Effect And Mechanism In Pig Parthenogenetically Activated Embryos After Delipation

Lipid droplet(LD)was important for oocyte maturation and embryonic development as the energy source,on the other hand,the LD content of pig oocytes through pre-hatching blastocysts is particularly large,while the physical damage caused by LD during cryopreservation leads to very low embryo survival rates.Several studies have reported that the delipation of pig embryos improves their survival rate with the non-affecting blastocyst formation and the total cell numbers.But others reported the delipation for cryopreservation of pig embryos with the reduced the developmental viability.However,these studies only assessed the viability of delipated embryos,and moreover very few.The present study was designed not only to measure the effect of delipation on the developmental viability of pig parthenogenetically activated(PA)embryos,but also to investigate the distribution pattern of LDs,mitochondria and endoplasmic reticulum,mitochondria DNA(mtDNA)copy number and adenosine triphosphate(ATP)content at different developmental stages after delipation.The brief procedure was as described the following that the matured oocytes were delipated by centrifugation after partial digestion of the zonae pellucidae,subsequently,they were subjected to parthenogenetic activation after total removal of zonae pellucidae by pronase,and then embryos were cultured in vitro in WOWs up to the blastocyst stage.1.Developmental competence of PA embryos was compromised after delipationThe in vitro developmental competence of the delipated and control oocytes was assessed by determining the percentage of embryos reaching the 2-cell(24 hours after PA),4-cell(36 hours after PA),and blastocyst(144 hours after PA)stages.To determine the total cell number,Day 6 blastocysts were randomly collected and stained with 10 mg/ml Hoechst 33342 for 10 min.Although the cleavage rate at the 2-and 4-cell stages of the PA embryos was not affected by delipation(95.83±2.25%vs.97.44±0.67%;79.17±4.47%vs.84.62±1.19%),the blastocyst rate was reduced significantly compared to the controls(21.67±3.78%vs.49.36±1.77%),but no significant difference was seen in the total number of cells per blastocyst(37.92 vs.41.88,respectively).2.LD distribution and amount in pig PA delipated embryos.MⅡ oocytes and PA embryos(1-cell,2-cell,4-cell,and blastocyst stages)were collected and fixed with 4%paraformaldehyde and then were stained with Oil Red O(ORO).Immediately after delipation,LD contents were significantly reduced with only smaller LDs remained in the delipated oocytes and were polarized to one side of the cytoplasm.After PA,the distribution pattern of LDs in the delipated embryos differed to the pattern of LDs in control embryos.At the early developmental stages(1-to 4-cell stages)in delipated embryos,they were distributed peripherally and formed a ring around the nucleus.However,by the blastocyst stage,homogeneous distribution of LDs was observed in both the inner cell mass and trophectoderm.Althoughthe content of LDs remained constant from MⅡ oocyte to the blastocyst stage in delipated embryos,but size of LDs became gradually smaller with the embryos development.3.Mitochondrial distribution and numbers in pig PA delipated embryos.MⅡ oocytes and PA embryos(1-cell,2-cell,4-cell,and blastocyst stages)were collected for staining with MitoTracker Red CMXRos(MTR)and then were fixed with 4%paraformaldehyde.Immediately after delipation,the mitochondria were partially polarized toward the peri vitelline space along with the LDs.The same distribution pattern of mitochondria was observed both in delipated and in control embryos at all developmental stages.At the early developmental stages(1-to 4-cell),a peripheral distribution of mitochondrial foci was observed in all of the blastomeres.And at the blastocyst stage,mitochondrial foci were homogeneously distributed throughout the inner cell mass and trophectoderm.Subsequently,the real-time PCR was used for mtDNA copy number assessment.Copy number of mtDNA decreased gradually from MⅡ to four-cell stages and subsequently kept consistent with blastocyst stage both in delipated and control embryos,but the copy number of mtDNA in delipated embryos was similar to that in the control groups no matter at which developmental stage was observed.4.Endoplasmic reticulum distribution in pig PA delipated embryos.MⅡ oocytes and PA embryos(1-cell,2-cell,4-cell,and blastocyst stages)were collected and stained with ER-TrackerTM Green.The distribution pattern of the endoplasmic reticulum at the MⅡ oocyte stage was affected seriously by the delipation.In the control MⅡ oocytes,the endoplasmic reticulum clusters were diffusely distributed throughout the ooplasm,but the endoplasmic reticulum clusters in the delipated MⅡoocytes were polarized to one side of the cytoplasm.The endoplasmic reticulum distribution pattern was the same for both delipated and control groups at the following developmental stages after PA.At the 1-to 4-cell stage,the endoplasmic reticulum clusters were diffusely distributed throughout the embryo cortex but a distinctive dense ring of fluorescence that enclosed the area of the nucleus was observed in the center of the embryo.At blastocyst stage,the endoplasmic reticulum clusters were homogeneously distributed in the inner cell mass and trophectoderm,additionally,the endoplasmic reticulum rings were also formed around the area of the nucleus in the both inner cell mass and trophectoderm.The study of addition inhibition of cytoskeleton suggested that the microfilament was essential for reorganization of endoplasmic reticulum at delipated 1-cell embryos,5.ATP content in pig PA delipated embryos.ATP content of delipated and control PA embryos(1-cell,4-cell and,blastocyst)was measured using luciferin-luciferase ATP assay system.Both in delipated and control embryos,ATP content progressive decreased from one-cell to blastocyst stages,while just at one-cell stage,a significant decrease of ATP level was observed in delipated embryos compared with that of control.