Study On Mechanisms Regulating Abnormal Development Of Cloned Pigs During Intrauterine And Neonatal Periods

The cloned pigs generated by somatic cell nuclear transfer(SCNT)offer valuable applications in agriculture,biomedicine,genetic resource protection and other fields.However,cloned pigs have a very high occurrence of malformation and mortality during intrauterine and neonatal periods,which severely limits the application of pig SCNT technology.The purpose of this study was to reveal potential mechanisms underlying abnormal development of cloned pigs during intrauterine and neonatal periods to provide scientific evidences for improving the somatic cell cloning efficiency in pigs.In this study we compared metabolic profiles of amniotic fluid and allantoic fluid between SCNT and artificial insemination(AI)-derived pig fetuses at day 65 of gestation by metabonomics,and coupled with placental morphological observation and transcriptome analysis to identify target metabolites that affect development of cloned pig fetuses.We also compared serum metabolic profiles among newborn SCNT piglets that died within 4 days(SCNT-DW4),AI piglets that survived over 4 days(AI-SO4)and SCNT piglets that survived over 4 days(SCNT-SO4)by metabonomics,and combined serum biochemical indexes detection and histopathological diagnosis to investigate potential mechanisms underlying abnormal development of cloned piglets.The major results were shown as follow:The body weight of cloned pig fetuses was significantly lower than that of the AI-derived pig fetuses.A total of 46 differential metabolites were identified by comparing amniotic fluid metabolic proflies between SCNT and AI pig fetuses,these differential metabolites were mainly involved in bile acid metabolism and steroid hormone biosynthesis.The bile acid level were increased and the steroid hormones levels were dysregulateds in amniotic fluid of cloned pig fetuses.Histomorphology analysis revealed that SCNT-derived placentas underwent poor fold development.A total of 632 differentially expressed genes(DEGs)were identified between SCNT and AI-derived placentas,these DEGs were mainly enriched in oxidative phosphorylation,steroid hormone biosynthesis and ABC transporters.The expression of steroid hormone biosynthesis-related CYP11A1,CYP19A1,HSD3B1 and HSD11B2 and the expression of bile acid transport-related ABCC3 and ABCB8 were significantly downregulated in placentas of cloned pig fetuses.Comparative metabolomics of allantoic fluid between SCNT and AI pig fetuses identified 73 differential metabolites that mainly concentrated in the fatty acids metabolic pathway.The levels of 25 fatty acids and their derivatives in the allantoic fluid of cloned pig fetuses were significantly lower than those of AI fetuses.Cloned pig fetuses had lower levels of 3 saturated and 15 unsaturated fatty acids in the muscle tissue,compared with AI fetuses.Cloned pig fetuses had poor development of placental folds and cytotrophoblasts.Cloned pig fetuses also had a lowerfatty acid transport protein 4(FATP4)expression level in placentas compared with AI fetuses.The mortality rate and malformation rate of newborn cloned piglets were significantly higher than those of AI piglets.The birth weight and placental index of SCNT-DW4 piglets were significantly lower than those of AI-SO4 and SCNT-SO4 piglets.Comparative serum metabolomics between SCNT-DW4 and AI piglets and between SCNT-DW4 and SCNT-SO4 piglets screened out 95 and 68 differential metabolites,respectively,and two comparisons had 31 common metabolites that mainly involved in purine metabolism and steroid hormone biosynthesis.The serum biochemical indexes of piglets showed that uric acid level,and creatinine and urea nitrogen level,two important indicators of renal excretion function,of SCNT-DW4 piglets were significantly higher than those of AI-SO4 and SCNT-SO4 piglets.Renal histopathological diagnosis showed that the kidney of SCNT-DW4 piglets exhibited marked pathological changes.The m RNA expression level of hepatic HPRT1,an important gene that regulate purine metabolism,and serum HPRT1 enzyme level of SCNT-DW4 piglets were significantly lower than those of AI-SO4 piglets.In summary,the placentas of cloned pig fetuses exhibited poor development of placental folds and cytotrophoblasts,downregulated expression of genes involved in bile acids transport,steroid biosynthesis and fatty acid transport,which probably lead to elevated bile acids levels and dysregulated steroid hormones levels in the amniotic fluid,and reduced fatty acids levels in the allantoic fluid and muscle tissue.These erroneous changes may cause abnormal intrauterine development of the cloned pigs.In addition,the downregulation of HPRT1,a key gene regulating purine metabolism,may give rise to elevated levels of purine metabolites in the serum of neonatal cloned pigs,which cause renal lesions and poor development of cloned piglets.This work elucidated some important mechanisms underlying abnormal development of cloned pigs during intrauterine and neonatal periods,which can provide the theoretical basis for the establishment of new method improving developmental efficiency of cloned pigs in the future,thereby promoting the application and development of pig cloning technology.