76 EX VIVO MODEL TO STUDY THE EFFECT OF ACUTE EXPOSURE TO DI-(2-ETHYLHEXYL) PHTHALATE ON BOVINE OOCYTE MATURATION AND DEVELOPMENTAL COMPETENCE

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolites are environmental toxicants that potentially affect mammalian health. However, their effects on ovarian function, and in particular oocyte developmental competence, are less known. We established a model of acute exposure to DEHP to examine its immediate and long-term effects on follicles and oocyte competence. Lactating Holstein cows were synchronized and gavaged with DEHP (OXPLAST^®O, ZAK, Kędzierzyn-Koźle, Poland; 100 mg kg^–1 per day, n = 4) or water (n = 5) for 3 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed high DEHP metabolite levels in both the urine and plasma during DEHP exposure (acute phase), followed by relatively low levels through the subsequent 34 days (chronic phase). In the chronic phase, cows were synchronized with prostaglandin (PG)_2α-gonadotropin-releasing hormone (GnRH) (day 0) and ovaries were monitored by ultrasonography (Aloka SSD-900, 7.5 MHz; Aloka, Tokyo, Japan) through an entire oestrous cycle. On Day 18 of the cycle, cows were administered with PG_2α and within 30 h, follicular fluid (FF) was aspirated from the preovulatory follicle by ultrasonic scanner connected to a vaginal sector transducer (7.5-MHz, PieMedical, Maastricht, the Netherlands). For each group, the FF were pooled and analysed for DEHP metabolites (MEHP, MEHOH, MEHHP) by LC-MS/MS. These FF further served as maturation medium for in vitro embryo production. For all cows, FF aspirated before DEHP administration did not contain any of the examined metabolites. However, during the chronic phase, the level of MEHP (but not MEHOH, MEHHP) was higher (22.31 v. 0.00 nM) in the FF aspirated from treated cows (FF-DEHP, n = 4) relative to controls (FF-control, n = 5). To examine the effect of MEHP on oocyte maturation and developmental competence, cumulus-oocyte complexes aspirated from abattoir ovaries (n = 250 × 5 replicates) were matured (22 h, 38.5°C, 5% CO₂) in FF-control or FF-DEHP, then in vitro fertilized (18 h, 38.5°C, 5% CO₂) and cultured for 8 days in KSOM (38.5°C, 5% CO₂, 5% O₂). The proportion of oocytes with expanded cumulus cells (84.5 v. 81.2%) and distribution of oocyte within cortical granule types (I–III) did not differ between groups at the end of maturation. The proportion of oocytes with metaphase II plate and first polar body (i.e. nuclear-matured) was lower in the DEHP-treated group relative to the control (34.7 v. 64.3%, n = 100/group; P < 0.0001). Moreover, a decreased proportion of 2- to 4-cell-stage embryos (53.12 v. 67.61%; P < 0.04) and 7-day blastocysts (4.2 v. 12%; P < 0.08) was noted for the FF-DEHP group. In summary, the findings reveal long-lasting effects of DEHP exposure, expressed by MEHP incorporation into the FF, suggesting potential deleterious effects on developmental competence of the follicle-enclosed oocyte.