Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.
Cell-cycle control is critical for the proper development of multicellular organisms. Two distinct types of cell division cycles, mitosis and meiosis, must be coordinately regulated both spatially and temporally in germline development to produce enough competent gametes. MPF, maturation-promoting factor, promotes the transition from G2 to M phase during mitosis and meiosis (Schmitt and Nebreda, 2002), and is composed of cyclin B and cyclin-dependent kinase 1 (Cdk1) (Doree and Hunt, 2002). MPF phosphorylates multiple proteins to promote chromosome condensation and other prophase and metaphase events (Forsburg and Nurse, 1991; Nebreda et al., 1995; Nigg, 1995; Norbury and Nurse, 1992). Phosphorylation also regulates the activity of MPF itself (Coleman and Dunphy, 1994). This process involves Cdk1, Weel and Cdc25 and is evolutionarily conserved from yeast to mammals. Cdk1 is phosphorylated by an inhibitory kinase, Wee1/Myt1, so that it is inactive during interphase (Jin et al., 2005; Lundgren et al., 1991; Mueller et al., 1995; Parker and Piwnica-Worms, 1992). At the G2-M transition, Cdk1 is activated by Cdc25 phosphatase, which promotes the G2-M transition by removing inhibitory phosphate residues from Cdk1 (Draetta and Eckstein, 1997; Edgar and O'Farrell, 1989; Edgar and O'Farrell, 1990; Gould and Nurse, 1989; Hofmann et al., 1998; Zheng and Ruderman, 1993).
Many animals express multiple isoforms of the phosphatase Cdc25 (Russell and Nurse, 1986). Drosophila has two Cdc25 genes, string and twine (Edgar and O'Farrell, 1989). STRING is required for mitosis and is expressed in both somatic cells and mitotic germ cells (Lehman et al., 1999; Reed, 1995), whereas TWINE is required for meiosis and is expressed specifically in germ cells (Alphey et al., 1992; Courtot et al., 1992). The Drosophila twine loss-of-function mutant fails to complete the G2-M transition during spermatogenesis (Alphey et al., 1992). Mammals have three Cdc25 genes, CDC25A, CDC25B and CDC25C (Sadhu et al., 1990). Cdc25B−/− knockout female mice are sterile because their oocytes cannot exit developmental arrest at meiosis prophase 1 (Lincoln et al., 2002), whereas Cdc25A−/− mice exhibit early embryonic lethality (Ray et al., 2007), indicating that they are required for the control of oocyte meiotic cell cycle and embryonic mitotic cell cycle, respectively. However, Cdc25C is dispensable for both cell cycles (Chen et al., 2001). Thus, cdc25 family members appear to have distinct functions during development and may differentially regulate mitosis and meiosis.
During oogenesis, meiosis is arrested at meiotic prophase 1 in most organisms (Masui and Clarke, 1979), probably to provide enough time for oocyte development. The period of arrest ranges from hours to years depending on the species. Once oocytes are fully developed, the meiotic arrest can be released by a species-specific external signal such as progesterone in Xenopus laevis (Masui, 1967), and major sperm protein (MSP) in C. elegans (Miller et al., 2001). As a result, the oocyte cell cycle resumes and transits from meiotic prophase 1 to metaphase 1, and eventually progresses through the meiotic divisions. This resumption of the oocyte meiotic cell cycle and accompanying events including nuclear envelope breakdown, chromosome congression, and cortical rearrangement are generally called oocyte maturation (Greenstein, 2005; McCarter et al., 1999; Miller et al., 2001). Studies of Xenopus oocyte maturation (Masui and Markert, 1971) led to the isolation of MPF, the crucial intracellular regulator of maturation (Lohka et al., 1988). MPF was eventually identified to be the cyclin B-Cdk1 complex, which led to a greater understanding of the universal control of the G2-M transition in mitosis and oocyte meiotic maturation (Maller et al., 1989; Nurse, 1990). Although genetic analysis of mitosis has been extensive, genetic studies of oocyte maturation have remained limited. Nevertheless, over the last decade, several factors regulating oocyte maturation have been identified in C. elegans (Boxem et al., 1999; Chase et al., 2000; Detwiler et al., 2001). Notably, depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1, causes precocious oocyte maturation, indicating that wee-1.3 is required for oocyte meiotic arrest in C. elegans (Burrows et al., 2006).
