Objective To establish a transgenerational rat model with in utero p, p'-DDE exposure and observe inherited characteristics and possible epigenetic mechanism.
Methods Only the pregnant rats (F0) were administered with p, p'-DDE (100 mg/kg per day according to body weight) or corn oil at the critical time of testis development (from gestation day 8 to 15). Male rats (90 days old) of F1 generation were 1:1 mated with female of the same maternal treatment to produce F2 progeny. To reveal whether the male or female germ line was responsible for the transgenerational phenotype, F3 progeny was generated by intercrossing the control and treated male and female of F2 generation and divided as following groups:1) control male plus control female (C ♂-C♀), 2) exposed male plus exposed female (DDE ♂-DDE♀), 3) exposed male plus control female (DDE ♂-C♀), and 4) control male plus exposed female (C ♂-DDE♀). Sperm count and motility of male offspring of F1-F3 were analyzed with the Computer-Aided Sperm Analysis (CASA). Igf2 DMR2 methylation was analyzed with bisulfite genomic sequencing. Gene expression was analyzed with real-time quantitative PCR.
Results p, p'-DDE decreased the sperm numbers and motility in F1 and F2 generations and DDE ♂-C♀ and DDE ♂-DDE♀ in F3 generation. In F1 generation, sperm numbers and motility decreased from (70.63±11.79)×106/mL and (76.75±7.57)% to (70.63±11.79)×106/mL and (62.42±9.40)% after p, p'-DDE treatment, respectively. In F2 generation, sperm numbers and motility decreased from (82.86±38.60)×106/mL and (82.14±6.44%)% to (43.75±25.04)×106/mL and (60.88±25.03)% after p, p'-DDE treatment, respectively. As for DDE ♂-C♀ in F3 generation, p, p'-DDE decreased the sperm numbers and motility from (68.89±41.37)×106/mL and (68.56±10.67)% to (21.66±4.83)×106/mL and (29.33±32.70)%, respectively. As for DDE ♂-DDE♀, the sperm numbers and motility decreased to (28.00±16.60)×106/mL and (34.60±31.60)%, respectively. And there were statistical differences in the two indicators of the above mentioned groups compared with the control group (P<0.05). Significant hypomethylations of site 1, 16, and 18 were observed in p, p'-DDE treated rats of F1 to F3 generation. The level of mRNA encoding the paternally expressed gene Igf2, positively correlated with Igf2 DMR2 methylation, was down-regulated in the sperm of the treated F1 to F3 generation. Maternally expressed gene H19 was up-regulated in the sperm of the three treated generations (r=0.806 5).
Conclusion Igf2 DMR2 methylation may play critical roles in the transgenerational reproductive toxicity in the male rats induced by p, p'-DDE.
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