| Early embryo development research is based on understanding the individual occurrence and development of specific species,and its mechanism analysis has made great contributions to modern biology and medical development.At present,human cognition of the early embryonic development of other mammals is mainly limited to a few model animal s such as mice,which hinders the application of life science research results in animal husbandry and medical fields.Pigs are of great value in both livestock production and biomedical models,so they are one of the most highly studied large-scale model animals other than small-scale animals such as mice.Embryonic cell fate determination and differentiation are the basis of embryoni c development,and its regulatory mechanism is an important research field in developmental biology.The formation of inner cell mass(ICM)and trophectoderm(TE)is determined by the first cell fate determination and differentiation of mammalian embryo development,and is also the mammalian-specific embryonic cell differentiation process.The developmental fate of the inner ce ll mass is the formation of all tissue cells and extraembryonic endoderm tissue cells of the embryo body itself.The developmental fate of the trophoblast is the ectodermal tissue cells that form the fetal placental chorion,which plays an irreplaceable role in embryo implantation.The in vitro culture system is an important method for obtaining preimplantation embryos.However,current culture systems are unable to efficiently obtain embryos which are similar to the status in vivo.It has been reported that Fetal bovine serum(FBS)has a significant effect on the blastocyst rate and blastocyst number in vitro.However,due to the complexity and unknown of its components,the mechanism of action and subsequent developmental capacity of FBS to promote in vitro embryo development remains unknown.This limited the application of FBS in in vitro culture of porcine embryos.In this experiment,10% FBS was added to the cultur e medium on day 4 after in vitro fertilization.The results showed that compared to the con trol group,the addition of FBS did not increase the blastocyst rate,but the hatching rate of blastocysts increased significantly(33±6% vs 19±12%;P<0.05).The results showed that the number of blastocysts with obvious ICM structure in the embryosclearly added with FBS on day 4,and the ICM cell number increased significantly.The number and the ratio of ICM cells to the total number of cells were significantly higher than those of the control group,and most of the blastocysts were found to be cultured in vitro until day 8.In addition,our study demonstrates that FBS can promote the development of porcine embryos in vitro by activating the ROCK(Rho A/Rho-kinase)signaling pathway.We added the ROCK signaling pathway inhibitor Y-27632 during in vitro culture,and the results showed that Y-27632 can inhibit the formation of blastocyst cavity.However,we also added FBS to save the effects of Y-27632 and obtained blastocysts normally.At the same time,we found that the expression levels of ROCK1 and ROCK2,the key factors of ROCK signaling pathway,were significantly increased in porcine blastocysts after FBS culture.Immunofluorescence experiments also showed that the enrichment of phosphorylation of Rho A/Rho-kinase in the nucleus of the experimental group was significantly increased.These results indicate that FBS can promote the in vitro development of porcine early embryos by activating ROCK signaling pathway,and also lead a foundation for the application of FBS in porcine in vitro culture system and further study its mechanism of promoting embryo development.Oct4(also known as Oct3)is encoded by the Pou5f1 gene and is a homologous domain transcription factor of the POU(Pit-Oct-Unc)family and is a key factor in pluripotency regulatory networks.During the early embryonic development of mice,Oct4 is considered to be a marker gene for ICM.Early studies in our experiments showed that most of the embryos that kno cked down OCT4 during the zygote phase were blocked in the 8-cell stage.In this study,we found that OCT4 protein is widely expressed in almost all cells of porcine blastocysts during early embryonic development in pigs.In order to investigate whether porcine blastocyst trophoblast OCT4 has function in preimplantation embryo development,we selected small cavity blastocysts on day 5.5 of in vitro culture,specifically knocking down the expression of trophoblastic OCT4(OCT4TE-KD)by using lentivirus.The morphology of the OCT4TE-KD blastocyst did not change significantly during the in vitro culture to day 7(224 ± 35 μm vs 247 ± 29 μm).We transplanted OCT4TE-KD blastocysts on day 6 to allow embryos to continue to develop in vivo until day 8(day 2 posttransplant).We found that the morphology of OCT4TE-KD embryos changed significantly during this period,which was smaller than that of the control embryos.The number of cell clusters and trophoblast cells in OCT4TE-KD blastocysts were significantly lower than those in control blastocysts(183±58 vs 83±39;161±54 vs 73±35;P<0.05).The expression level of apoptosis-related gene BCL2A1 was significantly decreased.TUNEL assay showed that OCT4TE-KD blastocysts had severe apoptosis.When the OCT4TE-KD embryo developed to day 9(day 3 post-transplantation),the embryo was only 1/2 the diameter of the c ontrol embryo and almost died.The trophoblast of blastocyst on day 6 OCT4TE-KD and the embryo on day 8 were collected for transcriptome sequencing analysis.RNA-seq analysis showed that knocked down OCT4 in porcine TE specifically,resulted in some changes in the transcriptome,apoptosis,and the embryo died finally.The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells,and plays an important role in the differentiation of inner cell mass during mouse embryonic development.However,little is kno wn about the functional studies of porcine NANOG.Therefore,we conducted a preliminary study on the ex pression pattern and function of Nanog in early porcine embryos.First,using Q-PCR and fluorescence in situ hybridization(FISH),we demonstrated that NANOG m RNA is expressed from porcine 1-cell stage to blastocyst stage embryo.The NANOG protein is expressed in the nucleus of 4-cell embryos,and the expression level is decreased.It is expressed in some cells of 8-cell embryos,and NANOG protein expression is not detected in early blastocyst stage and metaphase blastocyst.FBS was added to the culture system,and the embryos were cultured until day 8,and it was found that the NANOG protein was expressed only in epiblast(EPI).To explore the role of NANO G in early embryonic development in pigs,we knocked down and overexpressed NANOG,respectively.The results showed that the MII knockdown of NANOG,embryonic development arrest in the 8-cell phase,the blastocyst formation rate was significantly reduced(36 ± 2% vs 16 ± 9%;P < 0.05).We knocked out NANOG by viral infection at the 4-cell stage and found that there was no ectoderm formation in the blastocyst stage,knocking down NANOG affected t he differentiation of ICM cell clusters into EPI.This indicates that Nanog is indispensable in the formation of porcine embryonic EPI.On the other hand,over expression of Nanog does not affect embryonic cleavage,but also affects blastocyst formation.This indicates that porcine NANOG is not required during the establishment of porcine embryonic stem cells.These results laid the foundation for the study of the role of porcine NANOG in pluripotency regulation. |
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