Negative synergistic effect of mono-(2-ethylhexyl) phthalate and the absence of estradiol on oocyte developmental competence – the bovine model

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are reproductive toxicants. Previously we examined the accumulation of DHEP in the follicular fluid of the preovulatory follicle. Cows were gavaged with DEHP (100 mg/kg per day) for 3 days and follicular fluid (FF) was aspirated before and 26 days after treatment. DEHP metabolites were analyzed by LC-MS/MS and estradiol levels by solid-phase RIA kit. While MEHP was not detected in the FF before treatment, a residual level was detected in DEHP-treated group 26 days after treatment. In addition, estradiol level in the FF was lower in DEHP-treated group than in the control (P < 0.05). Based on these findings, we aimed to examine whether the effects of these changes in the intra follicle micro-environment affect oocyte developmental competence by using an in-vitro embryo production system. Cumulus-oocyte complexes (COCs) were matured in the presence of MEHP (0, 20, 200, 500 and 1000 nM) with or without estradiol (0, 400, 800 and 2000 ng/ml). COCs were then fertilized and cultured for 7 days. The findings revealed that estradiol levels did not affect oocyte developmental competence. However, MEHP reduced the proportion of oocytes that cleaved and developed to 2- to 4-cell-stage embryos and 7-day blastocysts, in a dose dependent manner with a prominent effect on oocytes cultured with 1000 nM MEHP (P < 0.05). A negative synergistic effect of MEHP (20 nM and 1000 nM) was noted when oocyte were cultured without estradiol, expressed by reduction in blastocyst formation (P < 0.05). The findings indicate that MEHP at low levels has a negative impact on oocyte developmental competence and that DEHP-induced reduction in estradiol concentration is suggested to enhance this effect. The current study explores the risk associated with residual level of MEHP in the FF i.e., changes in the microenvironment of the follicle enclosed oocyte deleteriously affect oocyte developmental competence.