| Background:Before fertilization process,the genome is regarded as transcriptionally silent with no transcriptional functional activities.However,following the fertilization process,the specific gametes known as egg and sperm share their genetic material by uniting with each other and consequently zygote formation occur that ultimately leads to development of entirely a new individual.The transcriptional quiescent genome now allowing the newly formed zygotes to reprogram into totipotent state where it can perform several functional activities.Gradually after fertilization,the quiescent genome is regulated by the specific process known as zygotic genome activation(ZGA),which further stimulates the zygotic genome associated genes that are essential for early embryonic developmental competency.ZGA is a reprogramming process that changes the genome from silent mode at fertilization to transcriptionally activated state at later stages.ZGA process activates the zygotic gene products to replace the maternal supply which is the key step to initiate the process of development in mammals and also in different vertebrate species.The dramatic changes in development during ZGA are conserved in mammals and such changes are variables based on species and cell developmental stage.It usually takes place at 2-cell-stage in mice and 4-8-cell-stage in humans.It is complex cellular process which regulates gene expression and consequently different possible outcomes could be obtained.All these reprogramming of gene expression occurs at transcriptional and translation level.ZGA is divided mainly into two phases based on functional and transcriptional activities called as minor ZGA and major ZGA.The initial ZGA that is known as minor ZGA that occurs during S-phase of embryo at 1-cell-stage and G1-phase of 2-cell-stage embryo,which brings the developmental process in slow mode of activation.In contrast,the rapid and abundant transcriptional process that occur at late 2-cell-stage and regulates several functional activates is recognized as major ZGA.Activation and regulation of both ZGA is an essential for proper gene activation that consequently plays a role in cell proliferation and early embryonic development.The role of minor ZGA for the development of embryo beyond the 2-cell-stage was not defined properly in details because the product obtained from minor ZGA were identified with unexpected outcomes with transcription relatively at low level and produces transcripts from huge number of genes that were spliced ineffectively.Contrarily,the role of major ZGA is well defined that is essential for the development of embryos after 2-cell-stage,as evidenced that inhabiting major ZGA causes 2-cell-stage arrest.Reprogramming and gene activation take place during regulation of major ZGA and this reprogramming is an essential for early embryonic developmental competency Error during ZGA disrupts the genes expressions which ultimately cause early embryonic developmental arrest.Defect in ZGA cause downregulation of number of genes which are associated with early embryonic development and as a result impairment of the embryogenesis take place which leads towards infertility.A number of genes and maternal associated protein involved in ZGA have been identified including,YAP1,TLE6,BTG4,MED 13 and OCT4,but the role the RNA-binding maternal protein which is highly associated with ZGA in mice still need to investigate.Here,we identified RNA-binding maternal protein IGF2 mRNA-binding protein 2(IGF2BP2),also considered as IMP2,derived from the RNA-binding proteins family and have shown its abundant expressions in oocytes,embryos,and gonads in mice and also in other mammalian species.The functional activities of IMP2 has been demonstrated in cancer cell,type-2 diabetes,cell metabolism and translation initiation,however the role of IMP2 in early embryonic development during ZGA in mice still need to elucidateOver the past few decades,the low pregnancy rates of women using assisted reproductive technology(ART)and frequent implantation failure have provided the impetus for research aimed at improving embryo culture systems,especially for the stages between fertilization and transfer.We know that in vitro culture conditions are directly associated with embryo viability,development,and implantation success,so optimization of such protocols should result in improved pregnancy outcomes.A possible major difference in the developmental potential of embryos between in vivo and in vitro culture systems is the absence of specific oocytes and embryos growth factors in vitro.The number of growth factors involved in female fertility has been extensively studied,but reluctance to add essential growth factors in culture media has limited progress in optimizing embryonic growth and implantation outcomes,a situation that has ultimately led to reduced pregnancy outcomes.Insulin-like growth factor 2(IGF2)is the most intricately regulated of all known reproduction-related growth factors characterized to date,and is perhaps the predominant growth factor in human ovarian follicles.The role of IGF2 during oocytes meiotic maturation in porcine was demonstrated previously,however its contribution during early embryonic development still need to investigateMethods:The Imp2-knockout mouse line was generated by following the instructions and guidelines of the animal care and research committee of Shandong University.To generate conventional knockout mice,exons 3 and 4 of the Imp2 mice were flanked with LoxP sites and then Imp 2flox/flox