In C. elegans, the functions and interactions of genes involved in germline development can be studied precisely and unambiguously through powerful genetic and reverse genetic approaches such as analyses of deletion mutants and RNA interference (RNAi). Moreover, C. elegans germline development is ideally suited to cytological study because the hermaphrodite gonads, which are transparent tubes, contain mitotic germline stem cells, early meiotic prophase germ cells, differentiating oocytes and sperm in a distal to proximal distribution (Hirsh et al., 1976; Kimble and Hirsh, 1979). C. elegans expresses four cdc-25 genes. Previous phylogenetic analysis suggests that the four C. elegans CDC25 proteins are more closely related to each other than they are to those from other organisms such as fly and mammals (Ashcroft et al., 1998). RNAi analysis of cdc-25.1 suggests that it is required for the completion of meiosis in newly fertilized eggs before the onset of embryogenesis (Ashcroft et al., 1999). Furthermore, the phenotype of cdc-25.1 loss-of-function alleles, nr2036 and bn115, revealed that it is essential for germline mitotic proliferation (Ashcroft and Golden, 2002; Kim et al., 2009). However, functions of the other members during development have not been studied extensively. In this study, we sought to determine whether cdc-25.2 functions similarly to cdc-25.1 or is required for other processes of germline development. Here, we report that cdc-25.2 has an essential role in C. elegans oocyte meiotic maturation and it competes with wee-1.3 in this process.
cdc-25.2 is predominantly expressed in the adult hermaphrodite germ line
In a previous study, we demonstrated that cdc-25.1 is predominantly expressed in the germ line during postembryonic stages. To test whether cdc-25.2 is also highly expressed in the germ line, and if so, to see in which germ cells it is expressed, we measured expression levels of cdc-25.2 mRNA by quantitative real-time RT-PCR. The expression levels were measured at different stages of postembryonic development in wild-type N2 hermaphrodites, in N2 males, and in several germline-defective mutant hermaphrodites; fem-1(lf) hermaphrodites producing only oocytes, fem-3(gf) hermaphrodites producing only sperm, and glp-1(lf) hermaphrodites which contain few germ cells (Fig. 1A). First, we found that cdc-25.2 mRNA was hardly detectable in glp-1(q224) hermaphrodites compared with N2 hermaphrodites, indicating that cdc-25.2 is preferentially expressed in the germ line. In N2 hermaphrodites, expression of cdc-25.2 mRNA was relatively low until L4 larval stage, but the expression increased significantly in young adult (YA; just after the L4 to adult molting but before egg laying has started) and adult (Ad; egg-laying gravid adult worms) stages, when oogenesis actively occurs in hermaphrodite worms (Fig. 1A). Furthermore, although cdc-25.2 mRNA was expressed in feminized fem-1(hc17lf) hermaphrodites, it was barely detectable in N2 males and in masculinized fem-3(q20gf) hermaphrodites. These results indicate that cdc-25.2 is expressed primarily in oocytes and not in sperm. However, compared with the robust expression in the N2 hermaphrodite germ line, the expression was much weaker in the feminized fem-1(lf) germ line, suggesting that the presence of sperm or fertilization positively affect the cdc-25.2 expression level. In conclusion, quantitative real-time RT-PCR showed that cdc-25.2 is predominantly expressed in oocytes of adult hermaphrodites.
cdc-25.2 hermaphrodite germ lines are defective in oocyte development
To investigate the functions of cdc-25.2, we examined the phenotype of its deletion allele, ok597, which lacks 2761 bp of cdc-25.2 genomic sequence extending from within the first intron to within the fifth intron (Fig. 1B). Before phenotype analysis, cdc-25.2(ok597) was balanced with nT1[qIs51], which expresses GFP in the pharynx throughout postembryonic development under the myo-2 promoter, which made identification of cdc-25.2 homozygotes easy and unambiguous. The cdc-25.2 homozygotes were also confirmed by identification of deletion by a single-worm PCR.
