The Effects Of Graphene Quantum Dots On The Maturation Of Mouse Oocytes And Development Of Offspring

Objective: Recently,Graphene nanomaterials have attracted tremendous attention and been utilized in various fields because of its excellent mechanical thermal,chemical,optical properties,and good biocompatibility,especially in the biomedical aspects.However,there is concern that the unique characteristics of nanomaterials might induce undesirable influence.Therefore,in this work,we sought to systematically investigate the effects of graphene quantum dots(GQDs)on the maturation of mouse oocytes and development of offspring via vitro and in vivo studies.The aim of this study is to provide experimental and theoretical basis for reproductive toxicity induced by biological factors and to provide new ideas for the application of graphene materials in biomedical field.Methods and materials: In vitro experiments,the effects of GQDs exposure on mouse oocyte maturation were investigated.First,the effects of GQDs exposure on the first polar body emission were observed.Secondly,immunofluorescence(GQDs)were used to observe the morphological changes of subcellular structures,such as chromosome,spindle microtubule and microfilament,which were closely related to meiosis,and to observe the production of intracellular reactive oxygen species and DNA damage in the GQDs-exposed oocytes.The distribution of GQDs in oocytes and the morphology of mitochondria were observed by transmission electron microscopy(TEM).In vivo experiments,the effects of GQDs exposure to the development of offspring were studied.The mice were exposed to different concentration of graphene quantum dots(GQDs)via the veil injection at the 15mg/kg or 30 mg/kg per mouse dosage on gestational day 8.5(GD8.5).On gestational day 19(GD 19),the weight,length and numbers of embryo were analyzed and evaluated.When the first pregnancy mice gave birth to offspring,the growth and reproductive ability of the second generation mice were observed.Results: While performing in vitro experiments,we found that the influence of acute exposure to GQDs(0.5,1.0 and 1.5 mg/ml,respectively)for 12 h,the first polar body extrusion rate of oocytes decreased significantly in the presence of 1.0 mg/ml or 1.5 mg/ml concentration of GQDs-exposed groups compared to control group and 0.5 mg/ml dosage of GQDs-exposed group.Immunofluorescence staining results showed that there is a failure of spindle migration and actin cap formation during oocyte meiotic maturation.Further studies in our work showed that the underlying mechanisms might be associated with reactive oxygen species(ROS)generation and DNA damage.Moreover,when we monitored the location of GQDs in the mouse oocytes,it showed that GQDs may have been internalized into oocytes and tend to accumulate in the nucleus.Furthermore,when TEM was examined after an acute GQDs exposure,it was found that there appeared a great difference in mitochondrial morphology,included swollen and vacuolization of mitochondria accompanied by cristae alteration with a lower amount of dense mitochondrial matrix.While doing in vivo experiment,pregnant mice were injected with different dosage of GQDs at 8.5 days of gestation(GD 8.5)via the tail vein injection and it was found that there was a significant difference in mean fetal length between high dose of GQDs-exposed group and rest groups.Interestingly,all the second generation of female mice grew up normal,attained sexual maturity and after mating them with a healthy male mouse,they gave birth to healthy offspring which were marked as third generation.Conclusion: Over the past few years,the widespread potential applications of graphene quantum dots(GQDs)have called for their biosafety studies.With adequate studies of toxicity,both in vitro and in vivo,our work revealed that the GQDs affected the maturation of mouse oocytes in vitro,however,did not cause apparent toxicity to the treated animals under the low dosage exposure in vivo.Thus,this study might greatly contribute to the comprehensive understanding of the interaction of GQDs with reproductive system and their adverse effects,which are essential for further development and considered for the safety use of GQDs in biomedical applications.