Hatoya S, Sugiyama Y, Torii R, Wijewardana V, Kumagai D, Sugiura K, Kida K, Kawate N, Tamada H, Sawada T, Inaba T
Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Theriogenology. 2006 Sep 15;66(5):1083-90. doi: 10.1016/j.theriogenology.2005.12.015. Epub 2006 Apr 18.
We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.
我们研究了小鼠胚胎成纤维细胞（MEF）和犬胚胎成纤维细胞（CEF）对犬卵母细胞体外成熟（IVM）、体外受精（IVF）和体外培养（IVC）的影响。通过切片从卵巢中采集卵丘-卵母细胞复合体，并在三种不同条件下评估体外成熟情况：仅培养基培养（对照）、与MEF共培养或与CEF共培养。将卵母细胞培养48或72小时。仅使用直径大于100微米、细胞质均匀深色且有两层或更多层卵丘细胞的卵母细胞。培养基为添加10%胎牛血清（FBS）、100 IU/mL青霉素和100微克/毫升链霉素的TCM 199。IVM 48小时后，用通过上浮法筛选的新鲜犬精子对卵母细胞进行体外受精，并将卵母细胞和精子在改良的 Krebs-Ringer 碳酸氢盐溶液（TYH）中于38.5℃、5%二氧化碳的空气中共培养长达20小时。受精后，将卵母细胞转移至三种不同条件（与IVM相同）并进行培养。体外成熟48或72小时后，在MEF和CEF共培养中培养的MII期卵母细胞的成熟率高于对照培养的卵母细胞（P<0.05）。尽管在48小时和72小时培养中达到MII期的比率没有差异，但在所有三个处理组中，72小时时退化卵母细胞的百分比更高。三个处理组中单精受精和多精受精卵母细胞的比例没有差异。MEF和CEF处理组的卵裂率高于对照组（P<0.05）。与CEF共培养可使胚胎发育至16细胞期，与MEF共培养可使胚胎发育至桑葚胚期。总之，胚胎成纤维细胞共培养增强了犬卵母细胞的核成熟和细胞质成熟。
