A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid–free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus®PicoPure®RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM® 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker® Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P < 0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P < 0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.
Heat Shock-derived Reactive Oxygen Species Induce Embryonic Mortality in In Vitro Early Stage Bovine Embryos
