National Institute of Animal Nutrition and Physiology, Hosur Road, Adugodi, Bangalore 560 030, India.
Arch Environ Contam Toxicol. 2010 Jan;58(1):194-204. doi: 10.1007/s00244-009-9342-7. Epub 2009 May 28.
The aim of the present study was to examine the effect of heavy metals, cadmium and lead, on buffalo oocyte viability and in vitro development. Oocytes were aspirated from ovaries of slaughtered buffaloes. Only viable and metabolically active oocytes with more than three layers of cumulus cell layers and homogeneous ooplasm were selected. Effects of nine concentrations (0, 0.005, 0.05, 0.5, 1.0, 1.5, 2.5, 5, and 10 microg/mL) of cadmium or lead on buffalo oocyte viability, morphological abnormities, maturation, and embryonic development in vitro were studied. Oocytes were cultured for 24 h and then checked for viability (0.05% trypan blue staining for 2 min), morphological abnormalities, and reduction assay by MTT test in experiment 1. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were determined (experiment 2). In experiment 3, viable oocytes were matured in vitro in media containing different levels of cadmium or lead and then inseminated in vitro with frozen-thawed spermatozoa, and the resultant cleaved embryos were cultured in a control embryo culture medium for 8 days. In experiment 4, oocytes were cultured in control oocyte maturation medium, then fertilized, and the resultant embryos were cultured in media containing different levels of cadmium or lead for 8 days. The number of cells in the trophectoderm and inner cell mass (ICM) and the total cell counts (TCN) of blastocysts derived by in vitro culture of two- to four-cell-stage embryos (produced in control medium) in media containing 0, 0.005, 0.05, 0.5, and 1.0 microg/mL of cadmium or lead were analyzed by differential staining technique (experiment 5). Cadmium and lead were found to have a dose-dependent effect on viability, morphological abnormities, maturation, cleavage and morula/blastocyst yield, and blastocyst hatching. A significant decline in viability of oocytes was observed at 1.0 mg/mL cadmium or lead compared to the control group. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were 18 and 32 microg/mL, respectively. Cadmium and lead at 1.0 and 2.5 microg/mL, respectively, caused a significant reduction of maturation of oocytes compared to the lower concentrations. No cleavage or morulae/blastocysts were produced when the oocytes/embryos were cultured in media containing 2.5 and 5.0 mg/mL of either cadmium or lead, respectively. Similarly, no morulae/blastocysts were produced from cleaved embryos cultured in media containing 2.5 and 5.0 microg/mL cadmium and lead, respectively. The developmental block, degeneration, and asynchronous divisions were higher in embryos exposed to cadmium than in those exposed to lead. TCN and number of cells in ICM were significantly lower in blastocysts derived from two- to four-cell-stage embryos cultured in media containing heavy metals. In conclusion, cadmium and lead lowered the viability and development of buffalo oocytes but at a concentration higher than that estimated in the body fluids and environment. Cadmium was found to be more ovotoxic than lead.
本研究旨在探讨重金属镉和铅对水牛卵母细胞活力和体外发育的影响。卵母细胞从屠宰水牛的卵巢中抽吸出来。只有具有超过三层卵丘细胞层和同质卵质的有活力和代谢活跃的卵母细胞才被选中。研究了九种浓度（0、0.005、0.05、0.5、1.0、1.5、2.5、5 和 10μg/mL）的镉或铅对水牛卵母细胞活力、形态异常、成熟和体外胚胎发育的影响。卵母细胞培养 24 小时，然后用 0.05%台盼蓝染色 2 分钟检查活力，用 MTT 试验进行形态异常和还原测定（实验 1）。确定导致 100%卵母细胞死亡（1 天培养）的镉和铅剂量（实验 2）。在实验 3 中，将有活力的卵母细胞在含有不同水平镉或铅的培养基中体外成熟，然后用冷冻解冻的精子体外受精，并将所得的分裂胚胎在对照胚胎培养基中培养 8 天。在实验 4 中，卵母细胞在对照卵母细胞成熟培养基中培养，然后受精，将所得胚胎在含有不同水平镉或铅的培养基中培养 8 天。通过差异染色技术分析在含有 0、0.005、0.05、0.5 和 1.0μg/mL 镉或铅的培养基中由两到四细胞期胚胎（在对照培养基中产生）体外培养的桑椹胚和囊胚的滋养外胚层和内细胞团（ICM）中的细胞数和总细胞数（TCN）（实验 5）。发现镉和铅对活力、形态异常、成熟、分裂和桑椹胚/囊胚产量以及囊胚孵化有剂量依赖性影响。与对照组相比，在 1.0mg/mL 镉或铅下观察到卵母细胞活力显著下降。导致 100%卵母细胞死亡（1 天培养）的镉和铅剂量分别为 18 和 32μg/mL。与较低浓度相比，镉和铅分别在 1.0 和 2.5μg/mL 时导致卵母细胞成熟显著减少。当卵母细胞/胚胎在含有 2.5 和 5.0mg/mL 镉或铅的培养基中培养时，均未产生分裂或桑椹胚/囊胚。同样，当在含有 2.5 和 5.0μg/mL 镉的培养基中培养分裂胚胎时，也未产生桑椹胚/囊胚。暴露于镉的胚胎中的发育阻滞、退化和非同步分裂高于暴露于铅的胚胎。在含有重金属的培养基中培养的两到四细胞期胚胎衍生的囊胚中的 TCN 和 ICM 中的细胞数显著降低。总之，镉和铅降低了水牛卵母细胞的活力和发育，但浓度高于估计在体液和环境中的浓度。与铅相比，镉被发现更具卵毒性。
