The Impact of Ethylene Glycol Monomethyl Ether on Ovarian Function May Extend to the Next Generation in Female Mice: A Preliminary Study
Date Issued
2012
Date
2012
Author(s)
Weng, Shao-Ping
Abstract
Background and objectives: Environmental hazards, such as ethylene glycol monomethyl ether (EGME) and numerous new chemicals cannot be excluded from the impact on the reproductive function. To date, there are guidelines, based on in-vivo animal studies, for reproductive toxicological tests. However, those studies were mostly focused on developmental results such as teratogenicity. Much less molecular information related to fertility had been gained. So far, no sufficient knowledge from in-vitro models can pinpoint the mechanism of hazards on reproduction system. Therefore, within the frame of reproductive toxicology in females, there is an urgent necessity for toxicity-testing systems which are prognostic at the early stage, provide a better approach into the mechanisms leading to reproductive failure, and detect quantitatively and qualitatively the toxic damage to the process of oogenesis. After the animal experiment of reproductive toxicity, we tried to introduce the preimplantation genetic diagnosis (PGD) technology in in-vitro fertilization (IVF) system to clarify the molecular impact of EGME. Thereafter, we established the PGD system by evaluating the patterns of chromosome abnormalities in embryos that derived from intracytoplasmic sperm injection (ICSI) in microsurgical epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) patients in comparison to embryos derived from naturally ejaculated patients (EJAC). Those sperm from severe male factors may be damaged by some reasons, including environmental hazards, before fertilization. Thus, the main aim of this study is to explore the effects of EGME on reproductive functions by means of in-vitro culture and fertilization systems. Then, PGD was performed in severe male infertility.
Materials and Methods: First, this study assessed the toxicity of EGME on oocytes and cumulus-oocyte-complexes (COCs) by analyzing the number of oocytes in the F0 and F1 generations and evaluating apoptosis in oocytes and COCs after treating the F0 generation with EGME. There was a dose-dependent increase in the apoptosis ratios in the COCs from F1 mice, which were not directly exposed to EGME, with apoptosis ratios of 0.065, 0.102, 0.184, and 0.212 for the 0%, 0.05%, 0.1%, and 0.2% EGME dose groups, respectively. The retrieved COCs and denuded oocytes were prepared for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. As a negative control, the cells were labeling solution without the TdT enzyme; as a positive control, the cells were treated with DNase I. Blue fluorescent nuclei were observed in non-apoptotic cells that were only stained with 4'',6-diamidino-2-phenylindole (DAPI). Greenish-blue nuclei were observed in apoptotic cells stained with fluorescein in the TUNEL assay. Second, PGD was performed by fluorescence in situ hybridization (FISH). Chromosome abnormalities were categorized into polyploidy, haploidy, aneuploidy, and complex abnormality, which involved more than two chromosomes. The relationship between the embryo scoring system, combining embryo grading and blastomere numbers, and chromosomal were studied.
Results: All of the mice gave birth. There were no significant differences among these groups; however, the variance in pup number among the mice was much higher in the highest dose (0.2%) group compared to the other groups. The apoptosis ratios in the F1 animals, which were not directly exposed to EGME, were significantly different (p=0.002) among the four groups and showed dose-dependent increases. In the PGD study, there was no difference in the rates of fertilization and pregnancy and the percentages of euploid embryos among the MESA, TESE, and EJAC patient groups. In all three groups, less than half of the embryos that were analyzed by PGD were normal (41 ± 31%, 48 ± 38%, and 48 ± 31%, in MESA, TESA, and EJAC, respectively). Complex chromosomal abnormality was significantly more frequent in the MESA group than in the EJAC group (48.3% versus 26.5%, p<0.001). When both blastomere numbers and grades were considered, the 7–8 A/B subgroup had the highest euploidy rates in the MESA/TESE (58.3%), ICSI/EJAC (61.3%) and IVF/EJAC (59.7%) groups.
Conclusion: The increased apoptosis of cumulus cells may play a role in the toxicity of EGME toward ovarian function. EGME toxicity seems to affect female offspring in future generation(s). This study model for reproductive toxicology appears useful in diagnosing environmental hazards but further studies including embryo transfer should be considered to bring out more solid results. PGD seems applicable to explore the molecular damage of environmental chemicals. However, advanced technology, such as array comparative genomic hybridization, for whole genome survey should be considered instead of FISH.
Subjects
environmental toxicant
germ cell
reproductive toxicology
ethylene glycol monomethyl ether
apoptosis
preimplantation genetic diagnosis
male infertility
Type
thesis
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