33258 (1 pg/ml) for 15 min in the dark. Embryos were washed three times in PBS-PVP

to remove excess Hoechst 33258, mounted on 10% (w/v) poly-L-lysine coated slides

using 3- to 4-pl drops of glycerol, and the slides affixed with coverslips. Labeling of

TUNEL and Hoechst 33258 nuclei was observed using a Zeiss Axioplan 2

epifluorescence microscope (Zeiss, Gottingen, Germany). Each embryo was analyzed for

total cell number (blue nuclei) and TUNEL-positive blastomeres (green nuclei) with

DAPI and FITC filters, respectively, using a 20x objective.

Differential Staining

 Zona-intact embryos were removed from culture and washed 3 times in 50 [tL

drops of PBS-PVP. To label trophectoderm cells (TE), embryos were placed into 500 [tL

of PBS-PVP containing 0.5% Triton X-100 and 100 [tg/mL propidium iodide for 30 s at

37C. Embryos were then washed immediately through 3 wells of a 4-well plate

containing 500 tL of PBS-PVP each. To fix embryos and stain inner cell mass cells

(ICM), embryos were then incubated in a 50 tL drop of PBS-PVP containing 4%

paraformaldehyde and 10 tg/mL Hoechst 33258 for 15 min at room temperature.

Embryos were then washed three times in PBS-PVP, mounted on 10% (w/v) poly-L-

lysine coated slides using 3- to 4-.l drops of glycerol, and then covered with coverslips.

Labeling of propidium iodide and Hoechst 33258 nuclei was observed using a Zeiss

Axioplan 2 epifluorescence microscope (Zeiss, Gottingen, Germany). Each embryo was

analyzed for the number of ICM (blue nuclei), the number of TE cells (pink nuclei), and

total cell number (blue + pink nuclei) with a DAPI filter using a 20x objective.

RT-PCR

 The relative abundance of 14 gene transcripts was determined using semi-

quantitative RT-PCR as described previously (Wrenzycki et al., 2001b). Primer