Effects Of ICR Pregant Mice Exposure To PCB118 On Male Offspring’s Spermatogenesis In Testis And Its Mechanism

Polychlorinated Biphenyls(PCB),especially PCB118(2,3’,4,4’,5-Pentachlorobiphenyl),as typical persistent organic pollutants(POPs)have a production history of nearly a hundred years on a global scale.Because of wide pollution and stable physical and chemical properties,recent research have noted that PCB as well their metabolites have many potential adverse effects on humans and wildlife.Coplanar polychlorinated biphenyls(Co-PCBs)have the properties of dioxin-like compounds that can cause endocrine disorders and reproductive damage.Whereas,non-coplanar polychlorinated biphenyls are neurotoxic and immunotoxin.Metabolites of polychlorinated biphenyls are metabolized in body to produce large amounts of OH-PCB and Me SO2-PCB homologues there are large amounts of OH-PCB and Me SO2-PCB homologues during the metabolic process of PCB in body,which can be accumulated in animal blood and adipose tissue for secondary damage.Everal reports described that endocrine disruptors lead germ cells to abnormal growth and functional damage through Circulatory system.Likewise,previous works in our laboratory have shown that the methylation levels of F1 male mice sperm in offspring have been was altered after the PCB118 exposure during embryonic.On the basis of existing literature data,in this paper,we carried out studies in an effort to investigate that whether the proliferative capacity of the testis and the reproductive ability of adult sperm of the F1 male mice are affected by PCB118 which F0 pregnant mice exposed during pregnancy.We would also aim to explore the extent of this influence and clear mechanism.Contents: 1)To explore the effects of PCB118 pregnant exposure on development of offspring;2)verifying whether PCB118 exposure during pregnancy lead the number of testicular spermatocytes changed;3)investigating the status of proliferation of offspring sperm cells after PCB118 treatment;4)whether it would happen that the sperm quality of offspring decline;5)through the above investigations to clarify the reproductive affects and its epigenetic mechanism of PCB118 on F1 generation.METHODS: In our experiment,7-week-old male and female mice were mating.The pregnant mice were divided into three groups(0 μg/kg、20 μg/kg、100 μg/kg).During six days of pregnancy(8.5 days to 13.5 days),three groups of F0 mothers were intragastrically inoculated with PCB118,and the offspring were labeled as F1 generation.Subsequent experiments contained: 1)collecting data including body weight,wet weight of viscera of F1 every week;2)making paraffin sections of7-week-old F1 male mice,performing HE staining and counting diameter of seminiferous tubules,thickness of spermatogenic epithelium,and spermatogonia cell count;3)By extraction of m RNA from testis tissue of 7-week-old mice,detecting m RNA expression levels of Sox2,Sox9,Plzf,through by real-time fluorescent quantitative PCR;4)The expression of PCNA protein in testis of 7-week-old male mice was detected by Western blot;5)the level of 5-m C and DNMT1 protein in fetal and 7-week-old mice testis were detected by immunohistochemistry.RESULTS: 1)PCB118 had no significant difference in body weight and sperm motility of F1 male mice(P>0.05),and the sperm deformity rate increased.2)Observation and statistics of testicular tissue paraffin sections revealed that the diameter of the seminiferous tubules and the thickness of spermatogenic epithelium as well as the number of spermatogonia;3)The results of q RT-PCR showed that the expression levels of sox2 and Plzf decreased,and the relative expression of sox9 did not differ.4)The expression of PCNA protein in the testis of 7-week-old male mice decreased,and the proliferation was abnormal.5)The level of 5-m C in fetal and7-week-old testis decreased,and the expression level of DNMT1 was decreased.In conclusion,the experimental results show that exposure to PCB118 during embryonic stage can lead to reproductive damage such as reduced diameter of testicular seminiferous tubules,decreased spermatogonial number and increased sperm abnormality in F1 male miceMoreover,the expression level of the marker gene Plzf of spermatogonia and the marker of PCNA protein of testicular cell proliferation activity decreased,and the expression level of Sox2 gene m RNA increased abnormally,indicating that the testicular development and spermatogonia cell proliferation of F1 male rats were affected.Further studies found that the expression of PCNA and DNMT1 protein in the embryonic testis of F1 male rats decreased,and the level of 5-m C decreased.This indicates that exposure to PCB118 during pregnancy reduces the proliferative capacity and overall methylation level of the testis of the F1 male rats during the embryonic stage.We speculate that F0 exposure to PCB118 during pregnancy can interfere with the expression of PCNA protein in the testis of F1 male rats in the uterus,thereby reducing the methyltransfer efficiency of DNMT1,thereby changing the DNA methylation level of mouse testis in embryonic stage,which may affect subsequent Growth and development of testis in male rats after birth and proliferation of spermatogonia.This provides new insights into the toxicity study of “parent-child”and provides a new explanation for epigenetic phenomena caused by exposure to PCB during pregnancy.