The Mechanism Of CSF₂ Regulates The Development Potential Of Yak Embryos Produced By In Vitro Fertilization

Yak is a kind of typical indigenous animal with good adaptability to high altitude and low oxygen environment in Qinghai-Tibet Plateau,and it is also one of the most important livestock breeds in the Qinghai-Tibet Plateau.However,yaks have a low reproductive rate,so they invariably breed only once every 2 years or twice in 3 years.Therefore,assisted reproduction techniques?ART?,such as in vitro maturation?IVM?,in vitro fertilization?IVF?,and in vitro embryo production?IVEP?,may be promising tools for enhancing the reproductive efficiency of yaks.Colony-stimulating factor 2?CSF₂?is known to promote the development and survival of rodents,ruminants and human pre-implantation embryos;however,the effect of CSF₂ on yak embryos has not been reported.The objective of this study was to investigate the effects of CSF₂ on the developmental competence of yak embryos cultured in vitro in modified synthetic oviduct fluid?m SOF?medium and on the expression pattern of apoptosis-,stress response-and development-related genes.This study will supply basic theory for the further understanding of the mechanism of CSF₂ on the development of yak pre-implantation embryo.The experiments and results in this study as follows:1.In each experiment,cumulus-oocyte complexes?COCs?were matured in vitro and fertilized with frozen-thawed semen.Zygotes were treated with varying concentrations of CSF₂?0,10,50,100 ng/m L?until day 8 after fertilization.Embryo development was calculated as the percentage of oocytes that formed embryos at the 2-cell,4-cell,8-cell,16-cell,morula and blastocyst stages.The total cell numbers?TCN?per blastocyst and their allocation to the ICM and TE lineages were determined using differential CDX2 staining.At day 8,the rate of blastocyst development was significantly higher in the cultures containing 50 ng/m L CSF₂ compared to the other treatment groups?P < 0.05?.There were no significant differences among the 0,10 and 100 ng/m L CSF₂ treatments?P > 0.05?.The percentage of oocytes that developed to the 2-cell,4-cell,8-cell,16-cell and morula stages were not significantly different among the treatment groups?P > 0.05?.At day 8,the mean TCN in blastocysts cultured with 50 ng/m L CSF₂ was significantly higher than in the other treatment groups?P < 0.05?,however no significant differences were observed among the other groups?0,10 and 100 ng/m L CSF₂ treatments;P > 0.05?.The TCN in the other stages was not different when evaluated by counting the TCN in the 2-cell-,4-cell-,8-cell-,16-cell and morula-stage embryos.Blastocysts cultured with 50 ng/m L CSF₂ showed increased numbers of both ICM?P < 0.05?and TE cells?P < 0.05?compared to the control group,resulting in greater TCN?P < 0.05?.However,there were no significant differences among the other treatments.Furthermore,the ratio of ICM/TE in the blastocysts did not differ significantly among the treatment groups?0,10,50 and 100 ng/m L CSF₂ treatments;P > 0.05?.We conclude that CSF₂ facilitates yak blastocyst formation and increase cell numbers.2.In each experiment,cumulus-oocyte complexes?COCs?were matured in vitro and fertilized with frozen-thawed semen.Zygotes were treated with varying concentrations of CSF₂?0,10,50,100 ng/m L?until day 8 after fertilization.The expression of apoptosis-related genes?BCL2,BAX and TRP53?and stress response-related genes?HSPA1A,HSPA5,HSPA9,HSPC1,HSPC3,HSPH1 and HSPH2?was examined by quantitative real-time PCR?q RT-PCR?to determine the m RNA levels.The expression of HSPA1 A protein was examined by immunochemistry to determine the protein levels.q RT-PCR analysis confirmed that treatment with 50 ng/m L CSF₂ significantly?P < 0.05?inhibited the expression of BAX,HSPA1 A,HSPA5,HSPC1 and HSPC3 m RNA in blastocysts cultured in vitro relative to the control group,but there were no significant differences between the other treatment groups?P > 0.05?.The expression of BCL2,TRP53,HSPA9,HSPH1 and HSPH2 m RNA in the blastocysts did not differ significantly among the treatment groups?0,10,50 and 100 ng/m L CSF₂ treatments;P > 0.05?.However,the ratio of BAX/BCL2 in blastocysts cultured with 50 ng/m L CSF₂ was significantly lower than in the other treatment groups?P < 0.05?,however no significant differences were observed among the other groups?0,10 and 100 ng/m L CSF₂ treatments;P > 0.05?.Immunocytochemical analysis confirmed that HSPA1 A protein accumulation was gradually reduced in yak blastocysts cultured in 0,10,100 or 50 ng/m L CSF₂,however,no significant differences were observed between the 10 and 100 ng/m L treatments?P > 0.05?.These findings demonstrate that CSF₂ inhibits the expression of HSPA1 A,BAX,HSPA5,HSPC1 and HSPC3 to facilitate yak blastocyst formation and increase cell numbers.3.In each experiment,cumulus-oocyte complexes?COCs?were matured in vitro and fertilized with frozen-thawed semen.Zygotes were treated with varying concentrations of CSF₂?0,10,50,100 ng/m L?until day 8 after fertilization.The expression of development-related genes?DNMT1,DNMT3 A,DNMT3B and IGF2?was examined by q RT-PCR.q RT-PCR analysis confirmed that treatment with 50 ng/m L CSF₂ significantly?P < 0.05?increased the expression of DNMT1,DNMT3 A,DNMT3B and IGF2 m RNA in blastocysts cultured in vitro relative to the control group.However,there were no significant differences between the other treatment groups?P > 0.05?.These findings demonstrate that CSF₂ increases the expression of DNMT1,DNMT3 A,DNMT3B and IGF2 to facilitate yak blastocyst formation and increase cell numbers.