Effects Of Heat Stress During Late Pregnancy In Sows And Mice On Placental Function And The Fetal Intestinal Development And The Underlyling Mechanisms

Heat stress induced by high temperature in summer has a long-term negative influence on animal health and livestock production.Pigs are highly sensitive to heat stress due to lack of sweat gland,thick subcutaneous fat and poor heat dissipation ability.Late gestation is a critical period of the fetal growth as well as the windows of fetal intestine development.The alterations of utero environment(such as fetal nutrient supply,glucocorticoids)affected the fetal intestine development.The placenta is an organ that connects the fetus to its mother.Besides providing nutrients for fetal growth and development,the placenta also serves as a protective barrier.However,the effects of heat stress during late gestation on the placental function and intestinal development of the offspring remained unclear.Therefore,late pregnant sows and mice were used in the present study and the heat stressed models were established.The study in pregnant sows was designed to explore the effects of heat stress during late gestation on intestinal function of neonatal piglets.The study of pregnant mice was designed to investigate the effects of heat stress during late gestation on the placental function and intestinal development of the offspring.1 Effects of heat stress during late gestation on the intestinal barrier function of neonatal pigletsIn the study,twelve healthy Landrace × Great White(LBD)primiparous sows were randomly assigned to two environmental treatments: thermos-neutral(TN,19-22 ℃,n = 6)and heat stress(HS,29-32 ℃,n = 6)conditions from day 85 of gestation until farrowing.Farrowing and litter data was recorded,and two piglets(one male and one female,respectively)from each litter at medium bodyweight were selected.According to the sow’s ambient temperature,their newborn piglets were divided into groups of in utero thermalneutral(IUTN)piglets and utero heat stressed(IUHS)piglets groups.The results showed that birth weights of neonatal piglets were not affected by maternal HS,but body weights of piglets at d 10 age and weaning were reduced by 18% and 17% respectively(P < 0.01).The length of small intestine,villus height and crypt depth in jejunum and ileum of newborn piglets were not different between IUTN and IUHS groups,but the activity of lactase in the jejunum and ileum was decreased in IUHS piglets(P < 0.05).Compared with the IUTN group,the serum diamine oxidase(DAO)content of newborn IUHS piglets was increased(P< 0.05).The m RNA expressions of intestinal barrier related genes,including zonula occludens protein 1(ZO-1),zonula occludens protein 2(ZO-2)and mucin 2(MUC2)in the jejunum of neonatal piglets were down-regulated by maternal HS(P < 0.05).The percentage of apoptotic cells and the m RNA expression of Fas cell surface death receptor(FAS)(P <0.05)in the jejunum of neonatal piglets were reduced in the IUHS group compared with IUTN group.Furthermore,the concentrations of serum cortisol and adrenocorticotropic hormone(ACTH)were raised in HS pregnant sows and neonatal piglets(P < 0.05).These results indicated that HS during late gestation decreased the activity of lactase,increased intestinal permeability and intestinal barrier dysfunction in newborn piglets,reduced the body weight of piglets at d 10 age and weaning,and these changes were associated with increased concentrations of stress hormones in pregnant sows and neonatal piglets.2 Effects of heat stress during late gestation on the intestinal metabolism of neonatal pigletsThe results of the previous chapter showed that HS during late gestation impaired intestinal barrier function of neonatal piglets.Intestinal barrier dysfunction was associated with the alterations of intestinal metabolism.However,whether heat stress during late gestation affected intestinal metabolism of neonatal piglets remained unclear.Therefore,the metabolomics was used in the study aimed to investigate the metabolic profiles in serum and jejunum of neonatal piglets.The experimental design was the same as above.The results showed that the concentrations of xanthine and PC(16:0/16:0)in serum of neonatal piglets were increased,but those of N-acetylglutamine and erythritol decreased in IUHS groups.The concentrations of choline,creatine and xanthine in jejunum of neonatal piglets were increased,but those of DL-tryptophan,taurochenodeoxycholate,glycocholic acid,chenodeoxycholate,glycohyodeoxycholic acid and glycodeoxycholic acid decreased in IUHS groups.Xanthine was the common differential metabolites between the serum and jejunum of neonatal piglets.The differential metabolites of the serum were enriched in the β-alanine and linoleic acid.And the differential metabolites of the jejunum were enriched in purine metabolism,bile secretion,primary bile acid biosynthesis and cholesterol metabolism.The lipid contents in serum and jejunum of newborn piglets indicated that the concentrations of serum total cholesterol(TC)in newborn piglets were significantly increased in the IUHS group compared with the IUTN group(P < 0.05),while triglyceride(TG)concentrations were not significantly different between the two groups;TG concentrations and the m RNA expression of apolipoprotein A Ⅳ(Apoa4)in the jejunum of neonatal piglets were significantly decreased in the IUHS group(P < 0.05),but TC concentrations were not affected by maternal HS(P ≥ 0.05).These results demonstrated that HS during late gestation obviously altered the metabolic profiles of the serum and jejunum in neonatal piglets,particularly lipid metabolism,which might be associated with the impaired intestinal development of newborn piglets under maternal HS conditions.3 Effects of heat stress during late gestation on the placental immune function and intestine development of fetal miceMaternal HS induced the intestinal barrier dysfunction of neonatal piglets,which might