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  • br Materials and methods br Results br Discussion PYR


    Materials and methods
    Discussion PYR-41 inhibits UBE1 activity that catalyses an initial step in the ubiquitination reaction. In the present study, we examined the role of this enzyme in sperm-egg fusion by inhibiting UBE1 enzymatic activity in eggs. Our results suggest that PYR-41 pretreatment disturbs the intrinsic fusion competence in eggs as follows: first, PYR-41 treatment reduced fertilization efficiency; second, this pretreatment specifically reduced the efficiency of sperm fusion with eggs; third, this pretreatment suppressed sperm enlargement leading to impaired meiotic chromosomal separation after fusion with the egg. PYR-41 is a derivative of pyrazone, the abbreviated term for the herbicidal substance 5-amino-4-chloro-2-phenyl-3 (2H) pyridazinone, which acts in vivo as an inhibitor of HPF photosynthesis in plants [13]. Since pyrazone is also able to remove excess uric acid, which causes gout or gouty arthritis, its treatment reduces the risk of complications in patients with conditions such as kidney stones. In mammalian reproduction, loss of p53 in female mice significantly decreases fertility because of reduced embryo implantation ability [14]. PYR-41 suppresses the degradation of p53 proteins and also activates its transcriptional activity by inhibiting UBE1 activity, resulting in differential killing of transformed HPF [[5], [6]]. Hence, PYR-41 is a potential anti-cancer drug. Pyrazone is useful for therapeutic treatment of some types of human diseases [15]; however, our results imply that it might have adverse effects for mammalian female reproduction. Our study contributes to the understanding of the molecular mechanisms of mammalian fertilization, and the cause of female infertility.
    Declaration of interest
    Author contributions
    Acknowledgement This study was supported by a Grant-in-aid for Scientific Research from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 26670733 and No. 26293363 to K. Miyado).
    Introduction Fluoride, as a member of the halogen group of elements, naturally existed in water, soil, animals, and plants around the world (Singh et al., 2018), and it is present in trace amounts in all mineralized tissues such as dentin, enamel and bone. It is documented that fluoride could enhance the stability of mineralized tissues and materials since it weakens inclusive mineral phase in tissues and materials (Tressaud and Haufe, 2008). Therefore, fluoride is used extensively to prevent dental caries; however, excessive ingestion may cause side effects of fluoride exposure and result in serious health hazards. Excessive fluoride intake may damage a variety of organs and tissues such as skeletal, nervous, digestive, respiratory, genitourinary and endocrine systems (Del Piero, 2013). Literatures show that fluoride affects diverse signaling pathways associated with proliferation and apoptosis, such as mitogen-activated protein kinase (MAPK), p53, activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) pathways (Iwatsuki and Matsuoka, 2016; Zhang et al., 2007, 2008). It is also reported that fluoride has an adverse effect on the cell cycle but it brings out various impacts which rest in different cell types (Ribeiro et al., 2017). Research data in hepatocytes cultured in vitro indicate that fluoride increased cell number at S phase and decreased cells at G2/M or G0/G1phase (Liu et al., 2018). p53 is activated in response to several malignancy-associated stress signals, giving rise to the inhibition of tumor cell growth (Garritano et al., 2013). Several responses can be elicited by p53, including cell cycle arrest, senescence, differentiation and apoptosis. The p53 protein is controlled by many different forms of post-translational modifications, including ubiquitylation, phosphorylation, acetylation, sumoylation, methylation, and neddylation (Lee and Gu, 2010; Muller and Vousden, 2013; Solomon et al., 2017). Acetylation is of vital importance to regulation of p53. It increases the protein stability of p53, binding to low affinity promoters, association with other proteins, antiviral activities, and is required for its checkpoint responses to DNA damage and activated oncogenes (Munoz-Fontela et al., 2011; Reed and Quelle, 2014; Zhang et al., 2015; Zhao et al., 2015). As an example, p300-mediated acetylation of p53 in human cancer cell lines has been implicated for p21 promoter transactivation and cell cycle arrest (Iyer et al., 2004; Li et al., 2006; Shi et al., 2016). Previous studies reported that p53/p21-mediated cell cycle leads to genetic mutations in mice (Kuroda et al., 2015).