Effect Of Ochratoxin A On Porcine Fibroblasts Mitosis And Oocytes Meiosis

Ochratoxin A(OTA)toxins are widely distributed in nature.Many crops and foods are susceptible to OTA contamination during storage,especially in long-term storage of livestock and poultry feed forage.When animals ingest contaminated feed forage,they accumulate OTA in the body and cause toxic damage to various organs.Human consumption of OTA by eating OTA-contaminated animals or plant foods which poses a potential threat to human health.Studies have shown that OTA produces significant hepatotoxicity,nephrotoxicity,immunotoxicity,neurotoxicity,mutagenicity,teratogenicity and carcinogenicity,but little studies have related to the reproductive toxicity of OTA in animals,especially to germ cells.In addition,the toxic effects are unclear currently.In this study,porcine fetal fibroblasts(pFF)were used as reference.The in vitro maturated porcine oocytes were used as the research object.The effects of OTA treatment on cell division and proliferation,subcellular structure and cell cycle progression were systematically studied.The effects of OTA treatment on cell cycle-regulated protein kinase expression were explored in order to investigate the toxic effects of OTA on porcine oocytes from the perspective of cell division cycle,and systematically compare OTA to mitosis of fetal fibroblasts and porcine oocyte reduction.The similarities and differences of several mitotic toxic effects,in order to reveal the reproductive toxicity and toxic mechanism of OTA on animal oocytes,provide theoretical and experimental reference for clinical prevention and treatment of OTA reproductive toxicity.The main research contents and results are as follows:Experiment 1.Effect of OTA on pFF mitosis and oocyte meiosisTo study and compare the effects of OTA on mitosis of pFF and meiosis of porcine oocytes,different concentrations of OTA(0,0.08 and 0.16 μg/mL)were used to treated pFF in vitro cultured firstly.The cell viability of each group of cells was detected by MTT assay,and the cell cycle of each group of cells was detected by flow cytometry.The results showed that the pFF in the treatmented group were irregular in shape and the number of adherent cells was significantly decreased.The cell viability decreased with the increase of OTA concentration,and the cell cycle arrest in G0/G1 phase.GV phase(germinal vesicle)oocytes were treated with different concentrations of OTA(0,3 and 6 μg/mL),matured in vitro for 44 h,and oocyte maturation was detected microscopically.Laser confocal microscopy Analyze the cell cycle progression.The results showed that the ovarian cell proliferation of pig oocytes was significantly inhibited,the first polar body excretion rate decreased in a concentration-dependent manner,and the number of oocytes developed to MII phase gradually decreased,while stagnating in the GVBD(germinal vesicle break down)period.The proportion of oocytes is significantly increased.These results indicate that OTA has a significant inhibitory effect on mitosis and oocyte meiosis in pFF cells,which is closely related to the toxic effects of OTA-blocking cell cycle progression.Experiment 2.Effect of OTA on the cytoskeleton during the division process of pFF and oocyteTo investigate the causes of pFF cells and oocyte cleavage after OTA treatment,compare the toxic effects of OTA on pFF and oocytes.In this study,indirect immunofluorescence staining combined with laser confocal microscopy to observe OTA on pFF mitosis and oocyte meiosis.The results showed that after OTA treatment,the nuclear border of most pFF was unclear and fragmented,accompanied by abnormal distribution ofα-tubulin;while in OTA-treated pig oocytes,most of the cell chromosomes were also found to be normal.Agglutination,but disordered,failed to be accurately arranged on the equatorial plate,a-tubulin was scattered and arranged disorderly,and failed to assemble into a typical medium-term spindle structure.These results indicate that OTA treatment can cause pFF and oocyte nuclear damage,and interfere with the normal assembly of tubulin into the spindle structure,resulting in mitotic arrest of pFF cells in G0/G1 phase,and oocyte meiosis arrest in GVBD phase.Experiment 3.Effect of OTA treatment on P1k1 mRNA expressionBased on the above findings,the toxic effects of OTA on pFF mitosis and oocyte meiosis are closely related to cell cycle arrest,while Plkl is an important serine/threonine protein kinase that regulates cell mitosis and meiosis cycles.To explore the toxic mechanism of OTA on pFF mitosis and oocyte meiosis,this study further explored the effect of OTA treatment on cyclin Plkl.Firstly,the dynamic expression and function of Plk1 during mitosis of pFF were analyzed by indirect immunofluorescence and laser confocal imaging.The results showed that Plk1 was expressed during mitosis of pFF cells.After inhibiting Plk1 kinase activity,the proliferation activity of pFF cells was significantly decreased.With the phenomenon of nuclear fragmentation and disorder of α-tubulin arrangement,this phenomenon is very similar to the results after OTA treatment.The effects of OTA treatment on the expression of Plkl were further studied by RT-PCR and Western blot.The results showed that the expression of Plk1 was significantly decreased in both pFF and porcine oocytes after treatment with OTA.We hypothesized that OTA may have a toxic effect on mitosis and oocyte meiosis of pFF cells by affecting the expression of cell cycle-associated protein Plk1,which ultimately leads to decreased mitotic activity of pFF cells and failure of oocyte maturation.