Requirement of V-ATPase for Ovulation and Embryogenesis inCaenorhabditis elegans

Toshihiko Oka,Masamitsu Futai

doi:10.1074/jbc.m002756200

Abstract

Immunofluorescence analysis indicated that VHA-11, the C subunit of Caenorhabditis elegans V-ATPase, was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage. Vital staining with acridine orange showed that the intestinal cells had acidic compartments generated by V-ATPase, consistent with the intracellular localization of VHA-11. RNA interference could efficiently silence vha-11 gene expression: introduction of vha-11 double strand RNA led to embryonic lethality. Worms injected with the vha-11 double strand RNA produced embryos that became lethal. The development of embryos was arrested at various stages. However, their numbers gradually decreased, and the worms eventually became sterile due to the failure of ovulation. Similar results were obtained for RNA interference of the V-ATPase proteolipid genes. These results suggest that V-ATPases, and thus inside-acidic organelles, are required for ovulation and embryogenesis.

Highlights

Vacuolar-type proton translocating ATPase (V-ATPase)1 is a ubiquitous enzyme responsible for the acidification of cellular compartments in eukaryotic cells
We observed that VHA-11, the C subunit of V-ATPase, was predominantly localized in the intracellular organelles of intestinal cells during late embryogenesis and that the generation of acidic compartments in the intestine was dependent on vha-11 expression
These results indicated that the antibodies recognized a 40-kDa vha-11 gene product

Summary

The abbreviations used are

V-ATPase, vacuolar-type Hϩ-ATPase; DAPI, 4Ј,6-diamidino-2-phenylindole dihydrochloride n-hydrate; dsRNA, double strand RNA; GFP, green fluorescence protein; kb, kilobase(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; UTR, untranslated region. Nantly expressed in H-shaped excretory cells contributing to the excretory system in adult worms. All vha genes are highly expressed in embryonic stages, their functions are unknown. We observed that VHA-11, the C subunit of V-ATPase, was predominantly localized in the intracellular organelles of intestinal cells during late embryogenesis and that the generation of acidic compartments in the intestine was dependent on vha-11 expression. Double strand RNA interference was used to silence V-ATPase gene-specific expression. Worms injected with vha-11 double strand RNA became sterile, due to the failure of ovulation. The oocytes underwent multiple rounds of DNA replication without cytokinesis and became polyploid, possibly because ovulation was not coupled with oocyte maturation in the injected worms, suggesting that V-ATPase activity or inside-acidic organelles are essential for oocyte maturation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION