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Objectives: To explore the effect of exposure to di (2-ethyl) hexyl phthalate (DEHP) in early pregnancy on endometrial decidualization in mice and its relation with lncRNA RP24 - 315D19.10 ., Methods: Early pregnancy mice were exposed to DEHP (1000 mg·kg －1 ·d －1 ) to construct the model. The uterus was collected on day 6 of pregnancy to detect its effect on decidualization by HE staining and immunofluorescence. A decidualization induction model of mouse endometrial stromal cells exposed to DEHP (0.1, 0.5, 2.5, 12.5, 62.5 μmol/L) was constructed. The changes of cell morphology were observed by light microscopy and phalloidin staining, and the expression of decidual reaction related molecular markers were detected by immunofluorescence, realtime RT-PCR and Western blotting. The expression of RP24 - 315D19.10 in decidua tissue and cells was detected by realtime RT-PCR. Cellular localization of RP24 - 315D19.10 was determined by lncLocator database and RNA FISH. AnnoLnc2 database was used to predict miRNAs bound to RP24 - 315D19.10 ., Results: The number of embryo implantation sites, uterine weight and uterine area were significantly lower in the DEHP exposed group than those in the control group, and the expression of the decidual reaction related molecular markers matrix metalloprotein 9 and homeobox A10 in the DEHP exposure group were also significantly lower than those in the control group (all P <0.05). With the increase of DEHP concentration, the expression of dtprp in decidua cells was gradually decreased. 2.5 μmol/L DEHP exposed stromal cells failed to be fully decidualized in vitro, andphalloidin staining showed abnormal cytoskeleton morphology. The expression levels of homeobox A10, bone morphogenetic protein 2 and proliferating cell nuclear antigen in the DEHP exposure group were significantly lower than those in the control group (all P <0.05). The expression of RP24 - 315D19.10 in DEHP exposed decidua tissue and cells was significantly reduced (both P <0.05). RP24 - 315D19.10 is mainly localized in the cytoplasm and RP24 - 315D19.10 might bind to 45 miRNAs, among them, miR-138-5p, miR-155-5p, miR-183-5p and miR-223-3p were associated with endometrial decidualization., Conclusions: DEHP exposure in early pregnancy may impair endometrial decidualization, and the damage may be associated with the down-regulation of RP24 - 315D19.10 .

Objective: To induce hypospadias in male rat offspring by maternal exposure to di-n-butyl phthalate (DBP) during late pregnancy and further investigate its mechanisms., Methods: We randomly divided 20 pregnant rats into a DBP exposure and a control group, the former treated intragastrically with DBP while the latter with soybean oil at 750 mg per kilogram of the body weight per day from gestation days (GD) 14 to 18. On postnatal day (PND) 1, we recorded the incidence rate of hypospadias and observed the histopathological changes in the genital tubercle of the hypospadiac rats. We also measured the level of serum testosterone (T) by radioimmunoassay and determined the mRNA and protein expressions of the androgen receptor (AR), sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor 8 (Fgf8) in the genital tubercle by real-time PCR and Western blot., Results: No hypospadiac male rats were found in the control group. The incidence rate of hypospadias in male offspring was 43.6% in the DBP-treatment group. Histological analysis confirmed hypospadiac malformation. The serum testosterone concentration was decreased in the hypospadiac male rats as compared with the controls (［0.49 ± 0.05］ vs ［1.12 ± 0.05］ ng/ml, P <0.05). The mRNA expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle were significantly lower in the hypospadiac male rats than in the controls (AR: 0.50 ± 0.05 vs 1.00 ± 0.12, P <0.05; Shh: 0.65 ± 0.07 vs 1.00 ± 0.15, P <0.05; Bmp4: 0.42 ± 0.05 vs 1.00 ± 0.13, P <0.05; Fgf8: 0.46 ± 0.04 vs 1.00 ± 0.12, P <0.05), and so were their protein expressions (AR: 0.34 ± 0.05 vs 1.00 ± 0.09, P <0.05; Shh: 0.51 ± 0.07 vs 1.00 ± 0.12, P <0.05; Bmp4: 0.43 ± 0.05 vs 1.00 ± 0.11, P <0.05; Fgf8: 0.57 ± 0.04 vs 1.00 ± 0.13, P <0.05)., Conclusions: Maternal exposure to DBP during late pregnancy can induce hypospadias in the male rat offspring. DBP affects the development of the genital tubercle by reducing the serum T concentration and expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle, which might underlie the mechanism of DBP inducing hypospadias.

