte maturation (the same as the control group) (Tukey test, P > 0.05) (Fig. 4). As illustrated in the figure, after incubated by 1 [micro]M A23187 for 60 min, about 26% of the oocytes became mature, whereas the GVBD rate of 10 [micro]M was about 38%. Therefore, 1-[micro]M and 10-[micro]M A23187 promoted oocyte maturation in a dose dependent manner.
Effect of Verapamil on 5-HT-Induced Oocyte Maturation (Fig. 5)
To investigate the function of calcium ions during GVBD, we used verapamil, a specific [Ca.sup.2+] channel blocker, to find its effect on GVBD in the northern quahog oocytes. ASW was the blank group and 5-HT (10 [micro]M) was the control group. As illustrated in Figure 6, verapamil (1 ~ 100 [micro]M) inhibited 5-HT-induced oocyte maturation significantly (two-way ANOVA, P < 0.001). Verapamil of 0.1 [micro]M could significantly restrain the maturation at 50 min (one-way ANOVA, P < 0.01), however, the results were not significant at 40 min and 60 min (one-way ANOVA, P > 0.05). Verapamil of relative higher concentration (100 [micro]M) could inhibit the maturation completely, with zero GVBD rate.
Effect of TFP on 5-HT-Induced Oocyte Maturation
To study the function of CaM during GVBD, TFP was used to find its effect on meiotic maturation in the northern quahog oocytes. As a Ca M antagonist, TFP blocks the binding of [Ca.sup.2+] to CaM. ASW was the blank group and 5-HT (10 [micro]M) was the control group. TFP (10 ~ 1,000 [micro]M) could inhibit 5-HT-induced oocyte maturation remarkably (two-way ANOVA, P < 0.001). Moreover, 1,000 [micro]M TFP restrained the maturation completely, like the effect of 100 [micro]M verapamil. Nevertheless, TFP of 0.1 [micro]M and 1 [micro]M promoted the maturation significantly only at 60 min (one-way ANOVA, P < 0.05), but not significantly at 40 min and 50 min (one-way ANOVA, P > 0.05).
[FIGURE 2 OMITTED]
5-HT plays a central role in several physiological processes in marine molluscs, especially in reproduction. It acts as a neurohormone to regulate spawning, parturition, and meiosis by reinitiating meiotic maturation in arrested oocytes (Garnerot et al. 2006). It often stimulates oocyte maturation of sevcral bivalve molluscs, such as Spisula solidissima (Hirai et al. 1988, Krantic et al. 1991), Spisula sachalinensis (Hirai et al. 1988, Varaksin et al. 1992), Crassostrea gigas (Osanai 1985, Kyozuka et al. 1997) and Ruditapes phillippinarum (Osanai & Kuraishi 1988, Guerrier et al. 1993).
In this paper, we have demonstrated that 5-HT (0.01 ~ 100 [micro]M) could induce oocyte maturation of the northern quahog effectively. GVBD occurred in the oocytes after incubating for 15~30 min, and the GVBD rates reached about 70% after 60 ruin incubation (Fig. 2). The efficiency of serotonin in triggering GVBD varied from 50% to 80%, depending on the degree of maturity of the oocytes. It could not be recognized on the basis of purely morphological criteria. In addition, we have proved that serotonin induced oocytes could be fertilized by sperms, and the embryonic development and larval development were normal (data not shown).
[FIGURE 3 OMITTED]
Our results indicate that an increase in extracellular [[Ca.sup.2+]]i can stimulate in vitro meiotic resumption in the northern quahog ooytes. In addition, calcium ionophore triggers GVBD in the northern quahog oocytes in calcium-containing artificial water. Thus, external calcium influx is apparently involved in meiotic activations. Moreover, extracellular [Ca.sup.2+] seems to be required for meiosis progression, because the exposure to verapamil, a specific [Ca.sup.2+] channel blocker, decreases in vitro maturation efficiency or inhibir the maturation completely.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Phosphatidylinositol signaling pathway comprises DAG (diacylglycerol)-PKC (protein kinase C) and [IP.sub.3] (inositol-1, 4, 5-trisphosphate)-[Ca.sup.2+] pathways. The two second pathways play key roles in amplifying extracellular signals and regulating various intracellular processes triggered by stimuli. In our previous study, we have demonstrated that phosphatidylinositol signaling pathway played a positive role and PKC pathway played a negative role in serotonin-induced maturation in the northern quahog oocytes (data not shown). Therefore, [Ca.sup.2+] pathway may play a positive role according to our previous study. With this study, we prove that [Ca.sup.2+] pathway plays a positive role in oocytes maturation of the northern quahog.