mice were crossed with Vasa-Cre(also known as Ddx-Cre,germ cell specific expression)transgenic mice to obtain Imp 2flox/+;Vasa-Cre mice which were intercrossed to generate conventional knockout mice.Pubertal female mice(24-28 days old)were superstimulted with hormones(PMSG and hCG),and oocytes and embryos were collected from control and Imp2-knockout mice and cultured in vitro in M16 medium to observe their early embryonic developmental competency.Fertility test was also performed to assess the total number of pups per female for the period of 6 months.RNA-sequence and quantitative proteomics analysis experiment of 2-cell-stage embryos were performed to observe the pattern of gene expressions in Imp2-knockout mice and compared with control mice embryos.Western blotting,immunohistochemistry,and confocal microscopy was also performed to observe the protein expression pattern in Imp2-knockout mice oocytes and embryos.qRT-PCR analysis was performed to examine the mRNA expressions of downregulated genes in Imp2-knockout mice by using oocytes and embryos samples.Cell culture,plasmid transfection,and luciferase assay was used to determine the relationship of IMP2 with IGF2 and to examined the gene expressions of relevant downregulated genes having IMP2 binding-sites in dose-depended manners.Translational and transcriptional machinery was also assessed by using 2-cell-stage embryos from control and Imp2-knockout mice.In addition,IGF2 was added in the culture medium containing control and Imp2-knockout zygotes to observe its effect on early embryonic developmental competency Finally,an embryo transfer experiment was also performed to determine the developmental fate of embryos obtained from control and IGF2-treatment in vivo.Spare human oocytes samples were also obtained and were cultured in vitro with or without IGF2 to observe their embryonic developmental competencyResults:The present study indicated that IMP2 is highly expressed in oocytes and embryos at different developmental stages and the expression pattern varies based on developmental stage.The maternal deletion of Imp2 in mouse embryos is dispensable for folliculogensis and oocytes meiotic maturation;however causes embryonic developmental arrest at the 2-cell-stage.Only few embryos(6%)progressed to the 4-cell-stage of development after in vitro culture.In vivo results also indicated that only 6%of the embryos from Imp2-knockout mice progressed to the blastocyst-stage.Analysis of proteomics and transcriptomics of 2-cell-stage embryos in mice revealed that deletion of IMP2 causes downregulation of several genes that are involved in RNA-binding and protein-binding activities and these parameters are essential for early embryonic development.Western blot and qRT-PCR analysis also have shown the reduced expression pattern of the down-regulated target gene in 2-cell-stage embryos sample from Imp2-knockout mice.Based on mRNA expressions and luciferase activity of downregulated genes,two genes known as Ccar1 and Rps14 were identified as target genes of Imp2,both of these are associated with embryonic developmental competency.Deletion of these two target gene Ccar1 and Rps14 in WT embryos cause developmental arrest and only 10%of the embryo progressed towards blastocyst-stage.Furthermore,total RNA-synthesis and global protein synthesis in Imp2-knockout 2-cell-stage embryos were reduced 2-fold compared to the control embryos,which suggest the impairment of translational and transcriptional machinery in Imp2-knockout mice.In addition,IGF2,a target of IMP2,when supplemented to the culture medium,improves the percentage of wild-type embryos which developed successfully to the blastocyst-stage:from 29%in untreated controls to 65%(50 nM IGF2).However,adding IGF2 in the culture medium had no effect on Imp2-knockout embryos regarding embryonic development and these embryos were still arrested at 2-cell-stage.Furthermore,the embryo transfer related protocol;foster mothers receiving IGF2-treated embryos showed more percentage of pregnancy and also have delivered more number of pups per female than females who received untreated control embryos.Based on luciferase assay,the data indicated that IMP2 binds with IGF2 at 5' UTR and the translational activity of downregulated genes were increased by adding IGF2 in dose-depended manners.Moreover,the IGF2 level in oocytes,embryos and in cultured medium was reduced up to 2-fold in Imp2-knockout mice samples compared to Imp2 control mice samples based on ELISA assay.Finally,for human clinical sample,IGF2 supplementation to the culture medium at 2PN-embryonic stage significantly enhanced the proportion of embryos that developed successfully from I-cell-stage to blastocyst-stage.Furthermore,it was observed that administration of IGF2 to the culture medium improved the human embryo scores compared to the embryos which were cultured without IGF2-treatmentConclusion:Collectively,our findings demonstrate the functional impact of RNA-binding maternal protein IMP2 during ZGA in mouse which is an essential for early embryonic preimplantation development.These findings revealed that IMP2 is critical for the stimulation and regulation of genes associated with ZGA and deletion of this protein caused developmental arrest of embryos mainly at 2-cell-stage and consequently impaired mouse embryo genes is.These findings also revealed the functional impact and usage of IGF2 as growth factor for animal biotechnology and for assisted reproduction in humans. |
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