Phthalate are high-production-volume synthetic chemicals with ubiquitous human exposures because of their use in plastics and other common consumer products.
Di-(2-ethylhexyl) phthalate (DEHP) is one of the most abundantly used phthalates and has been shown to induce developmental and reproductive toxicity in rodent models. In rodent and primates, DEHP is rapidly hydrolysed in the gut to produce its major metabolite mono (2-ethylhexyl) phthalate (MEHP). One possible way by which DEHP exerts its antifertility effect could, therefore, be by directly impairing the process of oocyte maturation.
The present study was conducted with the aim of examining the effect of DEHP and MEHP on resumption of meiosis, in vitro maturation and embryo development.
1) Collection of oocytes: For one experiment: female 4-6 week old NMRI mice were dosed daily with 2ml/kg bw/day, 4ml/kg bw/day DEHP (2.56 M) for 12 days and 8ml/kg bw/day DEHP for 8 days. For two experiments: The nontreated mice were used. Then animals were killed by cervical dislocation. Cumulus enclosed oocytes (CEO) at GV stage were harvested by puncturing the large antral follicles. Denuded oocytes were obtained by repeated pipeting of the CEO. Then denuded oocytes were transferred to the droplet medium.
2) Culture of immature oocytes: The basic maturation medium was MEM-a supplemented with 5% FCS, 7.5 IUhCG and 100 mIUrhFSH. In two experiments: Immature oocytes were matured in above medium with 0, 50,100,200 and 400mM MEHP. This maturation medium (MEM-a) was either used as control medium. Culture was carried out in an atmosphere of 5% co2 in air at 37oC for 24h. After 24h immature oocytes examined GV (Germinal Vesicle), GVB (Germinal Vesicle Breakdown) and MII (MetaphaseII) by inverted microscope.
3) In vitro Fertilization: Sperm were obtained from adult male NMRI mice by epididymal extraction. Fertilization and embryo development were accomplished in T6 medium.
In Experiment 1: The resumption of meiosis in 2ml/kg, 4ml/kg bw/day and 8ml/kg bw/day DEHP (2.56M) were 76.61%, 77.04%, 70% respectively. However the resumption of meiosis in experimental groups was significantly lower than control group (P<0.001). The in vitro maturation in 2ml/kg bw/day, 4ml/kg bw/day and 8ml/kg bw/day DEHP were 66.13%, 59.18%, 52.72% respectively. However the in vitro maturation in 4ml/kg bw/day and 8ml/kg bw/day DEHP was significantly lower than control group (P<0.001).
The percentage of embryo formation post 96h insemination in 2ml/kg bw/day, 4ml/kg bw/day and 8ml/kg bw/day DEHP were 27.85, 25.23 and 21.42 respectively. However the embryo formation in experimental groups was significantly lower than control group (P<0.03).
In experiment 2: The resumption of meiosis in 50, 100, 200 and 400 mM MEHP were 75.12%, 62.67%, 60.59%, 55.88% respectively. However the resumption of meiosis in experimental groups was significantly lower than control group (P<0.0001). The in vitro maturation in 50, 100, 200 and 400 mM MEHP were 53.23%, 48.88%, 42.36%, 36.76% respectively. However the in vitro maturation in experimental groups was significantly lower than control group (P<0.0001).
The percentage of embryo formation post 24h insemination in 50, 100, 200 and 400 mM MEHP were 60, 54, 44 and 27 respectively. However the embryo formation in 100, 200, 400mM MEHP was significantly lower than control group (P<0.03).
The results of the precent study indicate that DEHP and MEHP negatively influenced the ability mouse oocytes to undergo meiotic maturation and embryo development. Also results indicated that DEHP exerted its inhibitory effect in a dose-dependent manner.