Identification Of Differentially Expressed Genes In Conceptus With Distinct Morphology During Pre-implantation Stage And The Functional Study Of PLET1 Gene

Pig embryo/fetus mortality lead to the decreasing number of litter size,and intrauterine growth restriction(IUGR)occurs in pigs will result in the increasing number low birth weight of piglets.The low birth weight piglets exhibit a weaker resistant ability to infectious diseases and a lower feed conversion rate,thus it severely affects the reproduction efficiency of pigs.Two main processes are critical for successful pregnancy maintenance which directly associated to placenta efficiency:1)the embryo implantation;2)the placenta formation and development.Placenta efficiency is determined by the placenta size and transport efficiency.During the pre-implantation and the implantation stage,conceptus(mainly composed by inner cell mass,the endoderm,and the trophectoderm)will undergo a rapidly reprogramming process with the embryo expanding from a small spherical(10mm)to a long filamentous(150mm)conceptus,The elongated embryo will then take up enough endometrium space for implantation,therefore,further determined the size of placenta.Subsequently,trophoblast cells attached and adhered to endometrial epithelial cells to form a epitheliochorial placenta.So,spherical and filamentous conceptuses during the fast reprogramming stage were used as materials to screen the differentially expressed genes and the regulation pathway,which is related to implantion and the placenta development.Next,we choosed PLET1 as the candidate gene from these differentially expressed genes,and further investigated the role of PLET1 in placenta establishment and development,the main results were summarized as follows:1.Affymetrix chip were used to analyze the differentially expressed genes between the spherical conceptus and filamentous conceptus in Meishan and Yorkshire pigs respectively,further comparison analysis result to differentially expressed genes indicates that,410 genes were merely upregulated in Yorkshire embryos,and 634 genes were down regulated;288 genes were found to merely upregulated in Meishan embryos,and 292 genes were down regulated.It shows that the differentially expressed gene in Yorkshire pigs is far more than it in Meishan pigs,our results indicate that gene expression in Meishan pre-implantation conceptus tend to be steady.Moreover,totally 345 genes were up regulated,and 379 were down regulated in both Meishan and Yorkshire pigs,we hypothesized that these genes may be indispensable for regulation of embryo elongation.The gene ontology and KEGG pathway analysis of the differentially expressed gene shows that differentially expressed gene in Meishan and Yorkshire conceptus were both involved in angiogenesis,response to steroid hormone stimulus and regulation of epithelial cell proliferation,it revealed that these process may be indispensible for conceptus elongation.We also found that differentially expressed genes only in Meishan conceptus are mainly enriched in several signaling pathways associated to ECM-receptor interaction,focal adhesion and endocytosis,whereas differentially expressed genes only in Yorkshire conceptus are enriched in multiple signaling pathway including steroid biosynthesis,complement and coagulation cascades,glycosaminoglycan degradation and calcium signaling pathway,indicating that gene expression pattern changing in Yorkshire conceptus elongation may causes multiple functional changes in cell.2.PLET1 is an upregulated gene in both Meishan and Yorkshire reprogramming conceptus,therefore,it may be a critical gene for trophectoderm elongation.So,we firstly detect the PLET1 expression pattern in blastosyst,conceptus,and the placentas of whole pregnancy proceed,we found that it has a low expression in early blastosyst,but high in conceptus,this result is coincides with the microarray.We also found that PLET1 is also upregulated in the placenta during pregnancy,suggesting that PLET1 may participate in placentation.3.Further study to PLET1 shows that:(1)PLET1 m RNA and protein expressed only in conceptus and trophoblast cells of chorion tissues,therefore,PLET1 can be used as a trophoblast-specific cell marker in pig placenta.(2)PLET1 localization in trophoblast is changed during pregnancy by Immunofluorescence detection,the result indicates that PLET1 proteins were primarily localized at the apical side of the trophoblast cells from 50 gestational day,which trophoblast-endometrial epithelial bilayer become very characteristic and depend folds.PLET1 location is becoming more closely to endometrium,suggesting that it may regulate the trophoblast-endometrium interaction.(3)Two transcripts caused by variants of 3'-end were both expressed in pig placenta by 3? RACE,and alternative cleavage and polyadenylation is the reasons for 3? UTR shortening.Further q PCR detection of the transcripts result shows that ratio of long to short PLET1 m RNAs varied greatly in different gestational days,indicating that although the two transcripts encoding the same protein,they may play different roles during pregnancy.(4)The long 3? UTR segment result in a higher luciferase activity than the short one by an in vitro reporter study,and PLET1 expression is regulated by mi R-181a?mi R-181 c and mi R-365-3p,which target to 3? UTR segment.4.Bioformatic analysis of the amino acid predicted that porcine PLET1 contains two N-glycosylation modified sites,and we demonstrated that PLET1 is an N-glycosylation modified protein,and it is the main form in porcine placenta.5.Mouse plet1 is differentially expressed in placenta with different weights,A plet1 kouckout mouse was generated from genome by TALEN technology,and plet1 was confirmed to have a lower expression compared to the wild types by q PCR and western blots.Plet1 kouckout leads to a enlarge dimension in labyrinth,and the number of spongiotrophoblast cells were increased,in addition,maternal blood spaces become smaller and the blood content in labyrinth was increased.We found that mouse plet1 may be associated to the development of placenta.