alter immunoglobulin absorption and the establishment of innate immune function.The placenta plays an important role in the regulation of fetal innate immune function.We used pregnant mice to explore the effects of HS during late gestation on the placental immune function and intestinal development of fetal mice.Nineteen ICR female mice were randomly divided into two environmental treatments: the thermal-neutral group(TN),which was not heat stressed(24 ± 1℃,n = 11)and the heat stress group(HS)(35 ± 1℃,n = 8),which was heat stressed from gestation d 12.5 to 18.5.According to the ambient temperatures of the pregnant mice,the fetuses were divided into utero thermal-neutral group(IUTN)and heat stress group(IUHS).The results showed that the intestinal length and villus height were much shorter in IUHS group than IUTN group(P < 0.05).Transcriptome analysis showed that the genes involved in fetal intestinal(duodenum and jejunum)development were downregulated in the IUHS group compared with the IUTN group.According to protein-protein interaction(PPI)network and hub genes analysis of differentiated expressed genes,HS during late gestation had a profound effect on the cell cycle of fetal duodenum and jejunum,and expression of cell cycle-related genes was up-regulated in the IUHS groups compared with the IUTN groups.The m RNA expression and protein levels of cell division cycle 6(Cdc6)in the fetal duodenum and jejunum were increased in the IUHS group compared with the IUTN group(P < 0.05).We also found that HS during late gestation down-regulated the expression of innate immune system-related genes in the fetal duodenum and jejunum.Maternal HS also reduced the m RNA expression and protein levels of interleukin 1 alpha(IL1α)in the IUHS group compared with the IUTN groups(P < 0.05).Transcriptome analysis showed maternal HS had a profound effect on placental immune function,and the expression of placental immune response-related genes such as macrophage antigen CD68,Fc gamma receptor 1 and 3(Fcgγ1 and Fcgγ3)were increased in the HS group compared with TN group,while the m RNA expression and protein levels of colony stimulating factor-1(Csf1)were decreased due to HS(P < 0.05).These results revealed that heat stress during late gestation inhibited the fetal intestinal development,which was associated with increased expression of intestinal cell cycle-related genes increased.The development of intestinal innate immune system in the fetuses was also inhibited by maternal HS,which was associated with increased immune function and inflammatory response in the placenta.4 Effects of heat stress in mice during late gestation on the placental barrier function and fetal intestinal nutrients transport and metabolismMaternal HS increased serum cortisol levels in pregnant sows,but did not affect the serum cortisol levels in neonatal piglets.Placental 11β-hydroxysteroid dehydrogenase 2(Hsd11b2)can inactivate cortisol or corticosterone and serves as a protective barrier.The placenta provides nutrients to meet the demand of fetal growth and metabolism.The present study was aimed to investigate the effects of maternal HS on the placental barrier function and intestinal nutrients transport and metabolism in fetal mice.The experimental design was the same as the previous chapter.Maternal HS tended to decrease the placental weight(0.05≤ P ≤ 0.10),but significantly increased the numbers of dead fetuses(P < 0.05).The results of transcriptome analysis showed that maternal HS increased the transport and metabolism of glucose,amino acids and fatty acids in the placenta,but decreased the transport and metabolism of glucose,amino acids and fatty acids in fetal duodenum and jejunum due to maternal HS.PPI and hub genes analysis showed that maternal HS had an obvious influence on the lipid metabolism in the placenta,fetal duodenum and jejunum.Under maternal HS conditions,the concentrations of placental TG were increased(P < 0.05),but the concentrations of TG in the fetal jejunum and duodenum were decreased(P < 0.05).HS during late gestation also significantly reduced the m RNA expression and proteins levels of cluster of differentiation 36(CD36)and diacylgycerol acyltransferase(Dgat2)in the fetal duodenum and jejunum(P < 0.05).Furthermore,HS increased the concentrations of corticotropin releasing hormone(CRH),ACTH and corticosterone in serum(P < 0.01).Maternal HS also inhibited the m RNA expression and protein levels of Hsd11b2 and 5-hydroxytryptamine receptor 1D(Htr1d)in the placenta(P < 0.05),which was associated with increased corticosterone concentrations(P < 0.05)and increased gene expression of heat shock protein 90 alpha family class A member 1(Hsp90),hypoxia up-regulated 1(Hyou1)and corticotropin releasing hormone receptor 1(Crhr1)in the fetal brain.These results demonstrated that HS activated the hypothalamic-pituitary-adrenal(HPA)axis and increased corticosterone concentrations in pregnant mice.HS inhibited the m RNA expression and protein levels of placental Hsd11b2 and Htr1 d,which might in turn cause the corticosterone levels in the fetal brain increased.This might be the main cause of reduced intestinal nutrient transport and metabolism reduced in fetal mice.However,under HS conditions,the placenta may increase nutrients transport and metabolism to support the fetal growth.In summary,HS during late pregnancy in sows and mice increased maternal serum cortisol/corticosterone concentrations,inhibited the m RNA expression and protein levels of Hsd11b2 in the placenta,and altered the offpring response to stress,which in turn decreased the lactase activity and blocked the intestinal development in the offspring.Maternal HS also down-regulated the expressions of nutrient transport and metabolism and immune systemrelated genes in the fetal intestine,which were associated with increased the expression of nutrient transport and metabolism and immune function-related genes in the placenta under HS conditions.