Abstract
Juvenile hormone (JH) has a well known role in stimulating insect vitellogenesis (i.e. yolk deposition) and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear. The 78-kDa glucose-regulated protein (Grp78) is a heat shock protein 70-kDa family member and one of the most abundant chaperones in the endoplasmic reticulum (ER) where it helps fold newly synthesized peptides. Because of its prominent role in protein folding, and also ER stress, we hypothesized that Grp78 might be involved in fat body cell homeostasis and vitellogenesis and a regulatory target of JH. We report here that the migratory locust Locusta migratoria possesses two Grp78 genes that are differentially regulated by JH. We found that Grp78-1 is regulated by JH through Mcm4/7-dependent DNA replication and polyploidization, whereas Grp78-2 expression is directly activated by the JH-receptor complex comprising methoprene-tolerant and Taiman proteins. Interestingly, Grp78-2 expression in the fat body is about 10-fold higher than that of Grp78-1 Knockdown of either Grp78-1 or Grp78-2 significantly reduced levels of vitellogenin (Vg) protein, accompanied by retarded maturation of oocytes. Depletion of both Grp78-1 and Grp78-2 resulted in ER stress and apoptosis in the fat body and in severely defective Vg synthesis and oocyte maturation. These results indicate a crucial role of Grp78 in JH-dependent vitellogenesis and egg production. The presence and differential regulation of two Grp78 genes in L. migratoria likely help accelerate the production of this chaperone in the fat body to facilitate folding of massively synthesized Vg and other proteins.
Highlights
Juvenile hormone (JH) has a well known role in stimulating insect vitellogenesis and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear
We demonstrated in this study that loss of Grp78 function resulted in substantial reduction of Vg, accompanied with arrested oocyte maturation and ovarian growth
The maturation of pre-vitellogenic adult insects is characterized by the enhanced protein synthesis in the fat body required for oocyte maturation and successful reproduction [2, 13]
Summary
The sequenced locust genome [53] yielded two Grp genes, Grp (GenBankTM FJ472842) and Grp (GenBankTM FJ472843). To explore the spatial expression patterns of two Grp genes in locusts, qRT-PCR was conducted using total RNA from the fat body (Fb), ovary (Ov), midgut (Md), and brain (Br) of adult females collected at 8 days PAE. JHA-induced dimerization of FLAGMet and V5-Tai led to a 4-fold increase of wild-type Grp reporter activity without increase of the E-box-like motif mutant compared with the JHA-untreated group (Fig. 3D) These data suggest that the transcription of Grp is activated by the JH-receptor complex. Western blot analysis and subsequent quantification of band intensity demonstrated that depletion of both Grp and Grp caused 99.1 and 99.9% reduction of VgA protein levels in the fat body and ovary, respectively (Fig. 4D). Grp RNAi reduced VgA proteins to 7.8 and 1.1% of the control levels in the fat body and ovary, respectively (Fig. 4D).
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