Di-n-butyl phthalate disrupts the expression of genes involved in cell cycle and apoptotic pathways in mouse ovarian antral follicles

Abstract

Di-n-butyl phthalate (DBP) is present in many consumer products, such as infant, beauty, and medical products. Several studies have shown that DBP causes reproductive toxicity in rodents, but no studies have evaluated its effects on ovarian follicles. Therefore, we used a follicle culture system to evaluate the effects of DBP on antral follicle growth, cell cycle and apoptosis gene expression, cell cycle staging, atresia, and 17β-estradiol (E(2)) production. Antral follicles were isolated from adult CD-1 mice and exposed to DBP at 1, 10, 100, and 1000 μg/ml for 24 or 168 h. Follicles treated with vehicle or DBP at 1-100 μg/ml grew over time, but DBP at 1000 μg/ml significantly suppressed follicle growth. Regardless of effect on follicle growth, DBP-treated follicles had decreased mRNA for cyclins D2, E1, A2, and B1 and increased p21. Levels of the proapoptotic genes Bax, Bad, and Bok were not altered by DBP treatment, but DBP 1000 μg/ml increased levels of Bid and decreased levels of the antiapoptotic gene Bcl2. DBP-treated follicles contained significantly more cells in G(1) phase, significantly less cells in S, and exhibited a trend for fewer cells in G(2). Although DBP did not affect E(2) production and atresia at 24 h, follicles treated with DBP had reduced levels of E(2) at 96 h and underwent atresia at 168 h. These data suggest that DBP targets antral follicles and alters the expression of cell cycle and apoptosis factors, causes cell cycle arrest, decreases E(2), and triggers atresia, depending on dose.

FIG. 1

Effect of DBP on in vitro antral follicle growth. Antral follicles were mechanically isolated, placed in culture for 168 h, and growth was assessed as described in Materials and Methods. Data represent means ± SEM from four separate experiments, each with 8–16 follicles per treatment. Asterisks (*) indicate statistically significant differences (P < 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).

FIG. 2

Effect of in vitro treatment with DBP on the expression of Ccnd2 mRNA in mouse antral follicles treated in vitro for 168 h. Antral follicles were mechanically isolated and placed in culture for 168 h as described in Materials and Methods. At the end of 168 h, follicles were pooled per treatment group and processed for qPCR analysis of gene expression as described in Materials and Methods. All gene expression data were normalized to the expression of Actb. Data represent means ± SEM from three separate experiments each with 8–16 follicles per treatment. The asterisk (*) indicates a statistically significant difference (P ≤ 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).

FIG. 3

Effect of in vitro treatment with DBP on transcript levels of four major cyclin mRNAs in mouse antral follicles treated in vitro for 24 h: A) Ccnd2. B) Ccne1. C) Ccna2. D) Ccnb1. Antral follicles were mechanically isolated and placed in culture for 24 h as described in Materials and Methods. At the end of culture, follicles were pooled per treatment group and processed for qPCR analysis of gene expression as described in Materials and Methods. All gene expression data were normalized to the expression of Actb. Data represent means ± SEM from three to four separate experiments, each with 8–16 follicles per treatment. Asterisks (*) indicate statistically significant differences (P < 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).

FIG. 4

Effect of in vitro treatment with DBP on the expression of Cdkn1a mRNA in mouse antral follicles treated in vitro for 24 h. Antral follicles were mechanically isolated and placed in culture for 24 h and processed for qPCR as described in Materials and Methods. All gene expression data were normalized to the expression of Actb. Data represent means ± SEM from three to four separate experiments, each with 8–16 follicles per treatment. Asterisks (*) indicate statistically significant differences (P < 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).

FIG. 5

Effect of in vitro treatment with DBP on cell cycle staging of follicular cells. Antral follicles were mechanically isolated and placed in culture for 24 h and processed for flow cytometry as described in Materials and Methods. Data represent means ± SEM obtained from three separate experiments each with ≥30 follicles per treatment. Representative histograms from vehicle-treated follicles (A) and DBP-treated follicles (B). Summary data from all four experiments (C). Asterisks (*) indicate statistically significant differences (P < 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).

FIG. 6

Effect of in vitro treatment with DBP on the expression of Bid and Bcl2 mRNAs and on follicular atresia in mouse antral follicles treated in vitro. Antral follicles were mechanically isolated and placed in culture for 24 h and processed for qPCR or histological evaluation of atresia as described in Materials and Methods. All gene expression data were normalized to the expression of Actb. Data represent means ± SEM from three to four separate experiments each with 8–16 follicles (qPCR) or three to four follicles (follicle atresia) per treatment. Asterisks (*) indicate statistically significant differences (P ≤ 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO). A, B) Data for atresia rating at 24 and 168 h, respectively. C, D) mRNA expression data for Bid and Bcl2 at 24 h. Atresia rating scale: 1–4 for the presence of apoptotic bodies: 1 for healthy, 2 for 1%–10%, 3 for 10%–30%, and 4 for greater than 30% apoptotic bodies.

FIG. 7

Effect of in vitro treatment with DBP on levels of E₂ produced by mouse antral follicles. Antral follicles were mechanically isolated and placed in culture for 24 or 168 h and E₂ levels measured as described in Materials and Methods. Media samples from individual follicles were obtained at the end of the 24-h cultures (A) or during the 96-h media change of the 168-h cultures (B). Data represent means ± SEM from three to four separate experiments for a total of 12–18 follicles per treatment. The asterisk (*) indicates a statistically significant difference (P < 0.05) between DBP-treated follicles and vehicle-treated follicles (DMSO).