The Effects Of RASGRP3 Gene On Function Of Placental Trophoblast Cells And Its Regulation Of Signaling Pathways In Goat(Capra Hircus)

Improving the reproductive performance is an important guarantee to promote the economic benefit of goat industry.The main technical measures applied in the production are to increase the living litter size and survival rate.Placenta is the link between mother and lamb,which has important physiological functions such as hematopoiesis,transport,metabolism,endocrine,barrier action and immunology during the process of embryonic development.Goat placenta belongs to cotyledonary placenta.In early gestation,the most obvious changes are trophoblast tissue growth and placental cotyledon formation.Abundant placental development and function are the prerequisite for producing high-quality lambs.Trophoblast cells（TCs）are developed from the trophoblast ectoderm in the morula stage.TCs have migration and invasion functions and are closely related to the pregnancy recognition,embryo implantation,blood vessel formation and placental development at early pregnancy.However,for goat placenta,which genes or signaling pathways regulated these processes,and the specific regulatory molecular mechanism remains unclear.The purpose of this study was to screen out functional genes and related signaling pathways of placental trophoblast in goats during early pregnancy,and to reveal their effects on trophoblast cells and placental cotyledon functions and related regulatory mechanisms.We used the Dazu black goat as experimental animal,collected placental tissue as research object,built the research models in vivo and in vitro,used the transcriptomics,bioinformatics,histological and molecular biological methods to screen the candidate function genes and potential signal pathways in hircine trophoblast at early pregnancy,and identified the function of candidate genes in trophoblast cells and placental cotyledon.The following studies were carried out sequentially:1.Detection of tissue structure and screening of candidate functional genes of placenta trophoblast at early pregnancy in goat;2.Identification of expression patterns of RASGRP3 in placental tissues and analysis the correlation of its expression level with placental traits;3.Studying the function and regulation mechanism of RASGPR3 on hircine trophoblast cell;4.The effect of RASGPR3 on placental cotyledon development and growth of goat at early pregnancy.The detail research results are as follows:1.Detection of tissue structure and screening of candidate functional genes in placental trophoblast of goats at early pregnancyClarifying the normal growth and development process of trophoblast is an important prerequisite for exploring the formation and disease diagnosis of placenta in goat.In this chapter,the complete trophoblast samples of goat placenta were collected on days 20,25,and 30 of pregnancy（D20,D25,and D30）（3 biological replicates at each time point）,and the trophoblast tissue structure was detected and related growth indicators were recorded.Transcriptome sequencing was utilized to screen functional candidate genes and related signaling pathways.The results showed that the weight,length,width,area and thickness of trophoblast tissue and the number of capillaries per unit area were significantly increased with gestation（P<0.05）.Secreted phosphoprotein 1（SPP1）was detected as an attachment marker protein and its expression level was significantly increased（P<0.05）.A total of 23253 commonly expressed genes（CEGs）and 4439 differentially expressed genes（DEGs）were identified by trophoblast tissue transcriptome sequencing at three time points of early pregnancy.Among the top 100 common highly expressed genes（Ch EGs）,61 were ribosomal genes,21 were related to placental function,and the remaining 18 genes were not reported to be related to placental function.The common highly expressed genes（Ch EGs）were significantly annotated to ribosome generation and protein transcription（P<0.05）by gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis.In D30vs D20 group,DEGs were the most（including2372 up-regulated genes and 1343 down-regulated gene）.The number of DEGs in D25vs D20 group was slightly decreased,including 1768 up-regulated genes and 976down-regulated genes.In D30vs D25 group,DEGs was