Effect Of Follistatin On Development Of Pig Embryos Cultured In Vitro In Chemically Defined Culture Medium

As an necessary procedure in embryo in vitro production(IVP),in vitro culture(IVC)is of great importantance for breeding and reproduction of high quality animal,and uncovering mechanisms in animal reproduction and development.At present,development efficiency and quality of pig embryos after IVC are still unsatisfactory,which is mainly due to suboptimal medium.It has been shown that Follistatin(FST)could promote in vitro development of bovine and rhesus embryos by accelarating the first cleavage,enhancing quality and formation rate of blastocyst.Our previous study revealed that FST though is beneficial to swine embryo preimplantational development,the effect is not always consistant with cattle and monkey.Therefore,by supplementation of FST in chemically defined medium PZM-4 for IVC,the present study aimed to explore real physiological roles of FST in porcine embryonic development in order to provide basis for further optimization of pig embryo IVC system.Experiments are as follows:Experiment 1 Effects of different isoforms of FST(FST-315,FST-300,FST-288)on preimpantational development of parthenogenetic activation embryos(PAEs).The control group was FST-free PZM-4.PAEs were randomly added in embryo medium with10ng/mL FST-315,FST-300,or FST-288,respectively.The results showed that all isoforms of FST could significantly improve the early cleavage rate of embryos(P<0.05)(cleavage rate of 24 h after IVC);no significant difference among all groups in the total cleavage rate(cleavage rate of 48 h after IVC)was found.Additionally,FST-300 could improve blastocyst formation rate(P<0.05)of PAEs.Moreove,FST-288 mainly increased total cell number of blastocysts(P<0.05).Experiment 2 Functions of different isoforms of FST(FST-315,FST-300,FST-288)on in vitro development of somatic cell nuclear transfer Embryos(NTEs).Experimental group setting was similar to the Experiment 1.Reconstructed embryos were randomly assigned to each group,and cultured under the same conditions.It was found that FST-315 and FST-300 were beneficial for rates of early cleavage and total cleavage of NTEs,although no significant difference(P > 0.05).The improvement of the numbers,total cell number and TE cell number of NTEs blastocysts was not obvious(P>0.05).However,the improving trend of FST-300 was more effective.Experiment 3 Effects of different concentrations of FST-300 on in vitro development of NTEs.The control group was FST-free PZM-4.NTEs were randomly cultured in PZM-4 adding 10,20 or 50ng/mL FST-300,respectively.The results displayed that20ng/mL of FST-300 could significantly increase total cleavage rate of reconstructed embryos(P<0.05);no obvious difference was observed for the formation rate,total cell number and TE cell number of NTEs blastocysts(P>0.05).Experiment 4 To further investigated the optimum concentration and isoforms of FST protein on PAEs embryonic development,different concentration gradient of three isoforms of FST was added into PZM-4: FST-315(1,5,10ng/mL),FST-300(10,20,50ng/mL),FST-288(10,20,50ng/mL),and FST-free PZM-4 as the control group.It was shown that various concentrations of three isoforms of FST obviously strengthened the early cleavage rate of PAEs(P<0.05),among which 20ng/mL FST-300 was the best.Apart from this,the formation rate of blastocyst in FST-300(10ng/mL,20ng/mL),and the total cell number of blastocysts in FST-315(5ng/mL),FST-300(20ng/mL),and FST-288(10ng/mL,50ng/mL)could dramatically be increased(P<0.05).Experiment 5 To explore the combination of different isoforms and concentration of FST on the development of PAEs.FST-free as the control group,and the experimental group were set with different optimum concentration and isoforms of FST in PZM-4 as following.Group 1: 20ng/mL FST-300 + 5ng/mL FST-315 + 10ng/mL FST-288;Group 2:20ng/mL FST-300 +5ng/mL FST-315;Group 3: 20ng/mL FST-300 + 10ng/mL FST-288;Group 4: 5ng/mL FST-315 + 10ng/mL FST-288;Group 5: 20ng/mL FST-300 only.It was found that the combination of different isoforms of FST all could significantly improve the early cleavage rate of PAEs(P<0.05).On the formation rate of blastocysts,Group 1,2,3,5 play an important role(P<0.05).Group 4 and 5 made a contribution to the total cell number of PAEs blastocysts(P<0.05).However,the lever of GDF9,BAX,BCL-xl,p53,KLF4,SOX2,OCT4 and CDX2 among group 1,5 and control were similar.In conclusion,there were different effects on the preimplantational development of PAEs and NTEs by adding FST in IVC system,and the optimal concentration of different isoforms of FST were selected for improving porcine embryonic development of PAEs and NTEs.The research also enriched the formulation components in chemically defined medium of porcine IVC,and laid a better foundation for the mechanisms exploring of FST in embryonic development.