(105) Exposure to a Phthalate Mixture Alters Factors that Mediate Mouse Cumulus-Oocyte Complex Expansion and Stability In Vitro

Katie L. Land^1,2; Caroline V. Harper²; Madison M. Wilson²; Elizabeth T. Hayden²; Patrick R. Hannon^1,2

1. Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, United States


2. Department of Obstetrics and Gynecology, University of Kentucky, Lexington, KY, United States

Humans are ubiquitously exposed to phthalates due to their frequent use as solvents and plasticizers in many products, ranging from cosmetics and food packaging to medical devices and car interiors. Phthalates undergo rapid metabolism upon absorption, and these bioactive metabolites are detected in ovarian follicular fluid. As known endocrine disruptors, exposure to phthalates and phthalate metabolites may impair ovulatory processes like cumulus-oocyte complex (COC) expansion, which is vital for the expulsion of a fertilizable oocyte. Proper expansion of a stabilized COC relies on the formation of an extracellular matrix rich in hyaluronan (HA) and various stabilizing factors including tumor necrosis factor stimulated gene-6 (TNFAIP6) and versican (CSPG2). The stabilizing factor TNFAIP6 is dependent on prostaglandin E₂ (PGE₂) production via increased prostaglandin synthase 2 (Ptgs2) expression. Interactions between HA and matrix-stabilizing factors aid in secure transport of a mature oocyte through the reproductive tract and enable sperm and oocyte fusion for fertilization. This study used an environmentally relevant phthalate metabolite mixture (MPTmix), derived from urinary phthalate measurements in pregnant women, to best mimic human exposure and to investigate phthalate mixture effects on COC expansion and stability. Immature (PND 24) CD-1 mice were injected (IP) with 5IU pregnant mare serum gonadotropin (PMSG) to stimulate the development of multiple follicles. Mice were euthanized 47hr post-PMSG injection, ovaries were removed, and unexpanded COCs were isolated microscopically. Unexpanded COCs were plated in media treated with vehicle control (dimethylsulfoxide, DMSO) ± EGF (epidermal growth factor; potent COC expansion stimulus) and ± MPTmix (1, 10, 100µg/ml). Using the InFocus microscopy software, COC diameter was measured at 0 and 12hr to assess percent expansion (ovulation occurs around 12hr). COCs and culture media were collected at 12hr for gene expression analyses and ELISAs to measure the abovementioned genes/factors involved in COC expansion and stability (n=4-10; 19-36 COCs/replicate; p≤0.05). Relative to 0hr measurements, percent expansion of COCs at 12hr was significantly increased with EGF treatment (60%) compared to DMSO treatment (14%), although EGF+MPTmix treatment (all doses) did not significantly decrease percent expansion when compared to the EGF alone expanded controls. EGF+MPTmix decreased expression of Has2 (HA synthase; 100μg/ml) and HA levels (1μg/ml) compared to EGF treated controls. Ptgs2 expression was decreased with EGF+MPTmix treatment (10 and 100μg/ml) when compared to EGF, which also correlated with MPTmix-induced decreases in PGE₂ levels (100μg/ml) at 12hr. Expression of a PGE₂ receptor (Ptger4) was increased with EGF+MPTmix treatment (1μg/ml), while Tnfaip6 expression (prostaglandin-dependent) was decreased (100μg/ml) relative to EGF. Versican levels remained unchanged, despite MPTmix-induced decreases in Cspg2 expression (1 and 100μg/ml) compared to EGF. Egfr expression remained unchanged across all treatment groups suggesting that any MPTmix-induced effects are likely downstream of EGF receptor activation. When taken together, these data suggest that MPTmix exposure decreases HA and PGE₂ levels, likely through decreasing key synthases responsible for their production. The decreases in Ptgs2 expression and PGE₂ levels further lead to decreased expression of the HA binding partner Tnfaip6, while versican expression (Cspg2) was also decreased with MPTmix exposure likely via a separate mechanism. While MPTmix exposure did not alter percent expansion, this microscopy assessment is unable to evaluate matrix stability. Therefore, the MPTmix-induced changes in the factors that mediate COC expansion and especially stability could lead to defects in ovulation, impaired COC transport through the reproductive tract, and unsuccessful fertilization. Supported by R01ES033767, P30ES026529.