The Mechanisms In Altered Ontogenetic Development In Microcystin-leucine Arginine(MC-LR)-paternal-exposed Offspring Mice

Microcystin LR(MC-LR)is a kind of environmental pollutant produced by freshwater cyanobacteria,which is widely distributed in freshwater water in China and poses a great threat to the survival and health of aquatic organisms and mammals.Previous studies in our laboratory found that MC-LR exposure can lead to testicular tissue damage and sperm quality reduction in male mice.Therefore,it is of great practical significance to explore whether the toxicity of MC-LR can be transmitted to offspring through mammalian sperm and lead to abnormal growth and development of offspring.This research is divided into the following three parts:Part ?: We mated MC-LR-exposed males or control males with normal female mice.The number of offspring per litter,the total number of offspring,the pregnancy loss per group and the changes in the body weight of the offspring were recorded and analyzed.We also detected the tissue structure lesions of the main organs of the offspring mice.It was confirmed that the chronically exposure to MC-LR would lead to the disruption in the lung tissue of the offspring mice.We calculated the occurrence probability.The results showed that after mating with male mice exposed to MC-LR,the abortion rate of female mice increased significantly,the number of offspring per litter and the total number of offspring decreased significantly,and the weight of mice decreased significantly during development.The main organs of the progeny mice were observed by section staining.The results showed that the lung tissue structure of the progeny mice exposed to MC-LR was disordered and a large amount of collagen was deposited.Part ?: We clarified the expression of p16 and p21,and the important role of Wnt signal pathway in the process of lung structural lesions,and to screen Wnt protein with high specificity in this process.Furthermore,hairpinRNA lentivirus vector was used to target and inhibit Wnt protein to confirm its role in the process of abnormal lung tissue development in mice.The above experiments confirmed that the expression of p16 and p21 in the lung tissue of male mice was up-regulated after MC-LR exposure,and the abnormal activation of Wnt/?-catenin signal pathway in the lung tissue led to pathological changes in the lung tissue structure of mice.Part ?: High-throughput sequencing and bioinformatics analysis were used to detect the changes of non-coding microRNA expression profile in sperm of male mice exposed to MC-LR.We confirmed the decrease of Hsp90? expression in testis and the changes of piRNA expression profile in sperm.In addition,we used hairpinRNA lentivirus vector to target and interfere with the related signal pathway,and through cell line and mouse testis tissue specific knockdown model experiments,to prove the molecular regulatory mechanism of long-term MC-LR exposure leading to the change of piRNA expression profile in sperm.It was found that the expression of piRNA in mouse sperm decreased.In contrast,only a small amount of miRNA and tsRNA expression have been changed.At the same time,in the testis of mice exposed to MC-LR,the decreased expression of PP2A/B55 can increase the phosphorylation level of Hsf1 protein,and thus reduce the expression and activity of Hsf1,which inhibiting the expression of hsp90aa1,the coding gene of the piRNA related protein Hsp90?.However,after the testicular tissue-specific interference with the expression of Hsp90?,there was also a large amount of decreased expression of piRNA in mouse sperm,which was highly similar to the down regulated expression of piRNA caused by MC-LR.