[FIGURE 6 OMITTED]
In other bivalves, similar results have been testified. In Spisula, meiotic reinitiation stimulated by serotonin is completely blocked by the absence of external [Ca.sup.2+] (Kadam et al. 1990) and ah increase in intracellular [[Ca.sup.2+]]i is observed after the addition of serotonin in the presence of external [Ca.sup.2+] (Juneja et al. 1994). Similar results have also been found in Hiatella flaccida, a dose-dependent increase in [[Ca.sup.2+]i is observed by the application of different concentrations of serotonin (Deguchi & Osanai 1995). In Ruditapes phillippinarum, serotonin can also trigger an increase in [[Ca.sup.2+]]i and meiotic maturation in oocytes, which depend on external [Ca.sup.2+]. However, this maturation can be completely inhibited by an intracellular [Ca.sup.2+] chelator, BAPTA-AM (Guerrier et al. 1993). In oyster Crassostrea gigas, calcium ionophore A23187 can also induce GVBD in ooyctes (Osanai & Kuraishi 1988). In other animals, such as starfish (Tosuji et al. 2007), Xenopus (El-Jouni et al. 2005) and mammals (Boni et al. 2007), external [Ca.sup.2+] is also found to be required for meiotic maturation.
However, in some bivalves, internal [Ca.sup.2+] may also release during activation of meiosis in oocytes. In Ruditapes phillipinarum, GVBD and calcium transients can be triggered by various agonists in the absence of external [Ca.sup.2+] (Guerrier et al. 1993, Gobet et al. 1995, Lippai et al. 1995). Furthermore, [IP.sub.3] can also promote GVBD in surf clam (Bloom et al. 1988). In addition, serotonin normally can induce a transient rise in [IP.sub.3] levels prior to GVBD (Gobet et al. 1994), but it fails to cause GVBD in oocytes which are also incubated by heparin (Deguchi & Osanai 1995).
CaM is a ubiquitous intracellular mediator of calcium signaling. The binding of calcium to CaM enables it to activate various target enzymes, such as CaM dependent protein kinases, and therefore regulate many physiological processes (Vogel 1994). We demonstrated that TFP (a CaM antagonist) restrained the meiotic maturation remarkably or even completely, indicating that the inhibiting of CaM activity blocked resumption of meiosis maturation. Thus, the [Ca.sup.2+]/CaM compound could regulate the nuclear events of meiosis resumption in the northern quahog oocytes. This also suggests [Ca.sup.2+] and CaM are required for meiosis reinitiation. Studies have sug gested that [Ca.sup.2+]/CaM compound regulates the progress of mitosis by its downstream molecular CaMKII. CaMKII may exert its effect via regulation of MPF activity during the meiotic maturation of pig oocytes (Fan et al. 2003).
However, there are many studies show that no [Ca.sup.2+] influx occurs during meiosis process and [Ca.sup.2+] is not required for meiotic maturation. In oyster Crassostrea gigas, 5-HT does not trigger any changes in [[Ca.sup.2+]]i in oocytes (Kyozuka et al. 1997). In starfish, it has not been firmly established whether an increase of intracellular [Ca.sup.2+] is required for meiotic maturation in oocytes. Some investigators have shown no change in intracellular free [Ca.sup.2+] in response to 1-MA (1-methyladenine) in Asterias forbesi (Eisen & Reynolds 1984), Asterina miniata, Orthasterias Koehleri, and Pisaster oehraceus (Stricker et al. 1994). Thus, the question of whether [Ca.sup.2+] signaling pathway are needed for oocyte maturation remains controversial. This may be because of differences among species and between mechanisms governing spontaneous maturation in vitro and gonadotrophin-induced maturation in vivo.
In conclusion, we show that [Ca.sup.2+] is essential for the reinitiation of meiotic maturation in oocytes of the northern quahog and an increase in [[Ca.sup.2+]]i can promote meiotic maturation.
The authors thank Dr. Baozhong Liu, and the workers of the Bay Scallop Hatchery of Qingdao, for their help in the experiments. This work is supported by grants from Young Foundation of National 863 Program of China (No. 2001AA628040 and No. 2004AA603810 to Dr. Zhang.
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TAO ZHANG, (1) * QING WANG (1,2) AND HONGSHENG YANG (1)
(1) Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, People's Republic of China; (2) The Graduate School of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
* Corresponding author. E-mail: email@example.com; tao-zhang@ hotmail.com
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|Author:||Zhang, Tao; Wang, Qing; Yang, Hongsheng|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2009|
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