Effects And Mechanisms Of Sodium Fluoride On Meiosis Maturation Of Porcine Oocytes

Fluoride is an essential trace element closely related to human health,but long-term,excessive intake of fluoride can cause fluorosis in the body,leading to serious damage to tissues and organs,affecting human health.In this experiment,porcine oocytes were used as a toxicological model of NaF to study the effects of NaF on nuclear maturation and cytoplasmic maturation of oocytes and the DNA methylation pattern of imprinted genes.The main results are as follows:1.The COCs were treated with 0 mM,2 mM,5 mM and 10 mM NaF,respectively.It was found that the cumulus granule cells could not expand when the COCs were treated with 5 mM and 10 mM NaF,and the oocyte maturation rate showed a significant and extremely significant decrease.Therefore,subsequent studies used oocytes treated with 5 mM NaF.2.Immunofluorescence staining of mature oocyte DNA and α-tubulin of mature oocytes revealed that the number of oocytes in the normal spindle morphology of the NaF-treated group was significantly reduced(20.10% vs.56.27%,P<0.05).and the number of oocytes in the tail spindle was significantly increased(9.53% vs.1.37%,P<0.05),and the number of oocytes in the multipolar spindle was significantly increased(37.83% vs.0.80%,P<0.01).The cortical granules of mature oocytes were labeled with FITC-labeled peanut lectin,and cortical particle distribution was evaluated under laser confocal scanning microscopy.It was found that the number of oocytes continuously distributed in cortical particles in the NaF-treated group was significantly lower than that in the control group(67.20% vs.94.97%,P<0.05),while the number of oocytes in which the cortical particle incompleteness was discontinuously distributed was significantly increased(30.73% vs.4.20%,P<0.05).3.Using the mitochondrial membrane potential assay kit of JC-1 to determine the mitochondrial membrane potential of oocytes,it was found that there was no significant difference in mitochondrial membrane potential between the NaF-treated group and the control group(0.94 vs.1.00,P>0.05),indicating that NaF had little effect on the mitochondrial membrane potential of oocytes.Annexin V-fluorescein isothiocyanate(FITC)staining was performed using Annexin V-FITC apoptosis assay kit to detect early apoptotic of MⅡ oocytes,and it was found that the apoptosis signal of NaF treatment group was higher than that of the control group(Corrected value: 1.36 vs.1.00).The data is not stable enough for the quite different from different batches oocytes,and the difference is significant(P<0.05).4.The mature oocytes were subjected to parthenogenetic activation and in vitro culture.It was found that the cleavage rate of oocytes in NaF treatment group was significantly increased compared with control group(90.30% vs.94.89%,P<0.05),while the blastocyst formation rate was significantly decreased(11.18% vs.45.63%,P<0.01),indicating that NaF affects developmental potential of the embryos from these oocytes.5.The levels of imprinted genes GRB10,IGF2,PEG1,PEG10,H19,NNAT and XIST were detected.The results showed that the transcription level of NNAT in the oocytes of the NaF-treated group was significantly decreased.Methylation sequencing analysis of NNAT by bisulfite DNA sequencing(BSP)and combined bisulfite restriction analysis(COBRA)revealed 95% methylation in the control group.The unmethylated site was the 19 th site in the control group,while the NaF treatment group was 100% methylated.The protein level of NNAT was detected by immunofluorescence and it was found that NaF significantly reduced the expression of NNAT at the protein level.6.To determine whether NaF affects the glucose transport in porcine oocytes,2-NBDG fluorescence staining was performed on mature oocytes from the NaF-treated group,and NaF was found to significantly affect the glucose transport capacity of oocytes.NNAT antibody was injected into the cytoplasm of fresh MⅡ oocytes to block the function of NNAT.The results showed that the fluorescence intensity of 2-NBDG in the oocytes injected with NNAT antibody was significantly lower than that of the control group injected with IgG,indicating that the loss of NNAT affects glucose transport in oocytes.In conclusion,NaF affects cumulus expansion,polar body excretion,spindle morphology,cortical particle distribution,early apoptosis and development after parthenogenetic activation during porcine oocyte maturation.In addition,NaF increased the DNA methylation level of NNAT,but down-regulated its expression,affecting the quality of oocytes by disrupting the glucose transport capacity in oocytes.The results of this study provide a new toxicological mechanism for NaF to affect oocyte maturation.