177 EXPOSURE OF BOVINE OOCYTES TO A RESIDUAL CONCENTRATION OF MONO-(2-ETHYLHEXYL) PHTHALATE FURTHER IMPAIRS BLASTOCYST GENE EXPRESSION

Abstract

Di-(2-ethylhexyl) phthalate and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are reproductive toxicants. We recently documented a residual concentration of MEHP (~20 nM) and a 2-fold lower level of oestradiol in preovulatory-follicular fluid aspirated from cows gavaged with di-(2-ethylhexyl) phthalate (100 mg/kg/day; 3 days). To examine the effects of these changes on oocyte developmental competence, cumulus-oocyte complexes (COC; n = 3290; 14 replicates) were in vitro matured (22 h) in standard oocyte maturation media at 38.5°C and 5% CO₂. Oocyte maturation media was supplemented with MEHP (0, 20, and 1000 nM) dissolved in dimethyl sulfoxide (0.01% vol/vol), with or without oestradiol (0 or 2000 ng mL⁻¹). The COC were then fertilized (18 h) and further cultured in KSOM at 38.5°C, 5% CO₂ and 5% O_2, for 7 days. Matured oocytes (n = 20/sample; 4 replicates/group) and blastocysts (n = 4 embryos/sample; 4 replicates/group) were collected. Samples were then underwent mRNA isolation, followed by real-time PCR to analyse the expression of selected genes (Cyc-1, Mt-co1, Atp5b, Pou5f1, Sox2, and Dnmt3b) associated with early embryonic development. Gene expression was quantified and analysed by LightCycler®96 software; the ΔΔC_t method was used to calculate the relative expression; 2 genes (Ywhaz and Sdha) were used as internal reference. Statistical analysis was performed by one-way ANOVA followed by Student’s t-test. Findings revealed that maturation with oestradiol and 20 nM MEHP did not affect cleavage or blastocyst formation rate (80.3 ± 1.6 and 16.3 ± 2.2, respectively); however, maturation with 1000 nM decreased the proportion of oocytes that cleaved (70.6 ± 5.8 v. 83.2 ± 2.2, respectively; P < 0.01) and those developed to blastocysts, relative to the control (10.3 ± 1.2 v. 17.2 ± 1.2, respectively; P < 0.04). On the other hand, maturation with 20 or 1000 nM MEHP without oestradiol did not affect cleavage rate, but reduced proportion of developing blastocyst relative to control (15.9 ± 1.6, P < 0.09; 13.9 ± 1.5, P < 0.02; v. 20.6 ± 1.9; respectively). In addition, co-culture of COC with oestradiol and 1000 nM MEHP increased the expression of Cyc-1 and Mt-co1 in matured oocytes (P < 0.05); co-culture of COC with 20 or 1000 nM MEHP increased the expression of Cyc-1, Atp5b, and Dnmt3b in blastocysts (P < 0.05). However, culture with 1000 nM MEHP and without oestradiol increased the expression of Dnmt3b in matured oocytes but reduced the expression of Cyc-1, Mt-co1, Atp5b, Pou5f1, and Dnmt3b in blastocysts (P < 0.05). Although not significant, a similar pattern of gene expression was recorded for blastocysts developed from oocytes matured with 20 nM MEHP, and without oestradiol. The findings emphasise the potential risk associated with phthalate exposure: maturation with low MEHP concentrations deleteriously affected oocyte developmental competence, impaired gene expression in blastocysts developed from MEHP-treated oocytes indicated a carryover effect, and impaired expression of genes associated with early embryonic development suggested that these embryos are of inferior quality.