61 The role of CIDEA and dynamics of lipid droplets in bovine embryo development

Lipid droplets (LDs) in mammalian embryos function as an energy source for proper embryonic development, but little attention has been focused on the mechanisms of their dynamics and motion in early embryogenesis of domestic species. CIDEA, the cell death-inducing DNA fragmentation factor α-like effector domain-containing protein, is targeted to LDs in mouse adipocytes, where it promotes lipid storage. Moreover, Mau et al. (2022 Nat. Commun. 13, 3861) described how CIDEA promotes enlargement of LDs in mouse blastocysts through fusion. In our timelapse observations, LDs in live bovine embryos remained mobile and formed larger particles in the first few rounds of cell division. Thus, we hypothesized that CIDEA has a conserved function of LD enlargement in bovine early embryogenesis. Immunofluorescence and Fiji were used to analyze the size and number of LDs. Non-normally distributed data such as LD size were analyzed by two-sided Mann-Whitney U-test. Statistical differences were annotated with exact P-values: *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001. First, we profiled the size and numbers of LDs in whole bovine negative control (Ng) IVM/IVF embryos at 2- to 16-cell stages. The findings suggested that the size of LDs increased at the 2- to 8- cell stages, while the numbers conversely decreased. To understand the relation of CIDEA and LD dynamics, 1 mM morpholinos (Mor) and 25 μM siRNAs (si) were injected at the 1-cell stage to knock down (KD) CIDEA expression. Comparisons of relative CIDEA expression between Mor-Ng and Mor-KD embryos at 2-, 4-, 8-, and 16-cell stages were 1.00 versus 0.77 (**), 1.01 versus 0.91 (P = 0.29), 1.18 versus 0.98 (P = 0.19), and 0.76 versus 0.63 (P = 0.28). LD size comparisons of Mor-Ng and Mor-KD embryos at 2-, 4-, 8-, and 16-cell stages were 3.38 versus 2.67 μm (***), 3.37 versus 2.96 μm (****), 6.44 versus 2.47 μm (****), and 5.78 versus 5.28 μm (P = 0.069). Cleavage rates (54.5 ± 6.3% vs. 55.0 ± 6.5%) were not different, but blastocyst rates were impaired in the Mor-KD group (35.4 ± 4.3% vs. 2.40 ± 0.9%). Comparisons of relative CIDEA expression in si-Ng and si-KD embryos at 2-, 4-, 8-, and 16-cell stages were 1.00 versus 0.98 (P = 0.76), 1.04 versus 0.99 (P = 0.76), 1.14 versus 1.05 (P = 0.76), and 0.87 versus 0.90 (P = 0.76). LD size comparisons in si-Ng and si-KD embryos at 2-, 4-, 8-, and 16-cell stages were 2.42 versus 3.10 μm (****), 3.06 versus 1.20 μm (****), 4.60 versus 1.91 μm (****), and 3.00 versus 2.31 μm (*). No differences were observed in cleavage rates (75.1 ± 2.8% vs. 74.4 ± 6.0%) and blastocyst rates (1.7 ± 1.7% vs. 0.6 ± 0.6%) between si-Ng and si-KD. In the CIDEA induction experiment, 25 μM of trans-chalcone (TC) was supplemented in IVC culture medium and 0.1% DMSO as a vehicle control (VC) after denuding. Comparisons of relative CIDEA fluorescence intensity in VC and TC embryos at 2-, 4-, 8-, and 16-cell stages were 1.00 versus 0.86 (P = 0.06), 0.96 versus 0.80 (**), 0.92 versus 0.90 (P = 0.48), and 1.15 verus 1.01 (P = 0.06). LD size comparisons of VC and TC embryos at 2-, 4-, 8-, and 16-cell stages were 1.50 verus 4.80 μm (****), 4.25 versus 3.40 μm (****), 2.50 versus 3.80 μm (****), and 2.30 versus 4.65 μm (****). No differences were observed in cleavage rates (91.6 ± 3.1% vs. 93.0 ± 1.2%) or blastocyst rates (30.1 ± 3.0% vs. 30.4 ± 1.1%) between VC and TC. Data from this study demonstrate that LDs start to aggregate at fertilization and reach their maximum size at the 8-cell stage. The study also shows that CIDEA and enlargement of LDs are essential for bovine blastocyst formation.