Molecular Mechanisms Of Juvenile Hormone Regulation In Cell Polyploidization And Reproduction Of Locust

In addition to repressing insect larval molting and metamorphosis modulated by 20-hydroxyecdysone (20E), juvenile hormone (JH) plays essential roles in reproduction in adults. While the molecular mechanisms of JH action in insect reproduction remains largely unknown. The migratory locust, Locusta migratoria is a destructive agricultural insect because of its high fecundity. In many insect species including fruit fly, mosquito, cockroach and beetles, JH and 20E coordinate their reproduction. While in locust, JH acts independently of 20E to stimulate vitellogenesis, ovary development and oocyte maturation. Thus, locust is the ideal model to investigate the molecular mechanisms of JH regulation on insect reproduction. The migratory locust ovary consists of panoistic ovarioles without nurse cells in the follicles. Vitellogenin (Vg) for oocytes maturation is synthesized in the fat body, secreted into the hemolymph and processed into mature vitellin, taken by the developing oocytes through the patency among the follicle cells. Previous studies have demonstrated that fat body and follicle cells undergo endocycle during the first gonadotrophic cycle after locust adult eclosion. The high polyploidy cells work on locust reproduction and are regulated by JH, while the molecular mechanisms are unknown.To elucidate the regulation of JH on locust reproduction, we performed RNA-seq to identify differential genes expression (DEG) profiles between JH-deprived fat bodies and JH-restored via further treated with JH analog. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated DNA replication and cell cycle pathways were the top and fourth up-regulated by JH.16 genes enriched in DNA replcation pathway and 13 genes enriched in cell cycle pathway were all up-regulated via QRT-PCR detection. Here we first explored the six Mcm genes since they are essential to DNA replication. After we performed JH-receptor Met RNAi, we found Mcm3, Mcm4 and Mcm7 mRNA levels displayed signficant decrease in Met RNAi females. Analysis of upstream regions of Mcm4 and Mcm7, we identified E-box-like and E-box characteristic DNA motifs indicating the recognition by Met. Luciferase assays and electrophoresis mobility shift assays (EMSA) proved JH receptor complex Met/SRC could bind with the E-box or E-box-like motifs and directly activate their transcription. RNAi ofMcm4 and Mcm7 caused retardation of DNA replication and polyploidy in the fat body cells, inhibition of Vg expression in the fat body and further ovarian growth and oogenesis, which were similar to the phenotypes of JH deprivation and Met RNAi. Based on these results, we proposed that Mcm4 and Mcm7 make diffenreces in JH-involved locust reproduction. Met/SRC complex transduce JH roles through direct regulating transcription of Mcm4 and Mcm7 to affect DNA replication and polyploidy and further regulate vitellogenesis, ovarian development and oocyte maturation.Loading of the Mcm2-7 onto the origin of pre-replication complex to initiate DNA replication requires cell-division-cycle 6 (Cdc6), which was upregulated in the JH-induced DEG profiles. Given the essential functions of Cdc6 in DNA replication and cell cycle check, we observed the transcriptional regulation and importance in JH-involved locust reproduction. QRT-PCR informed that Cdc6 mRNA levels showed increase under JH induction but derease under Met RNAi. Analysis and clone of promoter region of Cdc6 gene revealed and E-box-like motif located from-1063 to-1058 bp. EMSA using nuclear extracts from dsMet-treated fat bodies and Drosophila S2 cells both demonstrated Met binds to the 20-mer E-box-like DNA motif located in the promoter of Cdc6. Luciferase reporter assays show that JH/Met/SRC complex directly activate Cdc6 expression. Cdc6 RNAi and cytometry experiments told that Cdc6 depletion led decrease of fat body and follicle cells ploidy as well as emergence of cell division. Cdc6-depleted locusts displayed inhibition of Vg expression, ovarian development and oocyte maturation, and were unable to restore to normal levels. Those data indicate that Cdc6 participates in JH-involved cell polyploidy and reproduction. Under JH induction, Cdc6 along with Mcm4 and Mcm7 increased DNA replication and cell ploidy to replicate gene copies including Vg, further promote massive synthesis of Vg to induce the maturation of primary oocytes.Besides, we carried out priliminary research on E2fl, a regulator on endocycle. E2fl RNAi induced multi-nuclei and dereased ploidy of fat body and follicle cells. Similar to Cdc6, Mcm4 and Mcm7, E2fl depletion led significant inhibition of Vg expression, ovarian development and oocyte maturation, as well as cell ploidy. Next, we will decipher E2fl transcriptional regulation, and how E2f1 functions on endocycle and polyploidy.In summary, our study has uncovered a key mechanism that JH acts via Met/SRC to directly modulate the expression of Mcm4, Mcm7 and Cdc6, further develop fat body and follicle cells polyploidy and promoter massive synthesis of Vg and oocyte maturation in the end.