The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development.

This study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 μM cysteamine (C+C); 100 IU catalase (CAT) or 100 μM β-mercaptoethanol (β-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66-100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6-48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6-93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.

This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

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SummaryWe examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P < 0.05) in the embryonic development rate (Control: 25.8% versus LA: 18.5%), but the proposed system was effective in promoting the decrease (P = 0.0130) in the intracellular lipid content (Control: 27.3 ± 0.7 versus LA: 24.6 ± 0.7 arbitrary fluorescence units of embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming (Control: 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.