PMC

Confocal immunofluorescence shows Cdx2 expression (pink) in outer cells of morulae and in Tr cells of wild-type blastocysts. In contrast, Nanog (red) was clearly detected in a subset of inner cells of morulae and primarily in the ICM of blastocysts. While Oct3/4 (green) was detected in all cells of wild-type morulae (D3.5) and early blastocysts (D4), the expression was downregulated in Tr cells, with sustained expression in ICM cells in late expanded blastocysts (D4.25). In contrast, the expression patterns of Cdx2, Nanog, and Oct3/4 in Faah^–/– embryos were aberrant. Unlike in wild-type embryos, Cdx2 expression was barely detected in Faah^–/– morulae, but the expression in asynchronously developing mutant morulae and blastocysts on day 4 morning was comparable to that of wild-type embryos. While the expression of Nanog and Oct3/4 was observed in most cells in Faah^–/– morulae, it was selectively downregulated in ICM cells, with sustained higher levels in Tr cells in mutant blastocysts in a stage-dependent manner (scale bar: 25 μm).

Supplementation with l-carnitine enhanced the OCRs of morulae. Thirty-eight 6–8-cell stage embryos, two from each 19 couples were cultured for 18 h without (n = 19) or with l-carnitine (LC; 1 mM; n = 19). Supplementation increased the mtOCRs of morulae (1.18 ± 0.13 fmol/s) compared with their control morulae (1.09 ± 0.1 fmol/s; P = 0.02.) (a). The maxOCRs of morulae cultured in l-carnitine were also improved but the difference did not reach statistical significance (3.07 ± 0.36 vs. 2.93 ± 0.23 fmol/s, respectively) (b). The OCRs without any mitotoxins of morulae cultured in l-carnitine were improved (P = 0.029, 3.95 ± 0.12 vs. 3.86 ± 0.11 fmol/s, respectively) (c1). l-carnitine did not affect the OCRs with FCCP of morulae (P = 0.155; with LC: 5.84 ± 0.35 vs. without LC: 5.7 ± 0.25 fmol/s, respectively) (c2) and the OCRs with cyanide of morulae (P = 0.06, 2.77 ± 0.03 vs. 2.77 ± 0.03 fmol/s, respectively) (c3)

Stereomicroscopic images of fresh and vitrified–warmed blastocysts (A–D) and morulae (A’–D’). (A,A’) Fresh blastocysts and morulae before vitrification. (B,B’) Blastocysts and morulae immediately after warming. (C,C’) Vitrified-warmed blastocysts and morulae after 24 h of in vitro culture. (D,D’) Vitrified-warmed blastocysts and morulae after 48 h of in vitro culture.