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. 2022 Jun 27;18(6):e1010292.
doi: 10.1371/journal.pgen.1010292. eCollection 2022 Jun.

Juvenile hormone promotes paracellular transport of yolk proteins via remodeling zonula adherens at tricellular junctions in the follicular epithelium

Hongyuan Zheng  1 , Ningbo Wang  1 , Jiaqi Yun  1 , Huijing Xu  1 , Jiebing Yang  1 , Shutang Zhou  1
Affiliations

Juvenile hormone promotes paracellular transport of yolk proteins via remodeling zonula adherens at tricellular junctions in the follicular epithelium

Hongyuan Zheng et al. PLoS Genet. .

Abstract

Juvenile hormone (JH) acts as a gonadotrophic hormone stimulating insect vitellogenesis and oogenesis. Paracellular transport of yolk proteins through intercellular channels (patency) in the follicular epithelium is a developmentally regulated and evolutionarily conserved process during vitellogenesis. However, the mechanisms underlying patency opening are poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH-regulated remodeling of zonula adherens (ZA), the belt-like adherens junction maintaining physical linking between follicle cells controlled the opening of patency. JH triggered phosphorylation of Partitioning defective protein 3 (Par3) via a signaling cascade including G protein-coupled receptor (GPCR), small GTPase Cell division cycle 42 (Cdc42) and atypical Protein kinase C (aPKC). Par3 phosphorylation resulted in its disassociation from β-Catenin, the cytoplasmic partner of ZA core component E-Cadherin. Release of Par3 from the β-Catenin/E-Cadherin complex caused ZA disassembly at tricellular contacts, consequently leading to patency enlargement. This study provides new insight into how JH stimulates insect vitellogenesis and egg production via inducing the opening of paracellular route for vitellogenin transport crossing the follicular epithelium barrier.

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Conflict of interest statement

The authors declare that they have no competing interests exist.

