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First published online 2 November 2005
doi: 10.1242/dev.02083

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Eph and NMDA receptors control Ca²⁺/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (50K): [in a new window]   Fig. 1. Models and MPK-1 MAPK activation. (A) In the proximal gonad, somatic ovarian sheath cells surround the oocytes, which are in diakenesis of meiotic prophase. Sperm are stored in the spermatheca and export the cytoskeletal protein MSP to induce oocyte meiotic maturation, MPK-1 MAPK activation and sheath contraction. The maturing oocyte signals the dilation of the spermathecal valve, and the oocyte is fertilized as it enters the spermatheca. (B) Simplified depiction of the block versus switch models for the control of oocyte maturation (MAT). See text for details. (C-I) MAPK-YT staining of wild-type and mutant gonads. (C) In wild-type gonads, sperm trigger MPK-1 MAPK activation in the oocyte(s) most proximal to the spermatheca. (D) In itr-1(sa73) hermaphrodites, more oocytes contain activated MAPK than in the wild type. (E) MAPK activation is not observed in unmated fog-3(q443) females. (F) By contrast, MAPK activation is frequently observed in unmated unc-43(n498gf); fog-3(q443) females. (G) More oocytes contain activated MAPK in vab-1(dx31) hermaphrodites, a phenotype identical to itr-1(sa73) hermaphrodites (D). (H) In itr-1(sy290gf) unc-24(e138) hermaphrodites, fewer oocytes contain activated MAPK than control hermaphrodites. (I) The MAPK activation frequency in vab-1(dx31); itr-1(sy290gf) unc-24(e138) hermaphrodites is similar to itr-1(sy290gf) unc-24(e138) hermaphrodites, suggesting that itr-1 acts downstream of vab-1. All gonads are oriented as shown in A. The MAPK activation pattern in G has previously been reported elsewhere (Miller et al., 2003). Scale bar: 10 µm.

View larger version (121K): [in a new window]   Fig. 2. DIC micrographs of itr-1 and unc-43 gain-of-function female gonads. (A) In unmated fog-3(q443) female gonads, the oocyte maturation rate is slow and prophase-arrested oocytes accumulate in the proximal gonad (pg) to the left of the spermatheca (sp). Few oocytes that have completed maturation and ovulation are observed in the uterus (ut; no oocytes are seen in A). (B) The maturation rate of young unmated itr-1(sy290gf) unc-24(e138); fog-3(q443) females is slow and their gonads resemble young unmated fog-3(q443) females. (C) In contrast to unmated itr-1(sy290gf) and control females, the maturation rate of young unmated unc-43(n498gf); fog-3(q443) females is high. Numerous oocytes that have recently completed maturation and ovulation are observed in the uterus (arrows). Scale bar: 20 µm.

View larger version (86K): [in a new window]   Fig. 3. MSP-binding site distribution and expression of GFP::NMR-1 and VAB-1::GFP in the proximal gonad. (A) Fluorescein-labeled MSP (MSP-FITC) binds to oocytes and sheath cells of proximal gonads. In deconvolved mid-focal plane images, fluorescent intensity is enriched at the oocyte/oocyte interface. (B) A 3D reconstruction of the stack shows similar results. (C) The fluorescent intensity is high at membrane sites between oocytes even when the sheath cells are removed. (D) By contrast, MSP-FITC appears uniformly distributed around the oocyte plasma membrane in isolated oocytes. (E-G) In GFP::NMR-1-expressing gonads, GFP fluorescence is observed at the oocyte plasma membrane (E). Extensive co-localization with F-actin (F) is observed in the merged image (G). (H-J) VAB-1::GFP expressing gonads show a similar pattern of fluorescence. (K-M) GFP fluorescence is not observed in control gonads. (N,O) By focusing on the sheath membrane, GFP fluorescence is detected in the sheath of GFP::NMR-1-expressing gonads (N), but not in control gonads (O). Arrows indicate the sheath cells. All gonads are oriented as shown in Fig. 1A. DAPI is included in E-M. The VAB-1::GFP expression pattern has been reported previously (Miller et al., 2003). Scale bars: 10 µm.

View larger version (90K): [in a new window]   Fig. 4. UNC-43 CaMKII activation in the proximal gonad. (A) The rat sequence used to generate CaMKII-pT is highly conserved in C. elegans UNC-43, including the phosphorylated threonine (*, T284 in UNC-43 and T286 in rat CaMKII). (B-D) CaMKII-pT stains the oocytes of wild-type gonads. In mid-focal plane images, staining co-localizes with F-actin at the oocyte cortex. (E-G) CaMKII-pT staining is not observed in unc-43(n1186)-null gonads. (H-J) At the sheath and oocyte interface of wild-type gonads, CaMKII-pT staining is observed in the same focal plane as sheath cell F-actin. (K) In contrast to CaMKII-pT, MAPK-YT staining is uniformly distributed throughout the cytoplasm, and sometimes in the nucleus. (L) CaMKII-pT stains sheath cells surrounding the sperm in fem-3(q20) gonads. The genetic requirements of UNC-43 T284 phosphorylation are shown in M-U. (M) No CaMKII-pT staining is observed in unmated fog-3(q443) females, indicating that sperm are required for UNC-43 T284 phosphorylation. (N,O) CaMKII-pT stains unmated fog-2(q71) female gonads microinjected with 100 nM MSP (N), but not buffer-injected control gonads (O). (P) CaMKII-pT staining is not observed in unmated vab-1 RNAi ceh-18(mg57) females, which undergo constitutive oocyte maturation independent of sperm presence. CaMKII-pT staining is not observed in vab-1(dx31) (Q) and nmr-1(ak4) gonads (S), but it is observed in vab-1(e2) kinase dead (R), itr-1(sa73) (T) and itr-1(sy290gf) (U) gonads. All images in B-M were processed with deconvolution software and include DAPI to visualize DNA. All gonads are oriented as shown in Fig. 1A. Scale bars: 10 µm.

View larger version (22K): [in a new window]   Fig. 5. Working models of the MSP signaling mechanism. MSP binds to multiple receptors on the oocyte plasma membrane (PM), including the VAB-1 Eph receptor. (A) When extracellular MSP is scarce or absent, ephrin/VAB-1- and CEH-18-dependent pathways negatively regulate MPK-1 MAPK activation and oocyte maturation. The ITR-1 IP3 receptor acts downstream of VAB-1, while the NMR-1 NMDA-receptor subunit prevents activation of UNC-43 CaMKII. (B) MSP binding to VAB-1 triggers a switch (SW) from negative to positive regulation. As a result, NMR-1 stimulates UNC-43 T284 phosphorylation and signaling at the oocyte cortex. MSP also binds to unidentified MSP receptors (MSPRs) that act redundantly to promote oocyte maturation. MSPRs could function in oocytes (shown), sheath cells or both. Broken lines indicate downregulated pathways. See text for details. Models are based on results from this study and previous studies. SW, switch; GJ, gap junction; TF, transcription factor; IP3R, inositol triphosphate receptor.