cdc-25.2(ok597) homozygous progeny produced by heterozygous mothers developed into sterile adults with very high penetrance (100% at 16°C, 85% at 20°C and 95% at 25°C, n=60 for each temperature). The penetrance of sterility was not different between cdc-25.2 homozygous worms produced by heterozygous mothers (maternal load +, zygotic product −) and cdc-25.2 homozygous worms produced by rare fertile homozygous mothers (maternal load −, zygotic product −), suggesting that incomplete penetrance of cdc-25.2 sterility was not due to maternal contribution from heterozygous mothers. We recently found that knockout of another cdc-25 family member increased the sterility of cdc-25.2 mutants (our unpublished results). Therefore, the incomplete sterility of the cdc-25.2 mutant was probably caused by genetic redundancy with another cdc-25 family member. Unlike cdc-25.1 mutants, which are defective in germline mitotic proliferation, cdc-25.2(ok597) hermaphrodite gonads contained well-proliferated germ cells as well as sperm and some diakinesis-stage oocytes (Fig. 2A,C). However, cdc-25.2(ok597) gonads also contained endomitotic oocytes (Emos) in the proximal gonadal region; Emo phenotype was identified by the intense staining of DNA with Hoechst 33342 DNA dye (Fig. 2C, arrows). The Emo phenotype is caused by endoreduplication of DNA, resulting in polyploidy in oocytes (Iwasaki et al., 1996). We also observed that cdc-25.2(ok597) oocyte nuclei often looked swollen compared to N2 oocyte nuclei under Nomarski DIC microscopy (Fig. 2B,D). We observed the same Emo phenotype when we treated wild-type N2 hermaphrodites with cdc-25.2 RNAi (supplementary material Fig. S1C), and in cdc-25.2(ok597)/sDf75 hemizygous hermaphrodites (supplementary material Fig. S1D), confirming that the Emo phenotype was indeed caused by depletion of cdc-25.2.
To confirm that cdc-25.2(ok597) hermaphrodite gonads produce differentiated gametes, we also immunostained mutant gonads with two antibodies, J67 (Strome, 1986) and SP56 (Ward et al., 1986), that specifically recognize an oocyte-membrane epitope and a sperm-polypeptide epitope, respectively (Fig. 3). Germ lines of both N2 (Fig. 3A-D) and cdc-25.2(ok597) (Fig. 3E-H) hermaphrodites expressed both the oocyte-specific antigen (Fig. 3B,F) and the sperm-specific antigen (Fig. 3D,H), indicating that cdc-25.2(ok597) hermaphrodites produce both sperm and oocytes and their oocytes are differentiated. The difference in immunostaining patterns for the oocyte marker between N2 (Fig. 3B) and cdc-25.2(ok597) hermaphrodite gonads (Fig. 3F) may reflect aberrant cortical cytoskeleton arrangement in the cdc-25.2(ok597) oocytes. Taken together, the results of nuclear and immunofluorescence staining indicate that oogenesis in the cdc-25.2(ok597) hermaphrodite germ line starts normally but defects occur in later stages, leading to the Emo phenotype.
cdc-25.2 is dispensable for male germline development
cdc-25.2(ok597) sperm looked normal and differentiated (Fig. 2C, Fig. 3G,H). Also, expression levels of cdc-25.2 mRNA in N2 males and masculinized fem-3(q20gf) hermaphrodites were very low (Fig. 1A), suggesting that cdc-25.2 is neither expressed nor required during spermatogenesis and male germline development. To test this possibility, we first compared the morphology of cdc-25.2(ok597) male gonads to that of N2 male gonads by Hoechst 33342 nuclear staining (supplementary material Fig. S2), and observed no obvious differences. Second, we tested the fertility of cdc-25.2(ok597) males and found that they were capable of producing cross-fertilized progeny as efficiently as N2 males (data not shown), indicating that cdc-25.2 is indeed dispensable for spermatogenesis and male germline development.
