Preliminary Study On Gonadal Development And Mechanism Of Oocyte Maturation Of Hard Clam Mercenaria Mercenaria

To identify the function of cAMP signaling pathway in 5-HT-induced oocyte maturation of hard clam Mercenaria mercenaria, effects of several modulators of transducers on 5-HT-induced maturation were studied in vitro. Results showed that 5-HT of 0.01μM-100μM induced the maturation of oocytes remarkably, and the GVBD rate of each concentration was about 70% after an hour induction. The results of different doses of 5-HT were close, showing no dosage-dependence relation but time-dependent. Caffeine, theophylline and IBMX could inhibit spontaneous oocyte maturation slightly, but not significantly. High concentration of phosphodiesterase inhibitors (5mM IBMX, 10mM caffeine and 10mM theophylline) inhibited 5-HT-induced oocyte maturation significantly. 1mM IBMX had no effect on 5-HT-induced maturation. In addition, dbcAMP could not only inhibit spontaneous oocyte maturation, but also could inhibit 5-HT-ingduced maturation. The results indicate that cAMP signaling pathway is invovled in 5-HT-induced oocyte maturation, and the rise of cAMP concentration can inhibit the maturation. In conclusion, cAMP signaling pathway plays probably a negative role in 5-HT-induced oocyte maturation of hard clam.Effects of activator/antagonist of PLC and protein PKC on serotonin-induced maturation were studied. Results show that high-concentration neomycin could block maturation, while DMBA could promote maturation in the presence of 5-HT. It was also found in this study that in the presence of 5-HT, PMA blocked maturation, while Sphingosine accelerated maturation. These results indicate that 5-HT-induced maturation requires the activation of the phosphatidylinositol pathway. Decrease in PLC inhibited 5-HT-induced oocyte maturation, whereas decrease in PKC stimulated the maturation. Thus, PLC played probably a positive role in the maturation,while DAG-PKC pathway maybe played a negative role.High concentration Ca²⁺ and A23187 (calcium carrier) could promote oocyte maturation.Verapami(1-100μM) could prohibit 5-HT-induced oocyte maturation, and Verapamil of 100μM inhibited oocyte matuartion completely. It is indicated that extracellular Ca²⁺ was necessary to oocyte maturation of hard clam. Trifluoperazin (TFP) inhibited 5-HT-induced oocyte maturation significantly, and 1mM TFP could inhibit the maturation completely. It is inferred that Calmodulin (CaM) could probably promote oocyte maturation.Artificial fertilization of 5-HT-induced oocytes was studied. Results showed that 5-HT-induced oocyte of hard clam could be fertilized and the subsequent development was normal, like natural fertilization and development. It was also found that theophylline and caffeine have no effect on fertilization of hard clam, whereas neomycin could inhibit the fertilization. Therefore, it is inferred that cAMP signaling pathway may not involve in the fertilization of hard clam, while phosphatidylinositol pathway involved in the course.Gonad development and gametal ultrastructure were studied. The gonad development of hard clam is similar to Ark shell (Tegillarca granosa). It was found that four cell parts of the oocyte, i.e. mitochondria, Golgi vesicle, endoplasmic reticulum and micropinocytotic vesicle, may be involved in the formation of yolk granule.