Preliminary Study On Lipid Metabolism In The Early Embryonic Developmental Stages Of Pig

Lipid droplets(LDs),as a lipid storage organelle,are widely existed in various kinds of cells.Porcine oocytes had a large number of LDs,which were important endogenous energy substance,with the energy provided by lipid metabolism for embryonic development.Studies showed that the PAT family proteinsare one kind of LDs surface proteins,which were mainlyrelated to the metabolism of LDs.LDs are not only closely linked tomitochondria,endoplasmic reticulum or other organelles,but alsoparticipate in a variety of cell activities.Therefore,it is necessary to investigate the LDs metabolism in pig embryos to provide more knowledge and further to improve the efficiency of embryo cryopreservation.The full text was carried out through the following three experiments:1 The affection of delipation on the fatty acid oxidation in pig embryosThis study was designed to measure the effect of delipation on the developmental viability of pig parthenogenetically activated(PA)embryos.Our results showed that no effect was observed on the cleavage after delipation,but a significantly decrease was observed on the blastocyst rate and cell number per blastocyst(P<0.05).The level of ROS in 1-cell and 4-cell embryos(P<0.001,P<0.05)was obviously increased,but no difference was observedat blastocyst stage.The expression level of genes related to fatty acids beta-oxidation(Acsl4,Acsl5,Cpt2,Acadl,Acaa2,P<0.05;Cpt1a,Cpt1b,Acadsb,P<0.01)were decreasedin 4-cell,while the expression level of genes related to glucose metabolism(Glut 1,P<0.05)was upregulated after delipation.After being treated with Etomoxir during embryos culture,the developmental viability of porcine embryos decreased both in delipated and normal pig embryos.This study indicated that both beta-oxidation and glucose metabolism was affected by the delipation of pig oocytes.2 The study of PAT family in pig oocytes and embryosFirstly,PAT family expressing pattern was checked in porcine oocytes.Our results not only confirmed the existence of PLIN2 and PLIN3,but also firstly showed the expression of PLIN1 and PLIN5 in pig oocytes,but PLIN4 was non-detectable in pig oocytes.Then the dynamic change of PATs in pigembryo during preimplantation development was investigated as the following experiments.After in vitro maturation of oocytes,the expression level of PLIN2,PLIN3 and PLIN5 decreased(P<0.01,P<0.01,P<0.001),while no change of PLIN1 expression was observed.For the parthenognetic activated embryos,transcripts of PLIN1,PLIN2 and PLIN5 decreased from 1-cell to 4-cell stage(P<0.05),but the transcript of PLIN3 kept invariant between 1-cell and 4-cell stages.Subsequent from 4-cell to blastocyst stages,transcripts of PLIN1 and PLIN3 were further decreased(P<0.05)but an obvious rise of PLIN2 was observed(P<0.05).By immunocytochemistry method,the presence of PLIN2 and PLIN3 was checked in Day 6 blastocysts,results showed that PLIN2 was around colocalized with large lipid droplets and PLIN3 was freely drstributed in cytoplasm.In conclusion,transcripts and proteins of PATs are firstly studied in very detailed in pig oocytes and embryos.3 Reconstruction of pig embryos with different lipidsEmbryos with different LDs were obtained by hand-made cloning(HMC)and delipation:W-L:whole LDs in embryos,H-L:half LDs in embryos,R-L:removed LDs in embryos.Subsequently,the developmental competence of these reconstructed embryos was assessed.Results showed that all of these three kinds of embryos could develop into blastocyst efficiencyly.To study connection pattern of LDs and mitochondria in pig early embryos,the H-L embryos were fused with one of the cytoplasm stained by MitoTracker Red CMXRos,while another half of karyocytst without staining.Following,1-cell(12h),2-cell(24h),4-cell(36h)and blastocyst(144h)were collected to observe the distribution of LDs and mitochondria.Results showed that LDs and mitochondria were successfully tracked from 1-cell to blastocyst stage.At 1-cell stage,both LDs and mitochondria were only visible in normal cytoplasm.At 2-cell stage,mitochondria were uniformly distributed in the cytoplasm in 28%of the total number.But the LDs are still not evenly distributed.However,both LDs and mitochondria were dispersed fully at 4-cell and blastocyst stage.In conclusion,with HMC and delipation,it is possible to reconstruct pig embryos with different LDs,which might be useful for lipid metabolism study and further to investigate the interaction among lipids,mitochondria and other organelles.