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First published online 18 January 2006
doi: 10.1242/dev.02241

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The C. elegans Myt1 ortholog is required for the proper timing of oocyte maturation

Anna E. Burrows¹, Bonnielin K. Sceurman¹, Mary E. Kosinski², Christopher T. Richie¹, Penny L. Sadler^1,*, Jill M. Schumacher³ and Andy Golden^1,

¹ Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Building 8, Room 323, Bethesda, MD 20892, USA.
² Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2175, USA.
³ Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA.

View larger version (80K): [in a new window]   Fig. 1. CDK-1 depletion impairs oocyte maturation. (A-F) Wild-type (WT; A,C,E) and CDK-1-depleted (B,D,F) animals stained with pH3 antibodies (A,B), NOP1 antibodies (C,D), and TOTO-3 to visualize DNA (E,F). Scale bar: 20 µm. (G) Quantitation of pH3- and NOP1-positive nuclei by oocyte position in the proximal gonad. In between the graphs is a table showing the number of oocytes analyzed. N2 is the wild-type strain. (H) DIC image of one-cell arrested embryos in the uterus of a CDK-1-depleted adult. (I) One-cell arrested embryos dissected from the uterus of a histone H2B::GFP transgenic animal depleted of CDK-1. The four embryos marked by arrows show the congression of the oocyte chromosomes, similar to metaphase of meiosis I. Older embryos remain arrested as one-cell embryos but their chromosomes have decondensed. spth, spermatheca.

View larger version (80K): [in a new window]   Fig. 2. WEE-1.3 depletion causes precocious oocyte maturation. (A-F) Wild-type (A,C,E) and WEE-1.3-depleted (B,D,F) animals stained with pH3 antibodies (A,B), NOP1 antibodies (C,D), and TOTO-3 (E,F). Scale bar: 20 µm. (G) Quantitation of pH3- and NOP1-positive nuclei by oocyte position in the proximal gonad. Below each graph is a table showing the number of oocytes analyzed. (H) Quantitation of the presence of an intact NE in the -1 oocyte from adult hermaphrodites untreated (WT) or depleted of WEE-1.3 or WEE-1.1 (control). Animals were analyzed 16-20 hours post-injection. n, number of gonad arms analyzed.

View larger version (30K): [in a new window]   Fig. 3. WEE-1.3 depletion causes an increase in the number of oocytes that stain for AIR-2. (A-D) Wild-type (A,B) and WEE-1.3-depleted (C,D) animals stained with an AIR-2 antibody (A,C) and TOTO-3 (B,D). Scale bar: 20 µm. (E) Quantitation of AIR-2-positive nuclei by oocyte position in the proximal gonad. Below the graph is a table showing the number of oocytes analyzed.

View larger version (65K): [in a new window]   Fig. 4. GFP::MBK-2 aggregates in WEE-1.3-depleted oocytes. (A,B) Fluorescence images of wild-type GFP::MBK-2 (A) and WEE-1.3-depleted (B) oviducts. The speckled pattern is present in the +1 embryo in A and in the -5 and -6 embryos in B. Scale bar: 20 µm. (C,D) The position of each oocyte with a speckled GFP pattern and a uniformly cortical pattern is indicated for eight representative animals of each. n=19 for the total number of untreated GFP::MBK-2 animals, and n=22 for WEE-1.3 depleted. spth, spermatheca.

View larger version (77K): [in a new window]   Fig. 5. CDK-1 depletion suppresses the infertility of WEE-1.3-depleted animals. (A-D) DIC images of uteri from wild-type (A), CDK-1-depleted (B), WEE-1.3-depleted (C), and co-depleted (D) adult hermaphrodites. Wild-type animals have embryos of all stages (A), whereas CDK-1-depleted animals contain only one-cell arrested embryos (B). WEE-1.3-depleted animals have uteri containing unfertilized `mushy' oocytes (C), whereas the co-depleted animals contain only one-cell arrested embryos (D). Embryos are 50 µm in length. (E,F) Quantitation of (E) pH3- and (F) NOP1-positive nuclei by oocyte position in the proximal gonad. Below each graph is a table showing the number of oocytes analyzed for each RNAi condition.