the least（including 124up-regulated genes and 86 down-regulated genes）.Down-regulated DEGs is mainly enriched in mitochondrial energy metabolism and degenerative diseases,including Oxidative phosphorylation,Parkinson’s disease,Alzheimer’s disease,Huntington’s disease and Cardiac Muscle contraction signaling pathways.The up-regulation DEGs is enriched in cell proliferation,adhesion and migration,which were related to PI3K/AKT,Focal adhesion,ECM-receptor interaction,RAP1 and CAM signaling pathways.The up-regulated guanine transformation factor related genes C3G,EPAC1,EPAC2 and RASGRP3 could affect downstream PI3K/AKT and Focal adhesion signaling pathway through promote RAP1 activation to regulate cell proliferation,adhesion and migration.2.Expression pattern of RASGRP3 gene in goat placenta and its correlation with cotyledon traitsBased on the results of trophoblastic transcriptome,the RAP1 signaling pathway was consider as the research object in this chapter.The expression patterns of DEGs including C3G,EPAC1,EPAC2 and RASGRP3 that regulated RAP1 activity in different placenta tissues and different pregnancy stages were determined,and the correlation between candidate genes expression and placental traits or lamb traits was analyzed.The results showed that the specific expression of RASGRP3 gene was high in the chorionic membrane and placental cotyledon,and low in the carumontum and endometrium.RASGRP3 protein expression was significantly increased in the first trimester of pregnancy（60 d）（P<0.05）,and then remained stable at medium and late pregnant stages.At the same gestation stage,with the increase size of cotyledon,the length and density of trophoblast in cotyledon were significantly increased（P<0.05）,the expression of RASGRP3 were significantly increased（P<0.05）,and there was a significant positive linear regression relationship between them（P<0.05）.The litter weight,cotyledon number,cotyledon average diameter and large cotyledon proportion（≥3cm in diameter）in three lambs were significantly higher than those in single lamb（P<0.05）.The mortality of lambs in<1.67kg group was significantly higher than that in 1.91-2.10kg group and>2.30kg group（P<0.05）,and the expression of RASGRP3in cotyledon trophoblast with low birth weight（<1.67kg）was significantly lower than that in normal（1.91-2.10 kg）or high birth weight（>2.30kg）groups（P<0.05）.The expression of RASGPR3enriched pathway related proteins was detected,result showed that RAP1 protein level and total GTP content in cotyledon trophoblast were significantly decreased（P<0.05）.The phosphorylation levels of PI3K,AKT and m TOR were significantly decreased compared with normal and high birth weight groups（P<0.05）.All the results indicated that RASGRP3 was specifically expressed in placental cotyledon,and its expression level was positively correlated with the growth density of the trophoblastic tissue in placental cotyledon,which affecting the size of the placental cotyledon and fetal growth.The role of RASGRP3 might contribute to trophoblast development and placental cotyledon growth,resulting in increased birth weight and decreased mortality through mediating RAP1 activity and affecting the downstream signaling pathway PI3K/AKT/m TOR expression in goat.3.Study on the function and mechanism of RASGRP3 on goat trophoblast cellsGoat trophoblast cells（GTC）occupy an absolute proportion in cotyledon tissue and are involved in placental development and nutrient transport.In this chapter,the regulation function of RASGRP3 gene on trophoblast cells was verified by knockdown technique,the main regulation pathway mediated by RASGRP3 was proved by activating agent to compensate downstream pathway activity.The results showed that CK7 and E-Cadherin proteins were specifically expressed in primary trophoblast and trophoblast cell line,while Fas protein was only expressed in fibroblasts.RASGRP3gene knockdown model was constructed by lentivirus vector in goat trophoblast cells,and RASGRP3 expression was significantly decreased at the transcription and translation level（P<0.05）.Knockdown of RASGRP3 expression resulting in significantly decreased proliferation,migration and invasion ability of trophoblast cells（P<0.05）,significantly increased apoptosis rate（P<0.05）,and significantly decreased intracellular mitochondrial