Mechanisms by which di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate disrupt ovarian folliculogenesis and steroidogenesis
Di(2-ethylhexyl) phthalate (DEHP) is a synthetic plasticizer used in the manufacturing of common polyvinyl chloride consumer, medical, and building products. Humans are exposed to DEHP on a daily basis because of its high production volumes, incorporation into widely used products, and its ability to leach out of the products. Daily exposure to DEHP results in measureable levels of the chemical and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in human blood, urine, amniotic fluid, umbilical cord blood, breast milk, and ovarian follicular fluid samples. This is of public health concern because DEHP and MEHP are known endocrine disrupting chemicals and reproductive toxicants in the female. The ovary is a primary regulator of the female reproductive and endocrine systems. Thus, proper regulation of ovarian function is required for reproductive health and steroid hormone controlled non-reproductive health. Two ovarian processes that are essential for normal ovarian function are folliculogenesis and steroidogenesis. Follicles are the functional units of the ovary containing the oocyte required for ovulation and fertilization and somatic cells responsible for synthesizing sex steroid hormones. Folliculogenesis is the process by which follicles undergo several irreversible developmental transitions starting from the most immature stage, the primordial follicle, to the most mature stage, the antral follicle. Steroidogenesis is the process by which somatic cells in the antral follicle enzymatically convert cholesterol to estradiol and other necessary precursor steroid hormones in a step-wise manner. Improper regulation of ovarian folliculogenesis and steroidogenesis can lead to reproductive and non-reproductive complications. Specifically, aberrant regulation of folliculogenesis, indicated by an accelerated depletion or activation of primordial follicles, can lead to premature ovarian failure and infertility. This is because the primordial follicle reserve is set at birth and constitutes a female’s reproductive potential. Additionally, an increase in atresia, an apoptosis controlled process of follicular death, can also cause premature ovarian failure and infertility. Further, aberrant regulation of steroidogenesis, indicated by an inhibition of steroid hormone synthesis, can lead to infertility. This is because the hormones produced by the ovary act on the hypothalamus-pituitary-ovarian axis and the reproductive tract to regulate ovulation and the initiation of fertilization and pregnancy. Additionally, the hormones produced by the ovary act on non-reproductive tissues to regulate the cardiovascular, skeletal, and brain systems. Thus, defects in steroidogenesis are also associated with non-reproductive complications. Very few studies have investigated the effects of environmentally relevant levels of DEHP and MEHP on ovarian folliculogenesis and steroidogenesis. Thus, the goal of my doctoral dissertation work was to investigate the ovotoxic effects of DEHP and MEHP on folliculogenesis and steroidogenesis and to elucidate the mechanisms by which these chemicals disrupt these essential processes. Specifically, I investigated whether and how DEHP and MEHP accelerate early folliculogenesis in vivo and in vitro. Further, I investigated how DEHP and MEHP disrupt the functionality of the antral follicle and inhibit estradiol production in adult antral follicles in vitro. I first tested the hypothesis that environmentally relevant levels of DEHP disrupt folliculogenesis in vivo by over-activating the phosphatidylinositol 3-kinase (PI3K) signaling pathway leading to the acceleration of primordial follicle recruitment. Factors within the PI3K signaling pathway are regulators of primordial follicle survival, quiescence, and recruitment. Specifically, factors that inhibit PI3K signaling maintain primordial follicle quiescence, and factors that drive PI3K signaling promote primordial follicle recruitment. I found that DEHP exposure for 10 and 30 days accelerated primordial follicle recruitment, evident by a decrease in the percentage of primordial follicles and an increase in the percentage of primary follicles. Additionally, DEHP exposure for 10 and 30 days at doses that accelerate primordial follicle recruitment increased factors that drive PI3K signaling and decreased factors that inhibit PI3K signaling. I next tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and the steroidogenic pathway in antral follicles in vitro to disrupt antral follicle functionality. Antral follicles must grow, survive from atretic demise, and produce steroid hormones to function normally. I found that DEHP exposure initially increased the levels of cell cycle regulators in a compensatory manner; however, this compensation does not rescue the follicle from growth inhibition. Further, I found that DEHP exposure caused atresia by increasing the levels of pro-apoptotic factors. In addition to the effects on growth and atresia, DEHP exposure also inhibited steroidogenesis. Specifically, DEHP exposure decreased the levels of precursor sex steroid hormones, in the relative absence of changes in steroidogenic enzyme levels, at time-points prior to decreasing estradiol levels. Further, I tested the hypothesis that MEHP directly mediates the DEHP-induced acceleration of primordial follicle recruitment in vitro, and MEHP directly inhibits the synthesis of sex steroid hormones in antral follicles in vitro via a different mechanism than DEHP. For the folliculogenesis experiments, I found that MEHP exposure decreased the percentage of germ cells and increased the percentage of primary follicles by using a neonatal ovary culture system enriched in primordial and primary follicles. Similar to the mechanism in vivo, MEHP exposure also over-activated PI3K signaling by decreasing a PI3K inhibitor and increasing a PI3K activator. Meanwhile, DEHP exposure did not alter follicular dynamics or PI3K signaling factors. Thus, MEHP mediates the effects of DEHP on folliculogenesis in vivo. For the steroidogenesis experiments, I found that MEHP exposure inhibits estradiol production later then DEHP exposure. Specifically, MEHP exposure decreased the levels of steroidogenic enzymes, in the relative absence of prior changes in steroid hormone levels, at the same time-point in which estradiol levels are decreased. Thus, DEHP and MEHP differentially inhibit steroidogenesis in the antral follicle. Collectively, my doctoral dissertation data suggest that DEHP and MEHP disrupt ovarian folliculogenesis and steroidogenesis, which are essential processes for normal reproductive and non-reproductive health.
reproductive toxicology, steroidogenesis, folliculogenesis, follicle, ovary, phthalate
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign