The Cloning And Expression Pattern Analysis Of Igf1ra And Igf1rb In Medaka

Medaka(Oryzias latipes)is a small freshwater fish,which has a series of advantages such as transparent embryos,small individuals and fast breeding.Medaka is a good model organism for the study of vertebrate development and reproductive biology.Moreover,it is the first non-mammalian vertebrate that the male sex-determining gene(dmy)has been identified.Many varieties of transgenic fish have been obtained in medaka,and these genes are specifically expressed in the gonads.It lays a foundation for the study of genes related to reproductive and gonadal development.We have investigated a lot of genes that regulate germ cells development.For example,knocking down piwi or vasa genes in medaka gives rise to defects in primordial germ cells(PGCs)migration;knocking down dazl or dnd genes affects the formation of PGCs.In addition,germ cell transplantation for surrogate production by germline replacement is also established in medaka.Therefore,medaka is an interesting model to study the gonadal development and reproduction.The insulin-like growth factor(IGFs)signaling system plays a key role in animal growth,metabolism,and self-renewal of embryonic stem cells.The IGFs signaling system mainly includes IGF ligands(IGF1,IGF2 and IGF3),IGF receptors(IGF1R and IGF2R),and IGF-binding proteins(IGFBPs).Many studies have described the role of IGFs in mammalian growth,development,metabolism,and tumorigenesis.However,there are a few studies on IGFs in gonadal development and reproduction of fish,especially in medaka.In this study,igf1 ra and igf1 rb were cloned in medaka for the first time,this is totally different from that in mammals.And the bioinformatics information of IGF1 R,including sequence alignment,phylogenetic tree and synteny were analyzed.In addition,the expression and the subcellular localization of igf1 ra and igf1 rb m RNA in gonads and embryos were analyzed.In this study,the open reading frame(ORF)of igf1 ra was 4197 nt,encoding 1398 amino acids(aa);while ORF of igf1 rb was 4224 nt,encoding 1407 aa.Bioinformatics analysis revealed that medaka IGF1 R is a homologue of human IGF1 R,and IGF1 R is relatively conservative in evolution.RT-PCR showed that medaka igf1 ra and igf1 rb m RNA were expressed in all tissues and embryos examined.Using chromogenic in situ hybridization(SISH)and fluorescent double-color in situ hybridization(FISH),both igf1 ra and igf1 rb m RNA in medaka ovary were found in the cytoplasm of I-IV oocytes,and igf1 rb m RNA was also found in many somatic cells,such as theca cells throughout oocytes development and granulosa cells of oocytes at stage V;however,igf1 ra m RNA was not found in somatic cells.In medaka testis,igf1 ra and igf1 rb m RNA exhibit completely different expression patterns.Igf1 ra m RNA was mainly expressed in somatic cells,such as Sertoli cells and Leydig cells.In addition,igf1 ra m RNA was also expressed in spermatocytes,spermatid,and sperm,but not in spermatogonia.However,igf1 rb m RNA was mainly expressed in spermatogonia and Leydig cells,weakly expressed in spermatocytes,spermatid and sperm.Whole mount in situ hybridization(WISH)showed that igf1 ra and igf1 rb m RNA shared overlapping,but distinct expression patterns in embryogenesis.Duing the early stages of embryonic development,igf1 ra and igf1 rb m RNA exhibit the same pattern of expression,both igf1 ra and igf1 rb m RNA were detected in all blastomeres of examined early stage embryos,and ubiquitously expressed during somitogenesis.It is worth noting that in the later stage embryos at seven days post-fertilization(7 dpf),igf1 ra and igf1 rb m RNA exhibit similar but differential spatial expression patterns.The signal of igf1 ra m RNA was strongly detected in the developing liver,brain and the whole developing pectoral fin,weakly detected in gonad.In contrast,igf1 rb m RNA was only weakly detected in the liver;it was abundantly detected in the tip of the tail,brain,gonad and the developing pectoral fin.The study of the expression pattern of igf1 r in medaka can help to understand the IGFs signaling pathway and its molecular mechanism in the reproductive development of vertebrates.It also provides a good theoretical basis for fish reproduction.