Objective: To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) on pregnant rat and the protection of zinc., Methods: Fifty rats were randomized equally into 5 groups consisting of blank (given 1 ml 0.9% sodium chloride), corn oil (given 1 ml corn oil), zinc (given 1 ml zinc gluconate including 1.2 mg zinc), DEHP (given 50 mg×kg(-1)×d(-1) DEHP) and DEHP + zinc (given 50 mg×kg(-1)×d(-1) DEHP + zinc gluconate). At the beginning of the experiment (about 7 d before pregnancy), the female rats were administered with corresponding drugs everyday. The pregnant rats were killed and the fetal rats were removed on 19th day. The following results in each group were recorded: the body weight and the organic weight of the female rats, the number and the weight of fetus rats and the placental weight., Results: The weight and the coefficient of female rats' kidney/body, spleen/body, brain/body, and heart/body in DEHP group compared with other groups were not statistical significance (all P > 0.05). The coefficient of female rats' liver/body, uterus/body, and ovary/body of blank group were (4.4 ± 0.7)%, (1.26 ± 0.09)%, (0.083 ± 0.009)% respectively, corn oil group were (4.5 ± 0.6)%, (1.29 ± 0.10)%, (0.084 ± 0.008)%, zinc group were (4.4 ± 0.4)%, (1.26 ± 0.08)%, (0.084 ± 0.009)%, DEHP group were (5.4 ± 1.0)%, (1.11 ± 0.08)%, (0.074 ± 0.012)%, and DEHP + zinc group were (4.4 ± 1.0)%, (1.28 ± 0.10)%, (0.082 ± 0.007)%; in DEHP group the coefficient of female rats' liver/body, uterus/body, and ovary/body compared with other groups was statistical significance (P < 0.05). The fetal quantity, fetal weight and placental weight of female rats of blank group were 12.8 ± 2.7, (6.03 ± 0.16) g, (1.00 ± 0.03) g respectively, corn oil group were 13.6 ± 3.1, (6.07 ± 0.20) g, (1.00 ± 0.04) g, zinc group were 13.3 ± 3.1, (6.16 ± 0.18) g, (1.00 ± 0.05) g, DEHP group were 9.2 ± 4.1, (4.03 ± 0.09) g, (0.95 ± 0.03) g, and zinc + DEHP group were 12.1 ± 2.9, (6.09 ± 0.17) g, (0.99 ± 0.03) g. In DEHP group the fetal quantity, fetal weight and placental weight of female rats compared with other groups were statistical significance (P < 0.05)., Conclusion: DEHP can damage female rats and fetal rats in gestation period. Zinc supplied before pregnancy can relieve the influence by DEHP.

Objective: To investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) exposure on pubertal development and reproductive endocrine function in prepubertal female rats and its possible mechanisms., Methods: 40 female SD rats at 3-week-old were randomly divided into a control group (corn oil) and three exposure groups, which were exposed consecutively for 28 days to DEHP by oral gavage at doses of 50, 150 and 500 mg/(kg x d). The onset of puberty was determined by daily examination for vaginal opening (OV), breast development and the first estrous cycle. By the end of the experiment, the rats were sacrificed during the diestrous stage. The gene expression interrelated with ovary function was detected by real-time PCR. Serum levels of follicle stimulating hormone (FSH) luteinizing hormone (LH), estradiol (E2), progesterone (P4) and testosterone (T) were measured by ELISA. The morphological change of ovaries was observed by HE staining under optical microscope. The expression of PPARgamma protein in ovary cells was measured by immuohistochemistry., Results: The age of vaginal opening was advanced by DEHP exposure in 500 mg/kg group, and the body weight was increased at the time of vaginal opening in 150 and 500 mg/kg groups (P < 0.05). Compared to the control group, the level of T in all DEHP groups was decreased; the levels of FSH and E2 were decreased and the level of P4 was increased in 150 and 500 mg/kg groups (P < 0.05). The gene expression of P4S0Aromin 150 and 500 mg/kg groups was significantly decreased compared to the control group. A significant increase of atretic follicles and a significant reduction of corpora lutea were observed in 150 and 500 mg/kg groups compared to the control group. The expression of PPARgamma protein in granulosa cells of follicle and corpora lutea in 150 and 500 mg/kg groups was higher than that in the control group and 50 mg/kg group (P < 0.05)., Conclusion: DEHP exposure can affect the pubertal development and reproductive endocrine function in prepubertal female rats, and its possible mechanism may be correlated with PPARgamma.

Objective: To use rat preantral follicle culture as a novel bioassay in order to study the effects of di-2-ethylhexyl phttmlate (DEHP) and its metabolite mono-2-ethylhexyl phthalate (MEHP) on folliculogenesis in vitro., Methods: The preantral follicles were mechanically dissected from ovaries and equally randomized to to 96-well plates. Six dose groups were setup to be acute exposed in 2.5, 5, 10, 20, 40 and 80 microg/ml MEHP concentration, 33.7, 67.5, 125, 250, 500 and 1000 nmol/L DEHP concentration on day 2,while another control group used solvent only, each group had 16 to 20 follicles. After subsequently cultured individually for 10 days, the follicles were induced to ovulate for 20h, follicle development and oocyte maturation were observed., Results: Day 11 follicle survival rate (54.76% +/- 3.37%) had significantly dropped after exposure to >10 microg/ml MEHP concentration, comparing to the control group (91.57% +/- 1.32%), the difference is statistical significant (P < 0.05). There is correlation between dosage and follicle survival rate, with correlation coefficient R2 = 0.92. Abnormal follicle rate (45.24% +/- 3.37%) is remarkably higher, and the difference is statistically significant (P < 0.05) comparing to the control group. At the concentrations of >20 microg/ml MEHP, follicle growth and differentiation were dependently impaired:antral follicles (46.18% +/- 1.67%) had significantly reduced (P < 0.05) comparing to the control group (85.91% +/- 5.03%). DEHP showed no adverse effects upon follicle development, there were no significant defference between DMSO and control., Conclusion: At the dose of >10 microg/ml, MEHP could inhibit the in-vitro rat follicle development, DEHP seemed no adverse effects upon follicle development.