The cdc-25.2 Emo phenotype is suppressed by fem-2 and fem-3 RNAi
To determine whether the defect in cdc-25.2 oogenesis occurs at or after oocyte maturation, we conducted RNAi experiments to knock down expression of fem-2 or fem-3 in cdc-25.2(ok597) hermaphrodites and observed the phenotypes, because this causes oogenesis arrest before maturation. Depletion of fem-2 or fem-3 feminizes hermaphrodite worms, causing a lack of sperm in their germ lines (Hodgkin, 1986). In the absence of sperm, oocytes are arrested at the diakinesis stage of meiotic prophase 1 without maturation because oocyte maturation is triggered by MSP provided by sperm (Miller et al., 2001). Both fem-2(RNAi) and fem-3(RNAi) in cdc-25.2(ok597) hermaphrodites significantly decreased the number of endomitotic oocytes in the gonads (Fig. 4). These results indicate that the defect leading to endomitotic oocytes in cdc-25.2 mutants occurs at oocyte maturation or later.
cdc-25.2 counteracts the action of wee-1.3 in oocyte maturation
The G2-M transition is regulated competitively by Wee1/Myt1 kinases and Cdc25 phosphatase through phosphorylation and dephosphorylation of Cdk1, the catalytic subunit of MPF (Fantes, 1979; Russell and Nurse, 1987). Therefore, we predicted that if cdc-25.2 regulates oocyte maturation, the phenotype of cdc-25.2 mutants would be suppressed by reducing the activity of wee-1.3, a C. elegans ortholog of wee1, which has already been shown to function as a negative regulator in C. elegans oocyte maturation (Burrows et al., 2006). We performed RNA interference of wee-1.3 on cdc-25.2(ok597) hermaphrodites and found that this treatment effectively suppressed the Emo phenotype (Fig. 4, Fig. 5C,G). Furthermore, by immunostaining, we examined the expression pattern of phospho-histone H3 (PH3) in N2 and cdc-25.2 mutant hermaphrodites before and after wee-1.3(RNAi) treatment (Figs 5, 6). Because histone H3 is strongly phosphorylated at metaphase (M-phase) in both mitosis and meiosis, PH3 can mark oocytes during the process of maturation (Hans and Dimitrov, 2001; Hendzel et al., 1997). In N2 hermaphrodites, some proximal diakinesis oocytes were positively immunostained by anti-PH3 (Fig. 5B, Fig. 6A). Compared with N2, fewer anti-PH3-positive diakinesis oocytes (Fig. 5D, Fig. 6A) were identified in cdc-25.2(ok597) hermaphrodites, indicating that oocyte maturation is repressed in cdc-25.2 mutants. In addition, cdc-25.2(ok597) hermaphrodites contained some PH3-positive Emos (Fig. 5D). however, wee-1.3(RNAi) hermaphrodites contained an excess number of PH3-positive oocytes as reported previously (Fig. 5F, Fig. 6A) (Burrows et al., 2006). We found that after treatment of cdc-25.2(ok597) hermaphrodites by wee-1.3 RNAi, the number of PH3-positive diakinesis oocytes, which were at the transition from prophase 1 to metaphase 1, was recovered nearly to the level of untreated N2 (Fig. 5B,H, Fig. 6A). These results indicate that cdc-25.2 counteracts the action of wee-1.3 to promote oocyte maturation in C. elegans.
cdk-1 and cyb-3 RNAi cause an Emo phenotype similar to that of cdc-25.2 mutants
To understand how cdc-25.2 promotes oocyte maturation, we examined RNAi phenotypes of MPF component genes. MPF is composed of cyclin B and Cdk1. In C. elegans, there are four cyb genes (cyb-1, 2.1, 2.2 and 3) and seven cdk genes (cdk-1, 2, 4, 5, 7, 8 and 9). Previously, both cdk-1 RNAi and simultaneous RNAi of all four cyb genes showed defects in oocyte maturation, suggesting that CDK-1 and CYB proteins probably function as components of MPF in C. elegans (Burrows et al., 2006). Moreover, RNAi of both cdk-1 and wee-1.3 led to the same phenotype as RNAi of cdk-1 alone, indicating that cdk-1 functions downstream of wee-1.3 in the oocyte maturation pathway (Burrows et al., 2006).
