Abstract
In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition. For successful insect vitellogenesis and oogenesis, vitellogenin (Vg) synthesized in the fat body (homologue to vertebrate liver and adipose tissue) must pass through the intercellular channels, a condition known as patency in the follicular epithelium, to reach the surface of oocytes. This process is controlled by juvenile hormone (JH) in many insect species, but the underlying mechanisms remain elusive. Previous work has suggested the possible involvement of Na+/K+-ATPase in patency initiation, but again, the regulatory cascade of Na+/K+-ATPase for patency initiation has been lacking. Using the migratory locust Locusta migratoria as a model system, we report here that RNAi-mediated knockdown of gene coding for Na+/K+-ATPase, inhibition of its phosphorylation, or suppression of its activity causes loss of patency, resulting in blocked Vg uptake, arrested oocyte maturation, and impaired ovarian growth. JH triggers G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate receptor (IP3R), and protein kinase C (PKC) to phosphorylate Na+/K+-ATPase α-subunit at amino acid residue Ser8, consequently activating Na+/K+-ATPase for the induction of patency in vitellogenic follicular epithelium. Our results thus point to a previously unidentified mechanism by which JH induces the phosphorylation and activation of Na+/K+-ATPase via a signaling cascade of GPCR, RTK, PLC, IP3R, and PKC. The findings advance our understanding of JH regulation in insect vitellogenesis and oogenesis.
Highlights
In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition
We demonstrated that juvenile hormone (JH) triggered a G protein– coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), IP3R, and protein kinase C (PKC) pathway to phosphorylate the ␣-subunit at Ser8, activating Naϩ/Kϩ-ATPase for the induction of patency during vitellogenesis
As the role of Naϩ/Kϩ-ATPase in locust vitellogenesis and oogenesis had not been previously determined by gene knockdown, we initially performed Naϩ/Kϩ-ATPase ␣-subunit (GenBankTM number MH450018) RNAi in vitellogenic adult female locusts. qRT-PCR showed that the mRNA levels of Naϩ/ Kϩ-ATPase ␣-subunit were reduced by 81% in the ovary of adult females at 6 days post-adult eclosion (PAE) (Fig. 1A)
Summary
Vitellogenin; JH, juvenile hormone; IP3, inositol trisphosphate; IP3R, inositol trisphosphate receptor; PKC, protein kinase C; PKA, protein kinase A; RTK, receptor tyrosine kinase; PLC, phospholipase C; IP3R, inositol trisphosphate receptor; CaMKII, calcium/ calmodulin-dependent protein kinase II; GPCR, G protein– coupled receptor; qRT-PCR, quantitative RT-PCR; PAE, post-adult eclosion; 2-APB, 2-aminoethoxydiphenyl borate; CC, chelerythrine chloride. Protein kinase C (PKC) mediates the phosphorylation of ␣-subunit at Ser, Ser, or Ser to enhance or repress the catalytic action of Naϩ/Kϩ-ATPase in vertebrates (26 –30). JH is reported to trigger receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate (IP3), and calcium/calmodulin-dependent protein kinase II (CaMKII) to stimulate the phosphorylation of Met for enhanced transcriptional regulation activity in A. aegypti (40 –42). We demonstrated that JH triggered a GPCR, RTK, PLC, IP3R, and PKC pathway to phosphorylate the ␣-subunit at Ser, activating Naϩ/Kϩ-ATPase for the induction of patency during vitellogenesis. These results shed some light on the regulatory mechanisms of JH-dependent vitellogenesis and oogenesis in insects
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