Thiamethoxam (TMX) is a neonicotinoid insecticide. Residues of TMX have been detected in various crops. Although it has specific high toxicity to insects and is designed to exterminate them, the toxicity has also found in mammals recently. Differ from acetylcholine toxicity, TMX has peroxide toxicity in mammals. Matured oocytes have the capacity of fertilization, but oocytes own abundant mitochondria and its maturation is vulnerable to reactive oxygen species (ROS). Excessive production of reactive oxygen species (ROS) can override antioxidant defenses, produce oxidative stress and DNA damage that triggers apoptosis and necrosis in organisms. However little is known about the harm of ROS induced by TMX during oocytes maturation. Here, bovine germ-vesicle (GV) oocytes were cultured to metaphase of the second meiosis (MII) stage in vitro with or without TMX. During this process, oocytes were evaluated by various methods. Microscopic examination showed that 1.6 mM TMX significantly inhibited the maturation process in which oocytes were arrested before MI stage or between MI and MII stage. Correspond to this two periods, immunofluorescence staining and enzyme activity analysis showed that active CDC25 and CDC2 reduced in TMX group compared to control; time lapse and immunofluorescence staining gave results that Cyclin B could not be degraded, actin cap could not form, and Bub3 could not be removed from kinetochores. In addition, MII oocytes exposed to TMX showed disordered chromosomes and spindle. To study further, oocytes cultured for 24 h were analyzed. On the one hand, these oocytes in TMX group accumulated more ROS and produced significantly decreased mitochondrial membrane potential and increased apoptotic signal compared to control by methods of quantities for dichlorodihydrofluorescein diacetate (DCHFDA), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide and Annexin-V, but the level of γH2AX protein in TMX group did not decline significantly compared with control. On the another hand, these oocytes were activated to be parthenogenetic embryos and cultured. Assessment for embryo development showed decreased rates of cleavage, morula and blastocyst in TMX group compared to control in vitro. In conclusion, these results suggest that ROS induced by TMX results in dysfunction of mitochondria and apoptosis, which block bovine oocytes to MI stage, trap them at AI/TI stage and trigger disordered chromosomes and spindle at MII stage. Additionally, MII oocytes with poor qualities result from TMX lose abilities to cleavage and develop to be morulae and blastocysts.

In oocytes from different species, MPF, a complex of Cdk1 and cyclin B, is the master regulator of cell cycle. The activity of MPF is regulated by the phosphorylations mediated by Wee1B kinase and Cdc25B phosphatase. Although a regulation of MPF activity by these inhibitory phosphorylations are well established, a dogma in the cell cycle is that MPF activity is regulated by the dynamics of cyclin B during the metaphase II (MII) arrest (also known as CSF arrest). However, growing evidences suggest that Wee1B-mediated Cdk1 phosphorylation is also critical to trigger the progression of cell cycle during the onset of anaphase. Therefore, in the present study, we investigated the role of Cdc25B phosphatase during MII arrest. Cdc25B is present in MII arrested oocytes as a hyperphosphorylated form and disruption of its function either by antibody or siRNA injection induces the progression of cell cycle to interphase. Moreover, the hyperphosphorylated form, which has been known as an active form of Cdc25B, is dephosphorylated during the anaphase onset. Interestingly, this dephosphrylation occurred ahead of cyclin B degradation. Conversely, overexpression of Cdc25B prevents metaphase to anaphase transition induced by calcium stimulation. Therefore, our findings provide novel paradigm in cell cycle that MPF activity during metaphase arrest is regulated by the balance between Cdk1 inhibitory kinases, Wee1, and the counteracting phosphatases, Cdc25. When cells exit from metaphase, Cdc25 is inactivated and Wee1 is reactivated and thereby Cdk1 kinase activity is rapidly and transiently decreased. This initial decrease of Cdk1 activity is further promoted by the proteolytic degradation of cyclin B, which ensures irreversible progression of cell cycle to interphase. Thus, the concerted effort of phosphorylation/dephosphorylation of Cdk1 and synthesis/degradation of cyclin B play roles in fine-tuning the activity of Cdk1 during metaphase to anaphase transition.

Keeping the intact germinal vesicle (GV) is essential for maintaining the capacity of mammals including human. It is maintained by very complex procedures along with folliculogenesis and is a critical step for getting competent oocyte. So far, a few mechanisms involved in folliculogenesis are known but GV arrest mechanisms are largely unrevealed. Cyclic AMP, a adenosine derived substance, have been used as inhibitor of germinal vesicle breakdown as a putative oocyte maturation inhibitor. In this study, we examined the potency of adenosine as GV maintainer and a possible signaling mediator for that. A1, A2b, and A3 were detected in cumulus cells of cumulus enclosed-oocyte (CEO). Intact of germinal vesicle was not kept like in follicle but the spontaneous maturation was inhibited by exogenous adenosine. It is inhibited with concentration dependent manners. Intracellular calcium level of cumulus was extensively increased after adenosine treatment. Based on these results it is suggested that one of the pathway for GV arrest by adenosine and its receptors is calcium mediated signaling pathway in CEO.