We performed RNAi of cdk-1 and cyb-3 because previous microarray analysis indicated that they were highly expressed in oocytes (Reinke et al., 2004). RNAi of cdk-1 and cyb-3 in N2 hermaphrodites caused an Emo phenotype similar to that of cdc-25.2(ok597) hermaphrodites (Fig. 7). To examine possible genetic interaction between cdc-25.2 and candidate MPF component genes, we conducted RNAi of cdk-1 or cyb-3 in cdc-25.2(ok597) hermaphrodites. RNAi of cdk-1 and cyb-3 caused the same Emo phenotype in cdc-25.2(ok597) hermaphrodites as in N2 hermaphrodites (Fig. 7, compare A, B with C, D, respectively). Taking into account the facts that cdc-25.2 mutants themselves displayed the Emo phenotype and no significant enhancement of the phenotype was observed after RNAi of cdk-1 and cyb-3, and based on studies in other systems, we consider that cdc-25.2 functions upstream of cdk-1 and cyb-3 to promote their activities in oocyte maturation (see Fig. 9).
To prove that cdc-25.2 positively regulates cdk-1 activity during oocyte maturation, we immunostained both N2 and cdc-25.2(ok597) hermaphrodite gonads with an antibody that specifically recognizes CDK-1 phospho-tyrosine 15 (Hachet et al., 2007) (Fig. 8). It is known that phosphorylation of Tyr15 by Wee1 or both Thr14 and Tyr15 by Myt1 blocks Cdk1 activity, and that this inhibition is counteracted by dephosphorylation by Cdc25 (Boxem, 2006; Mueller et al., 1995). Signals of inactive phospho-CDK-1 gradually increased in diakinesis oocytes up to the −2 position, but the signal was significantly diminished in the most proximally positioned oocytes (at the −1 position) in wild-type N2 gonads (Fig. 8B). By contrast, the phospho-CDK-1 signal remained strong in the most proximal diakinesis oocytes in cdc-25.2 mutant gonads (Fig. 8D). These results demonstrate that inactive phospho-CDK-1 is indeed dephosphorylated and activated by CDC-25.2 phosphatase during C. elegans oocyte maturation to promote the prophase 1 to metaphase 1 transition (see Fig. 9).
Cdc25 promotes progression through the cell cycle. Of the four cdc-25 genes in C. elegans, cdc-25.1 has already been shown to be required for germline mitotic proliferation (Ashcroft and Golden, 2002; Kim et al., 2009). In this study, we demonstrated that another family member, cdc-25.2, is also essential for C. elegans germline development. Phenotype analyses of cdc-25.2 mutants indicate that cdc-25.2 is required only for oogenesis and hermaphrodite germline development, and is dispensable for spermatogenesis and male germline development.
To understand the molecular mechanism underlying the Emo phenotype of cdc-25.2 mutants, we depleted several genes by RNAi in cdc-25.2 hermaphrodites. First, we found that feminization of cdc-25.2 hermaphrodites by RNAi of fem-2 and fem-3 significantly suppressed the Emo phenotype (Fig. 4). Feminization of the germline causes arrest of oogenesis at meiotic prophase 1, so this result indicates that the defect in cdc-25.2 oogenesis occurs at oocyte maturation or later. Second, we found that RNAi of wee-1.3 effectively suppressed the Emo phenotype of cdc-25.2 hermaphrodites (Fig. 4, Fig. 5C,G). Moreover, after RNAi of wee-1.3 in the cdc-25.2 hermaphrodites, the number of oocytes at the transition from prophase 1 to metaphase 1 was recovered from almost zero to nearly the level of wild-type N2 (Fig. 5D,H, Fig. 6). As wee-1.3 negatively regulates oocyte maturation, we concluded that cdc-25.2 functions to promote oocyte maturation by competing with wee-1.3 (Fig. 9). As it is already known in yeast that the G2 arrest phenotype of cdc25 mutants is suppressed by wee1 mutation (Fantes, 1979; Russell and Nurse, 1987), which causes premature cell division and production of small daughter cells (Nurse, 1975), competition between Cdc25 and Wee1 to regulate MPF seems to be evolutionarily well conserved.
Both cdc-25.2 loss-of-function mutation and wee-1.3(RNAi) caused defects in oogenesis but their phenotypes were different. wee-1.3(RNAi) oocytes contained a coalesced, indistinct mass of chromosomes in each nucleus, which appeared to be generated through additional rounds of DNA duplication as in Emos, although wee-1.3(RNAi) oocytes appeared to undergo far fewer cycles of endoreduplication than typical Emos (Burrows et al., 2006). In this study, we did not score these chromosome-coalesced oocytes as Emo, because the morphology was quite different from the typical Emo, and also because we assume that this coalesced mass of chromosomes is generated by a mechanism different from the one that produces an Emo phenotype in cdc-25.2 mutants. Nevertheless, the occurrence of the ‘coalesced mass’ phenotype in wee-1.3(RNAi) oocytes and the typical Emo phenotype observed in cdc-25.2 mutant oocytes indicate that both aberrant activation and inhibition of MPF activity can cause inappropriate endoreduplication in oocytes.
