Caenorhabditis elegans inositol 5-phosphatase homolog negatively regulates inositol 1,4,5-triphosphate signaling in ovulation

Abstract

Ovulation in Caenorhabditis elegans requires inositol 1,4,5-triphosphate (IP(3)) signaling activated by the epidermal growth factor (EGF)-receptor homolog LET-23. We generated a deletion mutant of a type I 5-phosphatase, ipp-5, and found a novel ovulation phenotype whereby the spermatheca hyperextends to engulf two oocytes per ovulation cycle. The temporal and spatial expression of IPP-5 is consistent with its proposed inhibition of IP(3) signaling in the adult spermatheca. ipp-5 acts downstream of let-23, and interacts with let-23-mediated IP(3) signaling pathway genes. We infer that IPP-5 negatively regulates IP(3) signaling to ensure proper spermathecal contraction.

Figure 1

IP₃-mediated ovulation pathway. Ovulation is dependent on an IP₃-mediated pathway that is activated by LIN-3 EGF and LET-23 EGFR, which likely stimulates hydrolysis of PIP₂ into IP₃. LFE-1 encodes an IP₃R homolog, which plays a known role in releasing intracellular calcium. LFE-2 encodes an IP₃K that phosphorylates IP₃ into IP₄ (Clandinin et al., 1998). IPP-5 encodes a 5-phosphatase (5-Ptase), which likely dephosphorylates IP₃ into IP₂.

Figure 2

Molecular characterization of ipp-5. (A) Genomic structure of ipp-5. CO9B8.1 corresponds to the predicted ORF, determined by the Genefinder Program, to encode a C. elegans homolog of the inositol polyphosphate 5- phosphatase. Exons are shown as white boxes. The sequence is inferred from the cDNA (generously provided by Y. Kohara). The numbers above each intron indicate the base pair position relative to itself. The indicated region deleted in sy605 spans 240 bp upstream of the start ATG codon through exon 3. (B) Protein sequence alignment of the Drosophila, dog, human, and C. elegans homologs of the type I inositol 5-phosphatase and the C. elegans and human homologues of the type II 5-phosphatase. A Drosophila homolog has not been identified genetically, but Genie has predicted an ORF (GenBank accession AAF56383) in the sequenced genome. Identical residues are darkly shaded, and conserved substitutions are lightly shaded. The signature motif present in all 5-phosphatase family members is underlined in black. The motif well conserved among all type II phosphatases is double underlined. The residues deleted in ipp-5 are indicated by a dashed underline.

Figure 3

ipp-5(sy 605) effects on ovulation. Still-frame series of Nomarski photomicrographs of a hermaphrodite (A–C) wild-type and (D–F) ipp-5 gonad during ovulation. The wild-type images have been flipped horizontally so the orientation matches that of the ipp-5 images. The uterus is toward the left and the proximal gonad is toward the right. The ipp-5 phenotype shows two oocytes, indicated by white arrowheads, being ovulated in the spermatheca per cycle; see Supplementary Information Videos 2 and 5. Nomarski photomicrographs of (G) lfe-1/itr-1; ipp-5 and (H) lfe-2; ipp-5. Double mutants between ipp-5 and lfe-X cause the endomitotic oocyte nuclei (Emo) phenotype, indicated by black arrowheads. “Sp” denotes the position of the spermatheca, and “Oo” denotes the oocyte. Animals were photographed using a ×100 objective and differential interference contrast optics. Scale bar, 20 μm.

Figure 4

Fate of secondary oocyte in ipp-5(sy605) animals. In sy605 mutants, the second oocyte is ovulated along with the proximal oocyte and is passed into the uterus. The secondary oocyte has not undergone maturation, as the nucleolus (arrowhead) and the nuclear envelope (arrow) are still present. The proximal oocyte that was ovulated is at the one-cell (1c) stage. A two-cell embryo is also shown (2c). Scale bar, 20 μm.

Figure 5

Expression of ipp-5. Nomarski photomicrographs of a transgenic worm and their corresponding GFP fluorescence photomicrographs. The white arrowhead indicates position of the distal spermatheca, and the white arrow indicates the sheath. (A and B) A worm before ovulation. (C and D) A transgenic sy605 worm during ovulation. The arrowhead indicates the distal spermatheca enclosing two oocytes in a sy605 transgenic animal. Strong expression is detected in the distal spermatheca, whereas faint expression is detected in the sheath. Fertilized embryos are to the left, and oocytes lining up in the proximal gonad are to the right. Scale bar, 20 μm.