The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

Abstract

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.

Fig. 1. Schematic diagram of a single adult hermaphrodite gonad arm. The distal gonad arm consists of a large syncytium with the circumferentially arranged nuclei on the surface and a cytoplasmic core, the central rachis (Hirsh et al., 1976). The nuclei in the distal arm are partially enclosed by plasma membranes. Each nucleus and its surrounding cytoplasm is called a germ cell (McCarter et al., 1999).

Fig. 2. LIP-1 expression and localization in the distal gonad arm. LIP-1 antibody staining (in red) and DAPI staining (in blue) in a dissected distal gonad arm of (A) a 2- to 3-day-old wild-type adult hermaphrodite and (B) a lip-1(0) mutant of similar age. (C) Higher magnification view of pachytene germ cells in a wild-type gonad stained with the LIP-1 antibody (in red), the nuclear pore complex antibody mAB414 (Yamada and Kasamatsu, 1993) (in green) and DAPI (in blue). (D) An xz-view of the same region as shown in (C). LIP-1 staining was concentrated in ∼2 µm long rod-like structures originating in the outer region near the pachytene nuclei and facing towards the cytoplasmic core. (E) Co-staining of LIP-1 with the hydrophobic dye Dil that labels the plasma membrane surrounding the pachytene germ cells. LIP-1 was concentrated at the junctions where membranes surrounding individual cells are connected. (A–C) Optical sections through the outer region of the distal syncytial gonad containing the germ cell nuclei. (D and E) Three-dimensional reconstructions of the entire data stacks (for details see Materials and methods). The scale bar in (B) is 20 µm and in (E) 5 µm.

Fig. 3. Activated DP-MPK-1 staining in lip-1(0) gonads. DP-MPK-1 staining (shown in red) and DAPI staining (shown in blue) in (A) wild-type, (B) lip-1(0), (C) fem-2(b245ts), (D) fem-2(b245ts); lip-1(0), (E) let-60(n1046gf) and (F) lip-(0) let-60(n1046gf) gonads. The gonads of 2- to 3-day-old adult hermaphrodites were dissected and stained with an antibody specific for the activated, di-phosphorylated form of MPK-1. The images were recorded with identical camera exposure settings, and contrast adjustments were applied simultaneously to all panels to allow a comparison of the relative signal intensities. The gonads shown in (C) and (D) are from animals that had been raised at 25°C and thus produced no sperm (Edgar and Hirsh, 1985). The gonad shown in (F) is from an animal with the full genotype lip-1(0) unc-24(e138) let-60(n1046gf); zhEx29[lip-1::gfp] that was segreg ated from a lip-1(0) unc-24(e138) let-60(n1046gf)/dpy-20(e1362) unc-22(e66); zhEx29[lip-1::gfp] mother. zhEx29 is a rescuing multicopy transgene that is expressed in most somatic cells including the somatic gonad but not in the germ cells (Berset et al., 2001). For each genotype, a minimum of 20 stained gonad arms were observed, and representative examples are shown. The scale bar in (F) is 20 µm.

Fig. 4. Accelerated oocyte development and endomitotic phenotype in lip-1(0) mutants. Nomarski micrographs of (A) a wild-type, (C) a lip-1(0), (E) a fem-2(b245ts) and (G) a fem-2(b245ts); lip-1(0) posterior gonad. DAPI-stained whole-mount preparation of (B) a wild-type, (D) a lip-1(0), (F) a fem-2(b245ts) and (H) a fem-2(b245ts); lip-1(0) animal. The arrowheads in (B) and (D) point at oocytes in diakinesis in the proximal gonad arm. Note in (C) and (D) the increased number of smaller oocytes that aligned in different planes of focus in the proximal gonad arm, and in (G) the formation of multiple endomitotic germ cells that (H) stained intensely with DAPI and often contained more than one nucleus. The animals shown in (E–H) had been raised at 25°C and thus produced no sperm (Edgar and Hirsh, 1985). The scale bar in (H) is 20 µm.

Fig. 5. Endomitotic phenotype in feminized lip-1(0) mutants. DAPI-stained condensed chromosomes in the nuclei of (A) fem-2(b245ts) and (B) lip-1(0) oocytes that have arrested in diakinesis. Both nuclei contain six bivalents that correspond to the six pairs of homologous chromosomes. (C) DAPI-stained chromosomes in the nucleus of a fem-2(b245ts); lip-1(0) oocyte that failed to arrest in diakinesis. The nucleus contains three bivalents (on the left side) and six univalent chromosomes, indicating that the homologous chromosomes have started to separate before the first meiotic division is completed. (D) A fem-2(b245ts); lip-1(0) oocyte that is undergoing endomitotic DNA replication. The decondensed chromosomes were stained intensely with DAPI. (A–D) Three-dimensional reconstructions of optical sections that were recorded and processed as described in Materials and methods. (E and F) Endomitotic oocytes in dissected fem-2(b245ts); lip-1(0) gonads were observed by Nomarski microscopy. Because of the continuous endomitotic replication, the nuclei were greatly enlarged. The nucleus in the upper oocyte started to divide, but no cytokinesis took place and karyokinesis was incomplete. The oocytes shown in (A) and (C–F) are from animals that had been raised at 25°C. The scale bars in (D) and (F) are 5 and 20 µm, respectively.

Fig. 6. A model for LIP-1 function. The dark grey color indicates the regions where MPK-1 is activated in the germline. (A) Wild-type germ cells that exit pachytene are no longer exposed to an unidentified signal (dark grey arrows) that induces MPK-1 activation during pachytene progression, but they contain LIP-1 (grey T-bars). Thus, MPK-1 is inactivated rapidly by LIP-1 during or before pachytene exit. (B) In lip-1(0) mutants, MPK-1 remains active after pachytene exit, leading to an acceleration of pachytene exit and the formation of endomitotic oocytes in the proximal gonad arm (indicated by uniformly black nuclei).