Exposure to di(2-ethylhexyl) phthalate inhibits luteal function via dysregulation of CD31 and prostaglandin F2alpha in pregnant mice

Abstract

Background: Di(2-ethylhexyl) phthalate (DEHP) exposure reduces embryo implantations, increases embryonic loss, and decreases fetal body weights. However, whether it is associated with the alteration of luteal function remains unknown. Thus, our aim in this study was to explore the effect and mechanism of DEHP on luteal function in pregnant mice in vivo.

Methods: Mice were administered DEHP by gavage at 125, 250, 500 mg/kg/day from gestational days (GD) 1 to 9 or 13. Levels of serum progesterone and estradiol were measured by radioimmunoassay. The numbers and sizes of corpora lutea were calculated by ovarian histomorphology. Steroidogenic enzymes were assessed by qRT-PCR. CD31 protein was detected by immunocytochemistry, and prostaglandin F2alpha (PGF2alpha) levels were evaluated by enzyme immunoassay.

Results: Treatment with DEHP significantly inhibited progesterone secretion in pregnant mice in a dose-dependent manner but did not inhibit estradiol production on GD 9 and 13. Treatment also showed concomitant decreases in transcript levels for key steroidogenic enzymes (CYP11A, 3β-HSD, and StAR) on GD 13. Furthermore, DEHP administration significantly reduced the numbers and sizes of corpora lutea on GD 13. No significant changes in the ratio of ovary weight vs. body weight were observed between the control group and treated animals on GD 9 and 13. In addition, treatment with DEHP significantly inhibited CD31 expression of corpora lutea, whereas plasma PGF2alpha levels in DEHP treatment groups were significantly higher compared with the control groups on GD 9 and 13.

Conclusions: The results show DEHP significantly inhibits luteal function of pregnant mice in vivo, with a mechanism that seems to involve the down-regulation of progesterone and steroidogenic enzymes message RNA, the decrease in CD31 expression, and the increase in PGF2alpha secretion.

Figure 1

Effect of DEHP exposure on serum estradiol and progesterone levels. (A) Serum estradiol levels on day 9 of pregnancy. (B) Serum estradiol levels on day 13 of pregnancy. (C) Serum progesterone levels on day 9 of pregnancy. (D) Serum progesterone levels on day 13 of pregnancy. The results are shown as the means +/− SD of 10 animals. Groups with different superscript letters are significantly different (P < 0.05, ANOVA followed by LSD multiple range test).

Figure 2

Effect of DEHP exposure on the ovarian weights, numbers and sizes of corpora lutea. (A) Ratio of ovary/body weight on day 9 of pregnancy. (B) Ratio of ovary/body weight on day 13 of pregnancy. (C) Numbers of corpora lutea on day 9 of pregnancy. (D) Numbers of corpora lutea on day 13 of pregnancy. (E) Sizes of corpora lutea on day 9 of pregnancy. (F) Sizes of corpora lutea on day 13 of pregnancy. The results are shown as the means +/− SD of 10 animals. Groups with different superscript letters are significantly different (P < 0.05, ANOVA followed by LSD multiple range test).

Figure 3

Ovarian histomorphology detected by H&E staining. (A) Representative images of ovary on day 9 of pregnancy. (B) Representative images of ovaries on day 13 of pregnancy. Squared areas at the top (×40) are presented at higher magnification (×200) at the bottom. CL, corpus luteum; F, follicle.

Figure 4

Effect of DEHP exposure on the expression of different steroidogenic enzymes mRNA. (A, B) StAR mRNA levels in the ovary on day 9 (A) and 13 (B) of pregnancy. (C, D) CYP11A mRNA levels in the ovary on day 9 (C) and 13 (D) of pregnancy. (E, F) 3β-HSD mRNA levels in the ovary on day 9 (E) and 13 (F) of pregnancy. mRNAs levels were quantified using quantitative PCR and were normalized to 18S. The data are represented as the means +/− SD of 10 animals. Groups with different superscript letters are significantly different (P < 0.05, ANOVA followed by LSD multiple range test).

Figure 5

Expression levels of CD31 protein detected by immunohistochemistry. (A, B) Representative images of ovaries on day 9 (A) and 13 (B) of pregnancy. Squared areas at the top (×40) are presented at higher magnification (×200) at the bottom. Inset is negative control.

Figure 6

Effect of DEHP exposure on prostaglandin F2alpha levels on day 9 and 13 of pregnancy. (A, B) Plasma concentrations of PGF2alpha on day 9 (A) and 13 (B) of pregnancy. The results are shown as the means +/− SD of 10 animals. Groups with different superscript letters are significantly different (P < 0.05, ANOVA followed by LSD multiple range test).