178 IN VITRO CULTURE OF BOVINE EMBRYOS WITH NEUROTROPHINS

Neurotrophins (NTs) mediate human embryonic stem (hES) cell survival and may also improve methods for hES cell derivation (Pyle et al. 2006 Nature Biotech. 24, 344–350) and quality of the inner cell mass (ICM). We searched published microarray data sets for tyrosine kinase receptors (TRK) (geo data base: GSM27469, GSM27470, GSM27471). The analysis suggested that bovine embryos in vitro at unspecified stages express TRKA, for nerve growth factor (NGF); TRKC, for neurotrophin-3 (NT3); and TRKB, for both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF). NTs functionally cooperate among them and also with basic fibroblast growth factor (bFGF) (Pyle et al. 2006; Logan et al. 2006 Brain 129, 490–502). Experiments in progress include detection of TRK expression by RT-PCR at defined development stages, and analysis of embryonic development with NTs and without bFGF. In this work we cultured embryos matured and fertilized in vitro from slaughterhouse oocytes for 8 days in SOF medium with 6 g L-1 BSA and 2 ng mL-1 bFGF (negative control). Development was monitored and cells were differentially counted in the ICM and trophectoderm (TE) of expanded and hatched blastocysts. NTs were used during the whole culture at 20 ng mL-1 as single (4 experimental groups: NGF, NT3, NT4, and BDNF) or as pooled (1 group) NT compounds. Data (5 replicates; 1403 oocytes) were processed by GLM and Duncan's test, and expressed as LSM � SE (a,b: P < 0.05). At Day 3, no differences were found at the 5- to 8-cell stage, but NT3 and NT4 increased the proportions of embryos at the 8- to 16-cell stage (19.1 � 2.2 and 20.5 � 2.2, respectively, vs. 12.9 � 2.2 to 13.7 � 2.2 within the other groups). On Day 6, NT4 yielded more morulae than controls, BDNF, and NGF (35.3 � 2.7 vs. 26.1 � 2.7, 27.4 � 2.7, and 27.8 � 2.7, respectively), and did not differ from the other groups. NT4 produced more total Day 7 blastocysts than NT3 and BDNF (12.5 � 2.2 vs. 8.1 � 2.2 and 9.9 � 2.2, respectively), whereas there were no differences within medium and expanded blastocysts and Day 8 blastocysts. Proportions of morulae that formed blastocysts were appreciably lower than in concomitant experiments without bFGF. Pooled NTs showed decreased values as compared to some single NTs within the ICM [13.0 � 4.0 vs. 29.1 � 4.6 (NT3) and 24.9 � 4.3 (NGF)], the TE [89.0 � 8.4 vs. 120 � 11.9 (BDNF)], total cells [102.0 � 8.5 vs. 134.0 � 9.9 (NT3), and 140.0 � 12.1 (BDNF)], and tended to differ (P = 0.08) within ICM/total cells [13.1 � 3.1 vs. 21.6 � 3.6 (controls) and 22.2 � 3.6 (NT3)]. Controls differed from BDNF (TE: 88.1 � 9.8 vs. 120.2 � 11.9; total cells: 110.8 � 10.0 vs. 140.0 � 12.1, respectively), and from NT4 for ICM/total cells (21.6 � 3.6 vs. 11.5 � 2.9, respectively). NT4 is likely to exert a role during early embryonic development. However, these blastocysts showed decreased cell counts in the ICM, probably reflected in the pooled NTs group. Targeting proliferation stimuli specifically to the ICM is difficult to get when the ICM is enclosed in the embryo, in contrast with the isolated ICM or the derived stem cells. This work was supported by Grant AGL2005-04479.