Figures

Fig 1
Fig 1. Dynamics of β-Cat distribution along with patency in the follicular epithelium.
(A) Subcellular localization of β-Cat in the follicular epithelium of adult females during the first gonadotropic cycle. PAE, post adult eclosion; Arrow heads indicate patency. Large square is the amplification of small square. Ol, ovariole; PO, primary oocyte. Scale bars: ovariole, 0.5 mm; follicular epithelium, 5 μm. (B) Patency in cultured follicular epithelia of 7-day-old adult females treated by JH at 0.1 μM for 1 h. Scale bars: 5 μm. (C) Statistical analysis of patency index in cultured follicular epithelium of 7-day-old adult females. **, P < 0.01 when compared to the DMSO control. n = 8–10. (D) Subcellular localization of β-Cat in cultured follicle epithelium of 7-day-old adult females under JH treatment. Arrow heads indicate patency. Scale bars: 5 μm.
Fig 2
Fig 2. Effect of β-Cat, Par3, aPKC and Par6 knockdown on patency and vitellogenesis.
(A) qRT-PCR and Western blot showing RNAi-mediated knockdown efficiency of β-Cat (iβ-Cat), Par3 (iPar3), aPKC (iaPKC) and Par6 (iPar6) in the ovary of adult females at 8 days PAE. *, P < 0.05. n = 6–8. (B) Representative phenotypes of follicular epithelium (FE), primary oocytes (Po), ovarioles (Ol) and ovaries (Ov) of adult females subjected to β-Cat, Par3, aPKC or Par6 RNAi. Arrow heads indicate patency. Empty arrow heads indicate break of bicellular junctions. Scale bars: ovary, 5 mm; ovariole, 0.5 mm; follicular epithelium, 5 μm.
Fig 3
Fig 3. JH-induced phosphorylation of aPKC and Par3 as well as zonula adherens disassembly.
(A) The levels of phosphorylated aPKC (p-aPKC) and Par3 (p-Par3) in the follicular epithelium of adult females during the first gonadotropic cycle. (B) Phosphorylation of aPKC and Par3 in cultured follicular epithelia of 7-day-old females treated by JH at 0.1 μM for 5–30 min. (C) Suppression of ACPD on JH-induced aPKC and Par3 phosphorylation in cultured follicle cells of 7-day-old females. ACPD was applied at 0.1 μM prior to JH treatment. (D) Distribution of p-aPKC in cultured follicular epithelium from adult females on day 8 and treated with DMSO, JH and ACPD+JH, respectively. Blue: nuclei. Green: F-actin. Arrow heads indicate patency. Scale bars: 5 μm. (E) Distribution of p-Par3 in cultured follicular epithelia from adult females on day 8 and treated with DMSO, JH and ACPD+JH, respectively. Arrow heads indicate patency. Scale bars: 5 μm. (F) Immunoprecipitation (IP) and Western blotting (WB) showing inhibition of ACPD on JH-induced disassociation of p-Par3 and β-Cat. (G) Distribution of β-Cat in the follicular epithelium treated with DMSO, JH and ACPD+JH. Arrow heads indicate patency. Scale bars: 5 μm.
Fig 4
Fig 4. Effect of ACPD treatment on JH-induced patency and vitellogenin transport.
(A) Effect of ACPD treatment on JH-induced patency in cultured follicular epithelium isolated from adult females on day 7. Arrow heads indicate patency. Scale bars: 5 μm. (B) Statistical analysis of patency index in cultured follicular epithelium as represented by Fig 4A. Means labeled with different letters indicate significant difference at P < 0.05. n = 8–10. (C) Effect of ACPD on patency, oocyte maturation and ovarian growth of 7-day-old adult females. Vitellogenic adult females were injected with 40 μg ACPD once per day from 5 to 7 days PAE and phenotypes were evaluated 6 h after the final injection. Ov, ovary; Ol, ovariole; Po, primary oocyte. Arrow heads indicate patency. Scale bars: ovary, 5 mm; ovariole, 0.5 mm; follicular epithelium, 5 μm. (D) Statistical analysis of patency index in the follicular epithelium of 7-day-old adult females subjected ACPD injection vs. the DMSO control. *, P < 0.05. n = 8–10. (E) Statistical analysis for the index of primary oocytes of ACPD- and DMSO-treated adult females at 7 days PAE. **, P < 0.01 when compared to the DMSO control. n = 8–10. (F) Western blot showing the abundance (upper panel) and quantitative analysis of band intensity (lower panel) of vitellogenin proteins in the fat body (FB), hemolymph (Hl) and ovary (Ov) of ACPD-injected adult females vs. the DMSO controls. * P < 0.05 compared to the respective DMSO control. n = 8–10. n.s., no significant difference.
Fig 5
Fig 5. Involvement of Cdc42 in JH-dependent patency and phosphorylation of aPKC and Par3.
(A) Inhibitory role of a Cdc42 specific inhibitor, ML141 (at 10 μM) in JH-induced patency of cultured follicular epithelium from 7-day-old adult females. Arrow heads indicate patency. Scale bars: 5 μm. (B) Statistical analysis of patency index in cultured follicular epithelia treated with JH, ML41+JH and DMSO. Means labeled with different letters indicate significant difference at P < 0.05. n = 8–10. (C) Effect of ML141 treatment on JH-stimulated phosphorylation of aPKC and Par3.
Fig 6
Fig 6. The role of GPCR in patency opening and phosphorylation of aPKC and Par3.
(A) Effect of GPCR inhibitor suramin (1 μM) as well as RTK inhibitors genistein (10 μM) and Su6668 (10 μM) on JH-induced patency of cultured follicular epithelium from 7-day-old adult females. Arrow heads indicate patency. Scale bars: 5 μm. (B) Statistical analysis of patency index in cultured follicular epithelia treated with DMSO, JH, suramin, genistein and Su6668. Means labeled with different letters indicate significant difference at P < 0.05. n = 8–10. (C) Effect of suramin, genistein and Su6668 treatment on JH-stimulated phosphorylation of aPKC and Par3.
Fig 7
Fig 7. A proposed model for JH-stimulated ZA disassembly and vitellogenin transport in the follicular epithelium.
JH promotes Par3 phosphorylation via a signaling cascade including GPCR, Cdc42, Par6 and aPKC. Phosphorylated Par3 disassociates from E-Cad/β-Cat complex, consequently causing ZA disassembly at tricellular vertices and patency opening. Patency allows yolk protein precursors to pass through the barrier of follicular epithelium and reach the surface of maturing oocytes.
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Grants and funding

This work was supported by the National Natural Science Foundation of China grants U1804232 (S.Z.) and U1904103 (H.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.