The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism

Abstract

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7 d to 19 d, experimental group was sustained to gavage DEHP 750 mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat's weight and AGD value, after maturation (PND80) recording testis and epididymis' size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 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 were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.

Fig 1. Control group and DEHP experimental group offspring (F1-F4) male rat’s testicular histology expression.

A:F1;B:F2;C:F3;D:F4 E: Control (200×).

Fig 2. Control group and DEHP experimental group offspring (F1-F4) male rat’s epididymis histology expression.

Normal control group of testicular seminiferous tubule epithelial developed normally, we can observe each stage sperm cells and a large number of sperm cells in the epididymis pipe. DEHP experimental F1 generation observed seminiferous tubule epithelial obvious atrophy, less sperm cells, pathologic hyperplasia of interstitial cells, number of sperm in the epididymis lumen scarce or lack, luminal epithelium cell falls off. F2 generation of rat testis partial seminiferous tubule epithelium atrophy, sperm cells less; F3 and F4 generation were tend to be normal, sperm cells grading well. A:F1;B:F2;C:F3;D:F4 E: Control (200×).

Fig 3. F1-F4 semen quality comparison.

* compared with control group, P<0.05.

Fig 4. Sperm abnormality.

This is illustrated by the results of the most serious damage of F1 generation of male rats’reproductive function, and with the increase of genetic algebra, damage degree showed a trend of decline. A:double head;B:head;C:curve;D:fold.

Fig 5. Control group and DEHP experimental group offspring (F1-F4) male rat’s DNMT1 expression.

High expression of dnmt1 in the F1, F2 generation significantly reduced, F3 and F4 generation basic without expression. A:Control;B:F1;C:F2;D:F3 E:F4 (×200)

Fig 6. Control group and DEHP experimental group offspring (F1-F4) male rat’s DNMT3a expression.

High expression of dnmt1 in the F1 and F2 generation, F3 significantly reduced and F4 generation basic without expression. A:Control;B:F1;C:F2;D:F3 E:F4 (×200)

Fig 7. Control group and DEHP experimental group offspring (F1-F4) male rat’s DNMT3a expression.

Result shows that with the increase of genetic algebra, the expression level of DNA methyltransferase gradually lowered, the testicular damage degree is reduced.

Fig 8. Control group and DEHP experimental group cryptochidism group compared to control group of biological process up regulation of GO Biological Process Classification.

Fig 9. Control group and DEHP experimental group cryptochidism group compared to control group of biological process down regulation of GO Biological Process Classification.

Fig 10. Control group and DEHP experimental group cryptochidism group compared to control group of biological process down regulation of pathway analysis.