| Background:Cryptorchidism is the most common urogenital malformation in pediatric period, and the incidence is as high as 3%-4% in the full-term baby boy. In Europe or the United States and the developed areas in China, the occurrence of cryptorchidism is obviously rising trend. Studies have shown that the occurrence and development of cryptorchidism is closely related to the endocrine, genetic, environmental factors, in which environmental factors may play a main role. Cryptorchidism is not only can lead to male reproductive dysfunction, infertility, and its malignant transformation rate is 20-60 times higher than normal testis. Therefore, the study of the pathogenesis of cryptorchidism has very important practical significance, become a research hotspot.Early research reports, exists in certain synthetic chemical pollutants in the environment, additives by interfering with biological Endocrine system affect the development and function of the urogenital system, now called this kind of material Environmental Endocrine Disruptors factor(EED). Phthalic acid diethyl has ester (Di (2Ethylhexyl) Phthalate DEHP) is a kind of environmental endocrine disruptors, belongs to the phthalic acid ester compounds (Phthalate esters, PAEs). DEHP is widely used in all kinds of plastic products, such as food packaging materials, production and processing of medicinal materials, it is also the important composition of other chemical industry, through various channels to the environment caused serious "white pollution", do harm to human health. Researches have proved that DEHP as a kind of environmental endocrine disruptors factor, parental (FO) exposure during pregnancy can induce offspring (F1) of cryptorchidism, the incidence of cryptorchidism is as high as 57%, and testicular volume significantly narrowed, testicular seminiferous tubule was dysplasia, sperm in epididymis tube cavity was rare, azoospermia, fertility declined obviously. Parental (FO) DEHP exposure during pregnancy, although there is no mutations in genes or DNA sequence change in the F1 generation, but can lead to the F1 DNA methylation modify status changes, the specific performance:(1) F1 generation of genomic DNA methylation levels were higher than normal control group about 10%; (2) three kinds of DNA methylation transferase (Dnmt1, Dnmt3a and Dnmt3b) expression have enhancement of different degrees. Findings suggest epigenetics research category of DNA methylation modification model change is one of the mechanism of cryptorchidism occurred. At the same time, F1 generation from the infected pregnant rat FO mating with normal female, the incidence of cryptorchidism in male F2 generation was significantly increased when compared with normal control group, the incidence of cryptorchidism F2 generation of male rat is about 12.5%.Environmental factors although there is no cause mutations in genes or DNA sequence change, but caused by the patterns of modification of DNA methylation and other epigenetic change could prompt response to the occurrence of diseases, theoretically more important is the "acquired" epigenetic modifications changed by reproductive cells (genetic imprinting) can passed to their children. Environmental pollution caused by DEHP that resulted in "acquired" cryptorchidism can be passed from the F1 to F2 generation. DEHP, however, is how to affect genomic imprinting through DNA methylation transferase methylation modification model changes, thus inhibiting the male reproductive system development the expression of key genes, inducing cryptorchidism occurred across generations? Mechanism is not clear at presently, the domestic and foreign information has been reported. This study will proposed DEHP in the critical period of development effect on pregnant rat (FO), the F1 to F4 generation of cryptorchidism inter-generational genetic evolution, screening key imprinting genes during the development of the male reproductive system, in order to ascertain the genomic imprinting modification in cryptorchidism inter-generational genetic role in the process of occurrence, development and mechanism, choose and clinical effective intervention for biopharmaceutical of cryptorchidism, improve the quality of the population in our country, provide scientific experimental basis. In this research, the main contents and results are listed as follows:Part 1 Establish "Acquired" cryptorchidism inter-generational genetic modelObjective:Parental (FO) DEHP exposure during pregnancy period induced offspring (F1-F4) occurring cryptorchidism, established "acquired" cryptorchidism inter-generational genetic model.Methods:1. Animal grouping and dosage:pregnant SD rats were randomly divided into 2 groups:normal control group and DEHP group, experimental group from E7 to E19 gavaged by 750mg/kgd DEHP, observing children’s cryptorchidism situation.2. Mating experiment showed conception rate was 50%in the F1 generation, in the F2 generation was 75%, in the F3 generation was 100%. HE staining showed that the seminiferous epithelium in F1 generation was atrophy and with a few spermatogenic cell, in F2 generation had improved, F3 and F4 generation was tend to be normal.3. Insulin-like factor 3, detection of testosterone (T) expression.Results:1. Successfully established cryptorchidism inter-generational genetic model. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there is no cryptorchidism occurrence in F3 and F4. Conception rate 50% mating experiment shows in the F1, F2 generation of 75%, the F3 and F4 generation by 100%..2. Conception rate 50% mating experiment shows in the F1, F2 generation of 75%, the F3 generation by 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation was tend to be normal.3. Insulin-like factor 3, testosterone expression decreased significantly in the F1 generation.Part 2 "Acquired" cryptorchidism genomic imprinting mechanism researchObjective:To observe the change of genome methylation status and genomic imprinting methylation modify difference, and confirm whether it is "acquired" cryptorchidism occurred across generations genetic mechanism.Methods:1. mRNA and protein expression of DNA methylation transferase.2. The genomic methylation detection and CpG methylation differences examination.3. Imprint genes screening and identification.Results:1. The Real Time PCR, immunohistochemistry and Western Blot showed that the expression of DNA methylation transferase was increased with the increase of genetic algebra, the difference was statistically significant.2. Fully sequenced genome methylation suggest F1 contrast with F4 generation and normal level of methylation has obvious difference.3. FGF9, RN5-8-S promoter region methylation differences clearly associated with the occurrence of cryptorchidism.Conclusion:1. The DEHP injured rats’ male reproductive function, and changed the expression of DNA methylation transferase.2. The abnormal expression of methylation transferase which in turn lead to genomic imprinting methylation modify pattern change and pass on to the next generation, so that the offspring of male reproductive system development key imprinting genes imbalances, and eventually lead to children cryptorchidism, could be one of the important mechanism of toxicology of reproductive system damage.3. The cryptorchidism inter-generational genetic has a tendency to repair itself, is involved in to DNA demethylation is still need further research. |
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