| In the process of pig breeding,sow fertility is an important factor to affecting the production benefits of pig farms.Ovarian dysfunction,miscarriage,stillbirth and mummified fetus caused by reproductive diseases have induced great economic losses to the pig industry.The fetal growth and development in utero during porcine pregnancy are affected by many factors,and the antepartum conceptus loss has a 40%rate excepted the genetic factors and diseased.R-spondin3（RSPO3）is the first identified member of the RSPOs family and plays an important role in embryonic development.Mice fetuses die before birth when Rspo3-deficient due to the defects of placental blood vessels ineffectively invaded into the chorionic membrane.Whether RSPO3 displays potential effect in the development of porcine placenta for fetal development has not been reported.In this study,we firstly cloned the porcine placenta RSPO3 m RNA sequence,and detect expression and localization of RSPO3 at sow maternal-fetal interface in different pregnant periods.Then we investigated the effect of RSPO3 on porcine placental trophoblasts and endometrial stromal by mediating the overexpression or down-regulation of RSPO3 using lentiviral system.The effect of RSPO3 on mouse embryonic development and fetal growth was also explored though a placenta-specific overexpression of RSPO3 by mouse blastocysts were infected with lentivirus.The main results are as following.（1）Expression of RSPO3 at the porcine maternal-fetal interface.The amplified fetal RSPO3 m RNA sequence was 1337 base pairs（bp）in length with a single base difference at the site 180（T→C）through blast analysis compared to porcine RSPO3m RNA sequence from NCBI.It shares high homology with those of the cattle and sheep,and a far genetic relationship to zebrafish and cynoglossus semilaevis.Porcine placenta RSPO3 m RNA encodes 275 amino acids,which is consistent with the amino acid sequence of porcine RSPO3 from NCBI.The protein structure consists of two adjacent Furin protein domains,TSR domain,N-terminal hydrophobic signal peptide and C-terminal residues.The expression of RSPO3 in maternal-fetal interface was different during porcine pregnancy.The expression of RSPO3 m RNA and protein was higher in uterine and fetal placentas on 40 gestation day（GD40）than those on GD25 and GD70.RSPO3 protein is mainly localized in villotrophoblasts,endometrial stroma,and glandular epithelium of the porcine maternal-fetal interface.（2）Effects of RSPO3 on the function of porcine trophoblast cells and endometrial stromal cells.In this study,the overexpression and down-regulation of RSPO3 in porcine trophoblasts cells and endometrial stromal cells were established.We found that RSPO3 overexpression significantly inhibited the trophoblastic proliferation and tube formation,and up-regulated the expression of the downstream targets of Wnt/β-catenin signaling pathway,including CCND1,c-Myc andβ-catenin,but significantly inhibited the expression of GSK-3βand Axin2.Meanwhile,down-regulation of RSPO3 exhibited the opposite effect in the porcine trophoblast cells.Further study showed that inhibition of Wnt/β-catenin pathway inhibited the proliferation,tube formation and the expression of CCND1,c-Myc andβ-catenin which were promoted by RSPO3 in the trophoblast cells.In addition,RSPO3 overexpressed in porcine trophoblast cells promoted the tube formation of the interaction between the trophoblasts and vascular endothelial cells,while the interaction between these two cells was inhibited when RSPO3 down-regulation.RSPO3 showed no influence on cell apoptosis of the porcine trophoblasts.The secretion of E2 and P4 was undetectable in porcine trophoblasts.However,RSPO3 overexpression increased the m RNA levels of hormone-related synthases 17β-HSD,3β-HSD and CYP19A1,while down-regulation inhibited the expression of 17β-HSD and 3β-HSD m RNA in porcine trophoblasts.In porcine endometrial stromal cells,RSPO3 overexpression promoted the cell proliferation,while down-regulated RSPO3 inhibited the proliferation.In RSPO3-overexpressed or down-regulated endometrial stromal cells,the interaction between the stromal cells and vascular endothelial cells was attenuated.In addition,RSPO3overexpression could activate the Wnt/β-catenin signaling pathway in porcine endometrial stromal cells.（3）Effects of placenta-specific overexpression of porcine RSPO3 on the development of placenta and fetus in mice.The expression of Rspo3 m RNA and protein in mouse placenta were decreased gradually during different embryonic days.RSPO3 protein was strong expressed in the endometrial stroma,placental trophoblast and labyrinth of the maternal-fetal interface in mice,higher expressed in the embryonic day 10 and 12,which was similar to those in porcine placenta.In mouse models,we found that placenta-specific overexpression RSPO3 increased the fasting blood-glucose and heart rate on D12,fetal weight and the crown rump length on D18,and the abdominal circumference on D15 and 18.No change was observed in blood pressure,urine protein as well histologic changes in liver and kidney.In summary,this study reveals the expression and location of RSPO3 on the porcine maternal-fetal interface.Meanwhile,we demonstrate that the roles of RSPO3on the porcine trophoblast cells and endometrial stromal cells and its molecular mechanism.The placenta-specific overexpression of RSPO3 promotes fetal development in mice was also determined.This study provides a theoretical basis for exploring the mechanism of embryo or fetal death during porcine pregnancy,and also provides an idea for the development of new technologies to improve fetal growth. |
