IMPROVED DEVELOPMENT OF RABBIT ONE-CELL EMBRYOS TO THE HATCHING BLASTOCYST STAGE BY CULTURE IN A DEFINED, PROTEIN-FREE CULTURE-MEDIUM
IMPROVED DEVELOPMENT OF RABBIT ONE-CELL EMBRYOS TO THE HATCHING BLASTOCYST STAGE BY CULTURE IN A DEFINED, PROTEIN-FREE CULTURE-MEDIUM
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DOI：
10.1530/jrf.0.0910113
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发表时间：
1991-01-01
期刊：
影响因子：
--
通讯作者：
FOOTE, RH
中科院分区：
文献类型：
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作者：
CARNEY, EW;FOOTE, RH
在Exp. 1、培养基199和培养基RD（RPMI-1640和Dulbecco's MEM，1：1 v/v）以2 x 2析因设计进行比较，分别补充15 mg牛血清白蛋白（BSA）/ml或1 mg聚乙烯醇（PVA）/ml。 所有培养基含有5 μ g胰岛素/ml、5 μ g转铁蛋白/ml、5 ng硒/ml（ITS）和10 ng表皮生长因子（EGF）/ml。 将单细胞胚胎在39 ℃、5%CO2的空气中培养65小时，然后用Hoechst 33342染色以确定卵裂球数。 当使用PVA代替BSA时，在培养基199中的胚胎发育较差（P < 0.001）（30 vs 76个细胞/胚胎），但在含有PVA或BSA的培养基RD中发育迅速（118和121个细胞）。 在Exp. 2在无BSA和PVA的培养基中。 在Exp. 3.在不添加蛋白质的RD培养液中培养65 h后的1-细胞胚胎发育率（囊胚率68%，117个细胞）与单独添加胰岛素、ITS或EGF的RD培养液中的1-细胞胚胎发育率无显著差异（P > 0.37）。 在无蛋白培养基RD中培养96 h，82%的1细胞胚胎形成囊胚，40%的1细胞胚胎通过透明膜孵化。 在活力的初步试验中，在该培养基中培养48或65 h并转移至同步受体的1细胞胚胎在第15天产生5/18（28%）和3/24（12%）活胎。 在RD培养基中培养1细胞胚胎65 h后，每个囊胚的细胞计数约为120个，表明细胞分裂比本实验室先前检测的所有其他培养基更快。 这是第一个报告的兔胚胎发育从1-细胞孵化囊胚期在一个确定的蛋白质无培养基。
In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml or 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5-mu-g insulin/ml, 5-mu-g transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39-degrees-C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P < 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P > 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.