LAUSR

Is it possible to increase the decreased levels of the sperm-oocyte binding protein, Juno, to restore the fertilization capacity of the oocyte, via the use of the CRISPR/dCas9 activation system? JUNO domain (in the oocyte) suppressed with melamine was opened using the CRISPR/dCas9 system and sperm-oocyte binding and fertilization capability have been restored. Melamine is a chemical that is widely used in the manufacture of laminates, plastics, etc. Evidence reveals that long-term exposure to melamine could damage reproductive systems in mammals leading to infertility. Izumo1 is the only cell surface protein expressed on sperm that is known to be essential for sperm-egg interaction in vivo. It was in vitro shown that high-dose feeding of melamine to female mice led to a significant decrease of JUNO on the plasma membrane of eggs. CRISPR/dCas9 system can provide the gene activation or repression to activate the target gene via Sam and Tet1 based systems. Six different gRNAs were designed for the transfection of oocytes. Six-week-old mice were fed with melamine (50mg/kg/day) for 2 weeks via gavage. Melamine gavage, water gavage, and control groups (n = 15 /each group) were created. CRISPR activation plasmids (SAM) were given by piezo microinjection into the GV oocytes (n = 100 oocytes/each group). Fertilization capacity was evaluated by sperm binding assay, qPCR, Western blotting, and IF staining. Two technical replicates were used in molecular studies. 293T cells were transfected (dCas9 SAM plasmids+gRNA) with Fugene. Mice randomly were assigned to 3 groups (n = 15), as each was given orally a dose of 50mg/kg/d of melamine, only water or no water via gavage. Microinjection of plasmids was performed. Post-injection, oocytes were incubated until MII stage. For binding and fertilization evaluation, motile sperms were incubated with oocytes, and pronuclei were checked. Juno and IZUMO1 levels were evaluated by qPCR, Western blotting, and IF staining. As the SAM system is more efficient compared to the Tet1 system when tested in 293t cells, the SAM system was used for mouse experiments. As a result of qPCR performed in oocytes collected at the end of gavage, it was observed that the JUNO expression levels were decreased by 40 folds in melamine fed mice (p < 0.05). The decrease in the level of JUNO protein was demonstrated by IF stainings, and a decrease in the oocyte count along with abnormal uterine shapes was also observed in this group. IZUMO1 expression in motile sperms was demonstrated by qPCR before sperm binding assay and the position of the IZUMO 1 domain before the acrosome reaction was demonstrated by IF stainings. Sperm binding assay has demonstrated a 70% reduction in fertilization competency of melamine-treated oocytes (p < 0.05). SAM plasmids and JUNO gRNA were given to oocytes by piezo injection. By sperm binding experiments conducted to evaluate fertilization capacities after microinjection, it was shown that the fertilization capacity was restored by 75% (p < 0.05). Re-gaining of Juno expression in the oocytes was supported by a 60 fold increase in qPCR results. Recovery of JUNO protein expression in the oocytes was also demonstrated by IF stainings. There is no known promoter region for the JUNO gene in the mouse. Therefore, we designed gRNAs targeting possible promoter regions. However, we have used two activation systems(SAM and Tet1) that are widely used to open a closed gene, but other activation systems (acetylation, etc.) can also be tried. Wider implications of the findings: This study is valuable since: -it presents a possible cure for unsuccessful fertilization in related cases. -it possibly reveals melamine’s unknown way of action. - it presents a new approach as a sperm-binding assay to be used in IVF clinics since Juno can also be expressed in somatic cell lines. non-clinical trials