The Toxicity Of Methoxychlor On In Vitro Maturation And Fertilization Of Mouse Oocytes

Methoxychlor (MXC) is an organochlorine pesticide, which is widely sprayed on fruits, vegetables, forage crops, and home gardens to prevent insects from attacking them. MXC is considered to be an alternative to dichloro- diphenyl-trichloroethane (DDT) due to its lower toxicity to mammals and its shorter half-life. Methoxychlor was detected in soil, river, animals and plants even in pregnant women,placenta,cord blood and newborn infant. Methoxychlor can disrupt function of male testiculus and epididymis, reduce spermatozoa; methoxychlor can also make ovarian follicle atresia, uterus protein expression changed. Therefore, in our present study, we selected an in vitro system in which we evaluated both oocyte maturation and postfertilization development. The aim of this study was to investigate the effect of different doses of Methoxychlor on in vitro maturation, fertilization, and post fertilization development of mouse oocytes. AIM: 1 To investigate the toxicity of Methoxychlor on in vitro maturation of mouse oocytes.2 To investigate the toxicity of Methoxychlor on in vitro fertilization of mouse oocytes.3 To investigate the toxicity of Methoxychlor on mouse embryos culture.Part 1 The toxicity of Methoxychlor on in vitro maturation of mouse oocytes.METHODS: 1,Immature oocytes were retrieved from mouse ovarian stimulated by PMSG. The oocyte in experimental groups were cultured in M2 medium with 7.25,14.5,29μg/mL MXC and the oocytes in the unexposed control group were cultured in M2 medium for 24 h. Germinal vesicle breakdown and the first polar body extrusion were observed with invert microscope during maturation.When the first polar body was found in the perivitelline space, the oocytes were classified as nuclear maturation (MⅡ). 2,Laser scanning confocal microscope(LSCM) was used to examine the meiotic spindle. 3,Oocyte extructed the first polar body were stained with Fluorescein isothiocyanate- peanut agglutinin to evaluate Cortical granule with Laser confocal microscopy for assess cytoplasmic maturation. 4,the intensity of cumulus expansion was estimated with invert microscope.RESULTS: 1,After 24 h of culture, the incidence of GVBD in experimental group occurred normally with the same efficiency when compared to the unexposed control, and the process of GVBD occurred without any significant delay. but the maturation rate decreased from 71.2%(unexposed control) to 22%(29μg/mL methoxychlor)and the significant differences in comparison to the unexposed control groups were seen also at concentrations of 14.5μg/mL methoxychlor (44.4%) and at 7.25μg/mL methoxychlor (52.8%). 2,Oocytes matured in the presence of 29μg/mL methoxychlor, displayed a first meiotic spindle smaller than that observed in the control experiment. 3,The percentage of a cytoplasm Cortical granule monolayer beneath the oolemma observed with LSCM in MⅡstage oocytes in experimental groups was not lower than those in control groups. 4,The degree of cumulus expansion was also influenced by methoxychlor: the higher the concentration of methoxychlor, the lower the intensity of cumulus expansion.CONCLUSION: 1,The toxicity effect of methoxychlor is inhibited maturation of oocyte in vitro , caused significant decrease of nucleus maturation rate. The administration of MXC, before and during pregnancy reduces the number of pregnancies and resulted infertilization. 2,Methoxychlor could affect oocytes maturation directly through altering the formation of the meiotic spindle .3,Methoxychlor could influence postfertilization development through inhibited intensity of cumulus expansion, but it is affected the distribution of CG non-significantly.Part 2 the toxicity of Methoxychlor on in vitro fertilization of mouse oocytes and on mouse embryos development METHODS: 1,The oocytes matured under 7.25 and 14.5μg/mL methoxychlor medium were fertilized in vitro(IVF) to examine the capacity of undergoing fertilization. 2,The obtained embryos were cultured in CZB medium, and the following changes were observed under microscope everyday. embryos were evaluated for the portion developing to 2-cell stage embryos, 4-cell stage embryos,8-cell stage embryos,morula and blastocyst, hatched blastocysts byχ2. 3,The total cell number of blastocyst was determined.RESULTS:1,There were no significant differences in fertilization, 2-cell rate, 4-cell rate,8-cell rate between 7.25μg/mL methoxychlor and control(P>0.05). There were no significant differences in fertilization, 2-cell rate and 4-cell rate between control and 14.5μg/mL methoxychlor treated group, but the number of 8-cell stage was significantly lower in methoxychlor than in the control group. 2,Compared with untreated controls, 7.25μg/mL methoxychlor significantly inhibited the rate of blastocysts and blastocyst hatching(P<0.05). 3,Cell numbers in developed blastocysts after treatment with methoxychlor at a concentration of 7.25 g/mL were not lower than control group.CONCLUSION:1 Fertilization and cleavage after in vitro maturation in the presence of 7.25μg/mL methoxychlor was not reduced in comparison to the unexposed control, methoxychlor also didn't suppressed embryonic development to morulae and blastocyst.2 Methoxychlor could inhibit blastocysts hatching, methoxychlor effect was possibly due to cytotoxicity and induction of apoptosis of preimplantation embryos.1,Methoxychlor significantly inhibited the rate of blastocysts and blastocyst hatching although it could not affect fertilization rate and 2-cell rate. It is established that the rate of blastocyst hatching decreased significantly, and then resulted in early pregnancy loss and adverse pregnancy results.