size,number,m DNA content and protein levels of electronic respiratory chain complex I,II,IV and V（P<0.05）.Co-immunoprecipitation（Co-IP）assay showed that RASGRP3 down-regulation significantly reduced RAP1GTP relative content（P<0.05）.Meanwhile,the total of intracellular GTP content and GTP enzyme activity were significantly decreased in RASGRP3 interference group（P<0.05）.RAP1 pathway activator(or PI3K/AKT pathway activator significantly compensated RASGRP3 knockdown affection on cell proliferation,migration and invasion functions（P<0.05）,and decreased apoptosis level（P<0.05）.The results proved that knockdown RASGRP3 gene inhibited cell proliferation,apoptosis,migration,invasion and mitochondrial function by regulating the RAP1GTP activity and phosphorylation level of PI3K/AKT/m TOR signaling pathway in goat trophoblast cells.4.Effect of RASGRP3 gene on placental cotyledon development in goatThe number and function of trophoblast cells in goat ensure placenta development and cotyledon growth.Based on the regulation function of RASGRP3 gene on trophoblast cells,the effect of RASGRP3 gene on placental cotyledon growth was determined in this chapter.An optimized adenovirus transfection system mediated by polypeptide hydrogel was constructed,and RASGRP3 gene interference and highly expressed adenovirus vectors were constructed to explore the structural and functional changes of trophoblastic layer in placenta cotyledon.The results showed that when the ratio of adenovirus titer and hydrogel RADA16 concentration was 1×10¹⁰pfu/m L and0.5 mg/u L,the injection volume was 50 u L,the cotyledon infection efficiency was the highest（58.19±4.2%）,the transfection efficiency was 2-fold higher than that of single adenovirus（P<0.05）.Western Blot analysis showed that RASGRP3 expression in GTCs was significantly decreased or increased in the RASGRP3-knockdown（ad-sh RASGRP3）and RASGRP3-overexpression（ad-RASGRP3）adenovirus groups compared with Na Cl and ad-ctrl groups（P<0.05）.At 45 days of gestation,the placenta was exposed by caesarean operation,and the cotyledon with average size（1.71 cm）was injected with hydrogel and RASGRP3 gene was successfully knocked down and highly expressed 7 days after transfection.About the cotyledon morphology and structure,the cotyledon diameter,weight and trophoblast density in ad-sh RASGRP3 group were significantly lower than those in Na Cl,ad-Ctrl and ad-RASGRP3 groups（P<0.05）.Meanwhile,these cotyledon indexes in ad-RASGRP3were significantly higher than those of Na Cl and ad-ctrl groups（P<0.05）,but there was no significant difference between Na Cl and ad-ctrl groups（P>0.05）.About protein expression in cotyledon,the relative protein level of Cyclin D1,P70,MMP9and MMP2 in trophoblast layer of ad-sh RASGRP3 group were significantly lower than those in Na Cl,ad-ctrl and ad-RASGRP3 groups（P<0.05）.The relative protein level in ad-RASGRP3 group was significantly higher than that in Na Cl and ad-ctrl groups（P<0.05）.In the activities of enzyme and signaling pathways,The RAP1GTP level,GTP,GTPase activity and phosphorylation level of PI3K/AKT/m TOR signaling pathway were significantly downregulated in ad-sh RASGRP3 group,and significantly upregulated in ad-RASGRP3 group（P<0.05）.These results suggest that RASGRP3 in placental cotyledon can activated RAP1 and PI3K/AKT/m TOR signaling pathway to regulate the growth of placental cotyledon by mediating downstream function proteins affecting the proliferation,apoptosis,migration and invasion of trophoblast cells.In conclusion,through transcriptome sequencing,bioassay and functional verification,the present study screened key genes（including RASGRP3）and signal pathways（including PI3K/AKT/m TOR）of placental trophoblast growth in goats during early pregnancy,and identified the specific expression,location and function of RASGRP3 in placental cotyledon trophoblast tissue.It was demonstrated that RASGRP3 gene regulates the proliferation,apoptosis,migration and invasion of goat placental trophoblast cells by activating RAP1 and promoting the phosphorylation level of PI3K/AKT/m TOR pathway.The results not only provide a theoretical basis for elucidating the molecular regulatory network of placental growth,but also provide a reference for screening genetic molecules that improve the efficiency of lambing in goats.