Role Of Epidermal Growth Factor Receptor(EGFR) In The Ovarian Development Of Mud Crab Scylla Paramamosain

The epidermal growth factor(EGF)signal system,playing a role in regulating cell proliferation,migration and differentiation,is involved in a series of important physiological processes,including organ development,body growth,reproduction and wound repair.In vertebrates,EGF receptor(EGFR)which is dimerized and further autophosphorylated with the specific binding of ligand,participates in the reproductive regulation by autocrine/paracrine mode.However,the related studies of EGFR were rarely reported in invertebrates other than Drosophila melanogaster,one of the model organisms.The mud crab Scylla paramamosain is an important economic crab in the south-eastern coastal area of China.In this study,we successfully identified SpEGFR from S.paramamosain by transcriptome sequence screening,gene cloning and bioinformatics analysis.Then we detected its temporal and spatial expression and cellular distribution by RT-PCR,qRT-PCR and in situ hybridization,respectively.Finally,we explored the role of EGFR on ovarian development by adding ligand protein and EGFR inhibitors in vitro.The main results are offered as follows:A full length cDNA sequence of SpEGFR was cloned from S.paramamosain.The results of sequence analysis showed that,after chopping the signal peptide,mature SpEGFR was a transmembrane protein and could be divided into three parts including an extracellular region with two ligand-binding domains,a hydrophobic transmembrane zone,and an intracellular region with tyrosine kinase domain(TyrKc).Amino acid sequence alignment and phylogenetic tree analysis revealed that SpEGFR possessed conserved domains and shared a high homology with that of other species.RT-PCR results showed that SpEGFR was widely distributed in all detected tissues and highly expressed in ovary.The qRT-PCR results showed that compared with the pre-vitellogenic stage,the expression of SpEGFR significantly increased to a high level at the early-vitellogenic stage and kept the status at the late-vitellogenic stage as well.In situ hybridization revealed that SpEGFR located in the oocytes of early vitellogenic stage with a notable positive signal in the perinuclear portion especially.These results indicated SpEGFR may take part in ovarian development in mud crab.In vitro experiments were performed to explore the reproductive mechanism of SpEGFR by adding human epidermal growth factor(hEGF).The results showed that the SpVgR and SpCyclin B of ovary explants from late vitellogenic stage crabs(summer season)were significantly increased when treated with InM hEGF for 1h,while there was no significant change towards SpEGFR.However,the SpVgR and SpCyclin B of ovary explants from the winter season crab in the same vitellogenic stage,did not show significant increase until treated with the higher concentration of 10 nM hEGF and the longer culture time of 12 h.We concluded that hEGF could activate SpEGFR,and stimulate the expression of SpVgR,SpCyclin B to promote ovarian development which may achieve through MAPK pathway.As for SpEGFR,a member of receptor tyrosine kinase family,made function by changing in molecular conformation and phosphorylation levels.Interestingly,the different physiological response to hEGF in the same vitellogenic stage crabs from different season,may be attributed to the decreased metabolism and physiological sensitivity in winter.In addition,hEGF-induced up-regulation of SpVgR and SpCyclin B were both suppressed by being pre-treated with EGFR inhibitor AG1478 and PD153035,respectively,which further indicated that EGF-EGFR pathway played a vital role in ovarian development in mud crab.Above all,hEGF-induced SpEGFR is involved in promoting ovarian development,which enriched the reproductive endocrine regulation network in S.paramamosain.