Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone production in cultured mouse antral follicles

Abstract

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant found in consumer products that causes ovarian toxicity. Antral follicles are the functional ovarian units and must undergo growth, survival from atresia, and proper regulation of steroidogenesis to ovulate and produce hormones. Previous studies have determined that DEHP inhibits antral follicle growth and decreases estradiol levels in vitro; however, the mechanism by which DEHP elicits these effects is unknown. The present study tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and steroidogenesis to inhibit antral follicle functionality. Antral follicles from adult CD-1 mice were cultured with vehicle control or DEHP (1-100μg/ml) for 24-96 hr to establish the temporal effects of DEHP on the follicle. Following 24-96 hr of culture, antral follicles were subjected to gene expression analysis, and media were subjected to measurements of hormone levels. DEHP increased the mRNA levels of cyclin D2, cyclin dependent kinase 4, cyclin E1, cyclin A2, and cyclin B1 and decreased the levels of cyclin-dependent kinase inhibitor 1A prior to growth inhibition. Additionally, DEHP increased the mRNA levels of BCL2-associated agonist of cell death, BCL2-associated X protein, BCL2-related ovarian killer protein, B-cell leukemia/lymphoma 2, and Bcl2-like 10, leading to an increase in atresia. Further, DEHP decreased the levels of progesterone, androstenedione, and testosterone prior to the decrease in estradiol levels, with decreased mRNA levels of side-chain cleavage, 17α-hydorxylase-17,20-desmolase, 17β-hydroxysteroid dehydrogenase, and aromatase. Collectively, DEHP directly alters antral follicle functionality by inhibiting growth, inducing atresia, and inhibiting steroidogenesis.

Early Prenatal Phthalate Exposure, Sex Steroid Hormones, and Birth Outcomes

“…In human gestation, E1 and E2 are maternal in origin until about week 8, when placental production rises through aromatization of fetal adrenal androgens (1). BBzP and DBP exposure leads to increased estrogenic activity in vitro in ovarian granulosa cells, while DEHP has been associated with reduced estrogenic activity in breast cancer cell lines (31,32,36,37). Although the mechanism underlying these observations is not known, these studies hypothesize that DEHP may stimulate peroxisome proliferator-activated receptors that suppress aromatization in the granulosa cell.…”

“…The few studies published to date suggest conflicting results and focus on estrogen production during adult life. DEHP metabolites have been negatively associated with estradiol concentrations in adult female rodents and in ovarian granulosa cells via decreased aromatase transcription (28)(29)(30)(31)(32), but this relationship has not been observed in human studies (27,33,34). In contrast, DBP and BBzP exposures have been associated with increased estrogenicity in breast cancer cell lines and in vivo in offspring exposure in utero or in adult female rodent models (35)(36)(37).…”

Mono(2-Ethylhexyl) Phthalate Accelerates Early Folliculogenesis and Inhibits Steroidogenesis in Cultured Mouse Whole Ovaries and Antral Follicles1

“…Previous studies have determined that DEHP exposure decreases estradiol levels in vivo [46][47][48] and in cultured ovarian cell types and follicles in vitro [16,[49][50][51]. Likewise, MEHP exposure has also been shown to decrease estradiol levels in cultured ovarian cell types and follicles in vitro [16,[19][20][21]53].…”

“…Previous studies have suggested that one potential mechanism by which DEHP and MEHP decrease estradiol levels is through a decrease in the ovarian mRNA levels of Cyp19a1, also known as aromatase, following exposure in vivo [46,78] and in vitro [16,[49][50][51]. However, not much is known about the direct effects of MEHP on the steroidogenic enzyme levels upstream of Cyp19a1 in the antral follicle.…”

“…Specifically, DEHP exposure has been shown to decrease estradiol levels and aromatase levels in vivo [46][47][48] and in vitro [16,49,50]. We have reported that the inhibition of steroidogenesis may be via a direct effect on antral follicle functionality because DEHP exposure in cultured mouse antral follicles directly decreased the availability of precursor sex steroid hormone levels at time points prior to decreasing estradiol levels [51]. This toxicity of DEHP may be mediated by MEHP, and MEHP exposure has also been shown to inhibit steroidogenesis in vitro [19-21, 52, 53].…”