Our long-term goal is to understand the effects of phthalates in the ovary and if possible, to reverse or prevent any adverse effects. Phthalates are a group ubiquitous environmental chemicals with anti-androgenic actions and are well-known male reproductive and developmental toxicants. Although their effects in adult ovary and females are also known, their developmental effects, and the mechanisms and the molecular players involved in females are much less known. One of the most common phthalates is di(2-ethylhexyl) phthalate (DEHP). Another phthalate, diisononyl phthalate (DiNP) is a less potent anti-androgen and is considered safe to replace DEHP. The safety of DiNP is debatable, because recent epidemiological studies suggest that DiNP is associated with male developmental pathologies. Some countries in the Western world have already banned or temporarily banned DiNP from children's items while others have not, which makes this study timely. The fact that effects of DiNP in the ovary are unknown adds further urgency to the matter. In this proposal, our objective is to study the development effects of DEHP and DiNP and identify the molecular players and the mechanisms involved, using environmentally relevant doses and route, several targeted exposure windows, and robust time course. Our hypothesis is that DEHP and DiNP cause dose-, exposure stage-, and age-dependent gene expression changes in the ovary that lead to altered folliculogenesis and female infertility. To test this hypothesis, we propose two Specific Aims. 1- To investigate when and how developmental exposures to DEHP and DiNP alter follicular dynamics and female fertility. We will expose pregnant and lactating female dams (F0) to environmentally relevant doses of DEHP or DiNP orally, a relevant route for DEHP; in three different exposure windows that span vulnerable ovarian developmental events, namely gonadal sex differentiation and early folliculogenesis. We will then examine age-dependent changes in ovarian folliculogenesis of F1 females at postnatal days 7, 21, 60, 180, and 360. We will also monitor fertility parameters to assess functional consequences of the exposures. 2- Identify molecular players that are altered by developmental exposures to DEHP and DiNP in the ovary and their precise relationships to alterations in folliculogenesis. To link changes in folliculogenesis and gene expression in the ovary, we will perform RNA-Seq, followed by differential gene expression and functional pathway analyses for the same time points as Aim 1. In histological sections, the localization of key ovarian proteins, including those detected in RNA-Seq will be examined with immunohistochemistry and quantified. Thus, important ovarian factors will be assessed at the protein levels, including their cell- and follicle stage- specific localization. Our studies will provide novel and significant insights into the effects of phthalates in the developing ovary and into possible adult health consequences of these effects. We will use a robust developmental study design, including global, unbiased gene expression analysis. These studies are important from a basic ovarian biology perspective and likely to have a strong positive impact on public health because its findings can be informative for future policy and clinical decisions.
Di(2-ethylhexyl) phthalate (DEHP) is one of the most common phthalates in the environment, and children are highly exposed to it. While DEHP is a well-known male developmental and reproductive toxicant, neither DEHP's nor its recent replacement diisononyl phthalate's actions in developing female gonad, the ovary, are well known. We propose to closely examine the harmful effects of phthalates in the developing ovaries and the adult health consequences in a well-focused and comprehensive manner, using physiological, anatomical, and molecular approaches. The results will help to better understand the ovarian biology, inform about the potential risks of phthalates in female infants and children, and develop protection or therapy against these and similar chemicals.
