The Effectand Mechanism Of Isobutyl Paraben On Porcine Oocyte In Vitro Maturation

With the development of industry,more and more chemicals are synthesized and used in various fields to adapt to different production and life.However,these products not only give us convenience and enjoyment but also lay a hidden danger in humans and animals.Isobutylparaben(IBP)is a synthetic chemical,which is widely used in many fields such as daily care products,medicine,feed,paper,printing due to its cheap and broad-spectrum antimicrobial activity.Recent studies have shown that IBP is an endocrine disruptor,posing a potential threat to the reproductive health of organisms.However,the effects of oocytes by this compound have not been reported.In this study,the acute toxicity of different concentrations of IBP(0 mmol/L,0.1 mmol/L,0.2 mmol/L,0.3 mmol/L,0.4 mmol/L?0.6 mmol/L)on porcine oocyte maturation was studied using an in vitro maturation culture system of porcine cumulus cell-enclosed oocyte complexes(COCs),and the possible mechanisms of its effects,including cumulus cell expansion,cytoskeletal distribution,oxidative stress,apoptosis,and histone methylation modifications were explored.The results of the study are as follows:(1)IBP inhibits cumulus expansion and polar body extrusion in porcine oocytes.Porcine cumulus cell complexes treated with different concentrations of IBP for 44 h in vitro revealed that the polar body extrusion(PBE)showed a linear decreasing trend with increasing concentration(r = 0.8410).Although 0.1 mmol/L of IBP treatment had no significant effect on the first polar extrusion ratio(P> 0.05).However,0.2 mmol/L of IBP significantly inhibited the first polar body extrusion of porcine oocytes(P< 0.05),and 0.4 mmol/L of IBP significantly inhibited cumulus cell expansion.The results indicate that IBP can affect porcine oocyte maturation by inhibiting cumulus cell expansion and first polar body extrusion.0.4 mmol/L IBP inhibits the expansion of cumulus cells and allow the extrusion of a small amount of the first polar body,which is conducive to the exploration of the relevant mechanism.Therefore,this concentration was selected in the subsequent test to explore the potential effect on porcine oocyte maturation.(2)IBP disrupted the oocyte cytoskeleton: Porcine oocytes were co-cultured with 0.4 mmol/L IBP for 27 h,and spindle assembly was examined using immunofluorescence staining for ?-tubulin.It was found that most of the porcine oocytes in the control group presented normal bipolar spindle morphology.Monopolar and multipolar morphologies appeared in the spindles of the treatment group,and the ratio of spindle abnormalities increased significantly(P< 0.05).In addition,the results of immunofluorescence staining of microfilaments revealed that the microfilaments of porcine oocytes in the control group were mainly concentrated in the cortical area and formed a circle,while the fluorescence signal of microfilaments in the treated group was significantly attenuated in the cortical area(P< 0.05)and mainly distributed in the cytoplasm.Therefore,IBP disrupts normal spindle assembly and the distribution of microfilaments in cortical regions,affecting the cytoskeleton of porcine oocytes.(3)IBP cause strong oxidative stress and apoptosis in porcine oocytes.Porcine oocytes were co-cultured with 0.4 mmol/L IBP for 27 h.Reactive oxygen species levels in porcine oocytes were measured using the reactive oxygen species detection kit DCHF-DA.The results showed that most of the porcine oocytes in the control group had no green fluorescence signal(the intensity of green fluorescence reflected the level of reactive oxygen species),while most of the porcine oocytes in the treatment group showed strong green fluorescence signal,and the fluorescence signal was significantly enhanced(P< 0.05),indicating that IBP could induce oxidative stress in porcine oocytes.Annexin V-FITC apoptosis detection kit was used to detect the early apoptosis of porcine oocytes.It was found that the green fluorescence of early apoptosis was rare in the control porcine oocytes,while more green fluorescence of early apoptosis was observed in the cortical periphery of some treated porcine oocytes.After statistical analysis,it was found that the ratio of early apoptosis was significantly increased(P < 0.05),indicating that IBP can induce early apoptosis and affect the survival of porcine oocytes.(4)IBP cause changed in epigenetic modifications in porcine oocytes: Porcine oocytes were co-cultured with 0.4 mmol/L IBP for 27 h.The fluorescence signal levels of H3K9me3 and H3K27me3 in porcine oocytes were detected using immunofluorescence staining technique,and the results revealed that most of the porcine oocytes in the control group showed weak red fluorescence for H3K9me3 and H3K27me3,while most of the porcine oocytes in the treatment group had significantly strong red fluorescence signals for H3K9me3 and H3K27me3.After fluorescence signal detection,it was found that the average level of fluorescence signal was significantly different between the control group and the treatment group(P< 0.05),indicating that the histone methylation level in porcine oocytes was significantly increased.The results suggest that IBP can alter epigenetic modifications in porcine oocytes.The results showed that 0.2 mmol/L IBP could inhibit the first polar body extrusion of porcine oocytes,0.4 mmol/L IBP could inhibit the expansion of cumulus granulosa cells,and 0.4 mmol/L IBP could affect the maturation process of porcine oocytes by disrupting oocyte spindle assembly,disturbing the distribution of microfilaments in the cortical region,inducing oxidative stress and apoptosis,and changing the mode of epigenetic modification.