View larger version (99K): [in a new window]   Fig. 6. WEE-1.3 depletion results in the appearance of aberrant microtubules in developing oocytes. (A-E) Live -tubulin::GFP animals were subjected to wee-1.3 RNAi. (A,B) Untreated animals. (A) Oocytes with typical cytoplasmic tubulin cytoskeletons. (B) A wild-type meiotic spindle; such a structure is normally observed in the oocyte upon fertilization in the spermatheca, or in the +1 embryo in the uterus, and is never observed in WEE-1.3-depleted oocytes in the spermatheca or uterus. (C-E) WEE-1.3-depleted oocytes in the oviduct (C,D) or the uterus (E). White arrows mark the perinuclear tubulin foci. Oocytes in D are outlined in black. (E) Tubulin clouds (marked by carets) form around the coalesced chromosomes of WEE-1.3-depleted oocytes that fail to be fertilized but nonetheless end up in the uterus. The spermatheca is to the right in each gonad shown. Scale bars: in A, 20 µm for A,C-E; 10 µm in B. (F) Quantitation of the number of oocytes with tubulin foci scored by oocyte position in the proximal gonad. spth, spermatheca.

View larger version (60K): [in a new window]   Fig. 7. Oocyte chromosomes do not maintain a diakinetic arrangement upon WEE-1.3 depletion. (A-F) Wild-type (A,B), WEE-1.3-depleted (C-E), and emo-1 (F) animals were DAPI stained to visualize oocyte chromosomes. Arrows and white boxes mark the normal diakinetic arrangement of chromosomes in growing oocytes; those marked with a white box are enlarged in B,D. White lines mark the spermatheca and the highly condensed sperm chromosomes. Carets (^) in C mark the most proximal oocyte chromosomes that have coalesced. (E) The -3 oocyte has stringy chromosomes and is flanked by normal diakinetic oocytes to the right and oocytes with coalesced chromosomes to the left. Asterisk in F marks an endoreplicating oocyte. (G-M) Images of live, untreated (G-J) or WEE-1.3-depleted (K-M) histone H2B::GFP transgenic animals. Diakinetic chromosomes are apparent in wild-type (G-I) and WEE-1.3-depleted (K) animals, and are marked with arrows and white boxes; the one in the white box in G is enlarged in H. The chromosomes of a wild-type fertilized oocyte congress during metaphase I (marked by a circle in I and enlarged in J). The coalesced chromosomes of WEE-1.3-depleted oocytes are marked by white arrowheads (K) and one is enlarged in L. (M) The uterus of a WEE-1.3-depleted animal in which the oocyte chromosomes have begun to endoreplicate. ut, uterus; spth, spermatheca. Scale bars: in A, 20 µm for A,C,F,G,I,K,M; in B, 5 µm for B,D,H,J,L; in E, 5 µm.

View larger version (56K): [in a new window]   Fig. 8. The chromosome coalescence phenotype of WEE-1.3-depleted oocytes is dependent on the presence of sperm. Diakinetic chromosomes (arrows) of oocytes in the proximal gonad are evident in DAPI-stained wild-type hermaphrodites (A), in unmated (C) or mated (D) fog-2 females, and in unmated WEE-1.3-depleted fog-2 females (E). Although the oocytes in fog-2 animals remain in diakinesis (C-E), the chromosomes of WEE-1.3-depleted fog-2 oocytes coalesce into one mass upon the introduction of sperm (F). This phenotype appears identical to that observed in wild-type hermaphrodites depleted of WEE-1.3 (B). The white carets (^) mark the oocyte chromosomes that have coalesced (B,F). The spermatheca (spth) is to the left in each panel. Scale bar: 20 µm.