Fertilization in vitro of mouse ova from inbred and outbred strains: Complete preimplantation embryo development in glucose-supplemented KSOM

Abstract

A new medium derived from the use of sequential simplex optimization methods (SOM) that overcomes the block to development beyond two cells in vitro in embryos of the CF1-cultured strain of mouse has recently been described. Contrary to previous reports, glucose was shown to have no significant inhibiting effect on embryo development to the blastocyst stage in SOM. A modification of SOM, designated KSOM, with an increased concentration of Na+ (95 mM) and K+ (2.5 mM), which has been described elsewhere, also supports growth beyond the two-cell block. KSOM produces a higher rate of compaction, a larger yield of blastocysts, and an increased rate of cell division of the trophoblast cells. We have reexamined the glucose effect by varying the concentration of glucose (either 0.2 mM or 5.56 mM) in KSOM and determined the ability of these media to support preimplantation development of CF1 ♀ x B6D2F1 ♂ zygotes through the blastocyst stage. Glucose is shown to have no significant inhibiting effect on development to the blastocyst stage. The yield of blastocysts is typically 85%-90%. A modification of KSOM derived from this study, designated modified KSOM, with an increased concentration of glucose (5.56 mM) and supplemented with 4 mg ml-1 BSA is now shown to support high rates of fertilization in vitro of CF1 ova with hybrid B6D2F1/CrIBR sperm and subsequent development of zygotes beyond the two-cell stage to blastocysts in high yield. A single medium, modified KSOM, thus supports both fertilization in vitro of CF1 ova and complete preimplantation embryo development without the need for a change of medium to overcome the two-cell block. The overall yield of blastocysts from CF1 ova is 80%-85%. Embryo transfer experiments at the pronuclear, two cell, and early blastocyst stages gave high rates of implantation and live fetuses from in vitro-fertilized CF1 oocytes. The general utility of modified KSOM for the fertilization of mouse ova in vitro is confirmed by obtaining high rates of fertilization in vitro and blastocyst development of two F1 hybrid strains: B6D2F1/J(C57BL/6J♀ x DBA/2J♂) and B6CBAF1/J(C57BL/6J♀ x CBA/J♂).