Roles Of Gonad-Inhibiting Hormone In Gonadal Development And Sexual Differentiation In Peppermint Shrimp,Lysmata Vittata

The peppermint shrimp,Lysmata vittata belongs to the genus Lysmata and its gonad is the ovotestis.The testicular part of the gonad matures first while the ovarian part is still immature.Whereafter,during the maturation of the ovarian part,the testis still has male function.This unique gonadal development pattern is called protandric simultaneous hermaphroditism(PSH).Gonad inhibiting hormone(GIH)plays an important role in regulating the reproductive process of crustaceans.Not only can it inhibit the ovarian development of crustaceans,but also indirectly inhibit the development of testis via regulating the androgenic gland.In this study,we selected the sequence of Lv-GIH1 from transcription database.Lv-GIH1 sequence was obtained by cloning and identification from L.vittata,and its distribution at different tissues and expression profiles in different gonadal development stages were detected.In addition,in vivo and in vitro experiments were conducted to explore its regulatory effects on gonadal development and secondary sexual characteristic development.The main results are as follows:1)Lv-GIH1 cDNA was 1238-bp in full length with a 233-bp 5’-UTR,a 666-bp 3’-UTR,and a 339-bp ORF encoding a 112-aa precursor.The precursor contained a 29-aa signal peptide and a 83-aa mature peptide.Sequence analysis showed that the cDNA sequence characteristics and specific functional sites of Lv-GIH1 were highly consistent with those of other crustacean species.Sequence alignment and phylogenetic analysis also showed that Lv-GIH1 was highly homologous with other crustaceans GIH.The RT-PCR results suggested that Lv-GIH1 was exclusively expressed in the eyestalk ganglion among other tissues in L.vittata.In addition,qRT-PCR results revealed that Lv-GIH1 had the lowest expression in the gonadal stage I,followed by significant up-regulation in the stage Ⅱ and stage Ⅲ,and later in stage IV,it was significantly reduced to the same level as stage I.2)The recombinant protein of Lv-GIH1(rGIH1)was obtained through prokaryotic expression and purification experiments.The result of applying rGIH1(10-8 M、10-7 M)to the hepatopancreas explants showed that the abundance of Lv-Vg in the hepatopancreas was significantly inhibited by rGIH1 at the concentration of 10-7M.Following short-term injection of GIH1-dsRNA,the expression of Lv-GIH1 in the eyestalk ganglion was significantly reduced,while the Lv-Vg expression in the hepatopancreas was found significantly up-regulated.These results suggested that Lv-GIH1 might inhibit vitellogenesis.3)Morever,long-term rGIH1 injection led to significantly reduced expression of Lv-Vg,Lv-VgR and Lv-CFSH1,while the level of Lv-CFSH2 did not change significantly.Furthermore,the histological and morphological results showed that diameter of oocytes and the ovarian size decreased after the administration of rGIH1.In addition,it was observed that rGIH1 injection inhibited the development of female secondary sexual characteristics i.e.the numbers of feathery setae surrounding the female gonopores.According to the histological characteristics of testicular section of the gonad,although the administration of rGIH1 inhibited the development of testicular spermatogenesis,the expression of Lv-IAG1 and Lv-IAG2 transcripts in AG showed no statistical difference.4)After the knock-down of Lv-GIH1,the expression of Lv-CFSH1 in the eyestalk ganglion was significantly up-regulated.Meanwhile,the histological and morphological analysis showed that silencing Lv-GIH1 increased the diameter of the oocytes and accelerated ovarian development.The feathery setae surrounding the female gonopore were denser when compared to those from the control.There were no significant difference in the expression level of Lv-IAG1 and Lv-IAG2 after long-term injection of GIH1-dsRNA,but the testicular spermatogenesis was promoted.Taken together,the results of this study indicate that Lv-GIH1 is involved in gonadal development,and might also participate in controlling secondary sexual characteristic development in L.vittata by inhibiting Lv-CFSH1 expression.