Effects And Mechanism Of Triclocarban On Oocyte Meiotic Maturation And Early Embryo Development In Mouse

As society continues to develop,environmental pollution is becoming more and more of a problem,posing a serious threat to the health of people and animals.There is considerable evidence that the adverse effects of environmental pollutants are an important influence on female fertility decline.Triclocarban(TCC),a broad-spectrum antimicrobial agent,is widely used in production and domestic applications.Its widespread use has led to its presence in the environment in large quantities.Humans are not only directly exposed to TCC through skin contact,but also absorbs TCC through diet and inhalation,resulting in its accumulation in the body.As the safety of TCC is still not fully understood and the potential effects on human health are not clear,there is an urgent need to assess the reproductive toxicity of TCC.The normal development of the oocyte and early embryo is a guarantee of fertility in humans/domestic animals.Oogenesis and preimplantation embryo development is a very sensitive process and is susceptible to adverse effects of environmental stress.However,the effect of TCC on female oocytes meiotic maturation and early embryonic development remains unclear.Therefore,in this experiment,mouse oocytes and early embryos were treated with different concentrations of TCC in an in vitro culture system,and immunofluorescence,laser confocal,chromosome spread,live cell staining and transcriptome sequencing were used to investigate the effects of TCC on the meiotic maturation of mouse oocytes and early embryonic development and the related mechanisms.The results of the experiment were as follows.1.Effects and mechanism of TCC on oocyte meiotic maturation in mouse(1)Oocytes were treated with 0,5,10,12 and 15 μM TCC for 14 hours in an in vitro oocyte maturation culture system to observe first polar body expulsion(PBE).The results showed that treatment with 12 μM TCC resulted in a significant decrease in the oocytes PBE ratio.When the 12 μM TCC-treated oocytes were continued in culture until 24 hours,almost all oocytes died.Alternatively,oocytes without PBE after 14 h of TCC treatment were transferred to a culture medium without TCC and continued to be cultured,most oocytes still failed to expel the first polar body and even died.The cell cycle results showed that the majority of the TCC-treated oocytes were blocked in the Pro-metaphase of Meiosis I(Pro-MI)and metaphase of Meiosis I(MI)phases of meiosis.(2)TCC caused abnormalities in meiotic spindle assembly and chromosome alignment in oocytes,changes in the localization of the microtubule organizing center(MTOC)associated proteins Pericentrin and p-ERK1/2,disruption of the Kinetochore-microtubule(K-MT)structure,and sustained activation of spindle assembly checkpiont(SAC).At the same time,the construction of the intracellular microfilament skeleton was disrupted.(3)Diethylstilbestrol and estradiol caused abnormal aggregation of estrogen receptorα(ERα)on the oocyte spindle,but no abnormal aggregation of ERα was observed in TCC-treated oocytes.(4)TCC disrupted mitochondrial function,reduced ATP production and decreased mitochondrial membrane potential in mouse oocytes.TCC increased oxidative stress in oocytes,lead to oxidative DNA damage and induced early apoptosis in oocytes.At the same time,TCC disrupted the distribution pattern of endoplasmic reticulum in mouse oocytes,affecting endoplasmic reticulum(ER)function.TCC affected the distribution pattern of Golgi marker GM130 in mouse oocytes,and the fluorescence intensity of GM130 was significantly decreased.In addition,the distribution pattern of oocyte lysosomes was also changed after TCC treatment.(5)TCC altered histone modification levels,as indicated by that H3K27me2 and H3K27me3 modification levels were significantly increased after TCC treatment.2.Effects and mechanism of TCC on early embryo development in mouse(1)Fertilized eggs were treated with 0,2,4 and 6 μM TCC in an in vitro developmental culture system of early embryos to observe early embryonic development.The results showed that TCC affected early mouse embryo development in a dose-dependent manner,with 6 μM TCC having no effect on the 2-cell development rate and a significantly lower 4-cell development rate in early embryos,and no embryos developed to blastocyst.(2)Sequencing of the transcriptomes of the control and TCC-treated 2-cell embryos yielded 7600 differentially expressed genes(DEGs),of which 3157 were up-regulated and 4443 were down-regulated.The GO analysis and the KEGG analysis of DEGs uncovered that the enriched DEGs were chiefly involved in redox,RNA synthesis,DNA damage,apoptosis,mitochondria,ER,Golgi apparatus,cytoskeleton,peroxisome,RNA polymerase and other components or processes.Venn analysis showed that the Zygotic Genome Activation(ZGA)process was affected;also,the degradation of ZGA-dependent maternal genes was significantly affected.(3)TCC affected epigenetic modifications in 2-cell embryos.Following TCC treatment,2-cell embryos showed a significant increase in DNA methylation levels,a significant decrease in H3K27ac,H3K9ac and H3K27me3 modifications and a significant increase in H3K4me3 and H3K9me3 modifications.(4)TCC induced oxidative stress and DNA damage in 2-cell embryos.In summary,TCC affected oocyte meiotic maturation,mainly by inhibiting first PBE,disrupting cytoskeletal assembly,leading to abnormal K-MT struction,activating the SAC and inducing cell cycle arrest.At the same time,TCC disrupted mitochondrial,ER,Golgi apparatus and lysosomal functions,leading to oxidative stress,DNA damage and early apoptosis in oocytes.In addition,TCC affected early embryonic development,disrupted early embryonic gene expression,interfereed with the progress of ZGA and maternal gene degradation,altered epigenetic modifications in oocytes and early embryos,and caused oxidative stress and DNA damage in early embryos.