MSP hormonal control of the oocyte MAP kinase cascade and reactive oxygen species signaling

Abstract

The MSP domain is a conserved immunoglobulin-like structure that is important for C. elegans reproduction and human motor neuron survival. C. elegans MSPs are the most abundant proteins in sperm, where they function as intracellular cytoskeletal proteins and secreted hormones. Secreted MSPs bind to multiple receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. MSP binding stimulates oocyte MPK-1 ERK MAP Kinase (MAPK) phosphorylation, but the function and mechanism are not well understood. Here we show that the Shp class protein-tyrosine phosphatase PTP-2 acts in oocytes downstream of sheath/oocyte gap junctions to promote MSP-induced MPK-1 phosphorylation. PTP-2 functions in the oocyte cytoplasm, not at the cell surface to inhibit multiple RasGAPs, resulting in sustained Ras activation. We also provide evidence that MSP promotes production of reactive oxygen species (ROS), which act as second messengers to augment MPK-1 phosphorylation. The Cu/Zn superoxide dismutase SOD-1, an enzyme that catalyzes ROS breakdown in the cytoplasm, inhibits MPK-1 phosphorylation downstream of or in parallel to ptp-2. Our results support the model that MSP triggers PTP-2/Ras activation and ROS production to stimulate MPK-1 activity essential for oocyte maturation. We propose that secreted MSP domains and Cu/Zn superoxide dismutases function antagonistically to control ROS and MAPK signaling.

Figure 1

The C. elegans proximal gonad and ptp-2 mutant phenotype. (A) Diagram of the wild-type hermaphrodite proximal gonad. The oocyte closest to the spermatheca (Sp) is called the −1 oocyte. Sperm (red) secrete the protein hormone MSP (black dots) to induce MPK-1 phosphorylation, oocyte maturation, and sheath cell contraction, which together facilitate ovulation. In fog-2(q71) and fog-3(q443) mutant females, all germ cells develop into oocytes, which arrest in diakinesis of meiotic prophase in the absence of male sperm. Oocytes mature sequentially every 20–25 minutes in the presence of sperm/MSP. (B) DIC micrograph of wild-type and ptp-2(op194) mutant gonads. The arrowhead points to the oocyte adjacent to the spermatheca (−1 oocyte). The arrow points to a fertilized embryo. Notice that the single fertilized embryo in the ptp-2 mutant uterus is large and has completed multiple cell divisions, contrasting with the smaller wild-type embryo that has not yet divided. (C) DIC and DAPI micrographs of an unmated ptp-2(op194); fog-2(q71) female. The arrowheads indicate diakinesis stage oocytes.

Figure 2

Molecular characterization of wild-type hermaphrodite, ptp-2 mutant hermaphrodite, and unmated female proximal gonads. The proximal gonad is in the same orientation as Fig. 1A, with the spermatheca to the right. The MAPK-YT, anti-Nop1, and anti-pH3 antibodies are described in the text. Bars, 20μm.

Figure 3

MPK-1 MAPK activity in wild-type and mutant gonads. Dissected proximal gonads were incubated with MAPK-YT antibodies, which recognize the diphosphorylated form of MPK-1. Panels are (A) wild type, (B) ptp-2(op194), (C) ptp-2-(op194); let-60(n1046gf), (D) let-60(n1046gf), (E) unmated let-60(n1046); fog-2(q71) female, (F) ptp-2(op194);gap-1(ga133);gap-2(tm748), (G) vab-1 RNAi, (H) vab-1 RNAi ptp-2(op194), and (I) ptp-2(op194); Pie-1p::gfp::ptp-2. Gonad orientation is with spermatheca to the right. Bars, 20μm.

Figure 4

Genetic relationship between ptp-2 and the innexin inx-22. (A, B) DIC micrographs of (A) unmated inx-22(tm1661); fog-2(q71) females and (B) unmated ptp-2(op194); inx-22(tm1661); fog-2(q71) females. Arrowheads indicate unfertilized oocytes in the uterus that have undergone oocyte maturation and ovulation. Gonad orientation is with spermatheca to the right. (C) Quantification of oocyte maturation rates. N is shown above error bars, which represent standard deviation.

Figure 5

ptp-2 expression. (A–E) Genomic DNA (~5kb) upstream of the ptp-2 translational start site drives GFP expression in embryonic cells (A), the developing vulva (B, arrow), adult body wall muscle (C, arrow), adult head neurons (D), and adult gonadal sheath cells (E). Bars, 10 μm.

Figure 6

Germline GFP::PTP-2 expression rescues ptp-2(op194) phenotypes. (A, B) GFP::PTP-2 expression, driven with the pie-1 promoter, is faint, but detectable in the germ line of adult hermaphrodites (A). GFP is observed throughout the germ cell and oocyte cytoplasm, but not in the nucleus or at the cell surface. In dissected gonads, GFP appears to localize to vesicles in the oocytes (B). Gonad orientation is with spermatheca to the right. (C) The transgene partially rescues the oocyte maturation rate, embryonic lethality, and larval lethality defects of ptp-2(op194) mutants. N is shown above error bars, which represent standard deviation. Bars, 10 μm.

Figure 7

Cu/Zn superoxide dismutase activity antagonizes oocyte MPK-1 phosphorylation. (A) Multiple oocytes in sod-1(tm776) hermaphrodite gonads contain phosphorylated MPK-1, detected using the MAPK-YT antibody. (B–C) In ptp-2(op194) mutants, sod-1 RNAi (B) and sod-1 sod-5 double RNAi (C) partially rescue the MPK-1 phosphorylation defect. Phosphorylated MPK-1 is not observed in ptp-2(op194) gonads (see Fig. 3B and H). Gonad orientation is with spermatheca to the right. (D) Quantification of oocyte MPK-1 phosphorylation levels in proximal gonads. N is shown above error bars, which represent standard deviation. Bars, 20μm.

Figure 8

Sperm promote increased reactive oxygen species levels. (A) Unmated females are more resistant than wild-type hermaphrodites to paraquat-induced lethality. Paraquat generates intracellular superoxide that becomes toxic depending on endogenous ROS levels and superoxide dismutase activity. (B–G) The ROS indicator H₂-DCF-DA shows enhanced fluorescence in wild-type dissected gonads (B, D) and live animals (F) compared to unmated fog-2(q71) female dissected gonads (C, E) and live animals (G). Dissected gonads in panels D and E were lightly fixed prior to mounting, causing a reduction in fluorescent intensity (see Methods). Gonad orientation is with spermatheca to the right. Bars, 20μm.

Figure 9

Oocyte mitochondrial function in the presence and absence of sperm. (A, B) Dissected proximal wild-type (A) and unmated fog-2(q71) female (B) gonads stained with Mitotracker CMXRos. Gonad orientation is with spermatheca to the right. The bright fluorescence in the wild-type spermatheca (panel A) is sperm mitochondria (arrow). Bar, 20μm. (C, D) Oxygen consumption rates in wild-type hermaphrodites (WT) and unmated fog-2(q71) females normalized to worm number (C) and protein content (D). The P values calculated using a Student’s T-test are 0.02 for panel C and 0.5 for panel D.