MPF promotes the transition from G2 (prophase) to M phase (metaphase) in both mitosis and meiosis, and is composed of cyclin B and Cdk1. Among the four cyclin B and the seven Cdk genes in C. elegans, cyb-3 and cdk-1 were reported to be highly expressed in oocytes, as determined by microarray analysis (Reinke et al., 2004). We found that RNAi of cyb-3 and cdk-1 caused an Emo phenotype, similar to that of cdc-25.2 mutants (Fig. 7). As their RNAi caused the same level of Emo phenotype in cdc-25.2 mutants as in N2 and no significant enhancement of the cdc-25.2 phenotype was observed after the RNAi, we assume that cdc-25.2 functions upstream of cdk-1 and cyb-3 in C. elegans oocyte maturation (Fig. 9).
During germline development, two distinct types of cell cycles, mitosis and meiosis, must be coordinately regulated to produce enough haploid gametes. In addition, different cell cycle control mechanisms seem to function between spermatogenesis and oogenesis, as the meiotic cell cycle is arrested and resumed only during oogenesis. In this situation, the germ line may require multiple cell cycle regulators for proper development. Function of cdc-25.1 is required for germline mitotic proliferation in both male and hermaphrodite germ lines (Ashcroft and Golden, 2002; Kim et al., 2009), whereas cdc-25.2 is required only for oogenesis as described in this study. We found that cdc-25.3 and cdc-25.4 are also preferentially expressed in the germ line (J.K., I.K., Y.H.S., unpublished results), so we expect that they also have important functions in C. elegans germline development. Studies of cdc-25 family genes may shed new light on the diversified cell cycle control mechanisms in C. elegans germline development and provide the basis of a link between the developmental process and cell cycle.
Materials and Methods
Strains and alleles
C. elegans strains were maintained and manipulated as described previously (Brenner, 1974). C. elegans variety Bristol, strain N2 was used as the wild type for all experiments. Most strains were maintained at 20°C on nematode growth medium (NGM) agar plates containing Escherichia coli strain OP50, unless noted otherwise. Larval stages were discriminated by time after hatching, and young adult (YA) and adult (Ad) stages were distinguished by the presence or absence of embryos in the uterus. Mutant strains included: BA17: fem-1(hc17) IV (Nelson et al., 1978), JK816: fem-3(q20) IV, JK1107: glp-1(q224) III (Austin and Kimble, 1987), VC402: +/eT1 III; cdc-25.2(ok597)/eT1 V, and YHS25: +/nT1[qIs51] IV; cdc-25.2(ok597)/nT1[qIs51] V. Temperature-sensitive mutants were maintained at the permissive temperature, 16°C, and examined at the non-permissive temperature, 25°C. The cdc-25.2(ok597) deletion mutation was separated from balancer eT1 in the original VC402 strain, out-crossed four times with N2, then balanced again with nT1[qIs51] (IV;V) to construct YHS25 strain for phenotype analysis. nT1[qIs51] (IV;V) is derived from nT1 (IV;V) and expresses GFP in the pharynx throughout postembryonic development under the myo-2 promoter. BC3958: dpy-18(e364)/eT1 III; sDf75 unc-46(e177)/eT1[let-500(s2165)] V was used to construct cdc-25.2(ok597)/sDf75 V hemizygous mutant strain. sDf75 is a small deficiency which uncovers the cdc-25.2 genetic locus.
Staining of the nuclei
To characterize germ lines, nuclei were stained: 20 young adult animals were dissected in 7 μl of M9 buffer on a poly-lysine-coated glass slide, covered with a coverslip, freeze-cracked in liquid nitrogen, and fixed, first in 100% cold methanol for 10 minutes, then in fresh 3% paraformaldehyde for 10 minutes. Specimens were then washed in PBS containing 0.5 mg/ml Hoechst 33342 DNA dye and mounted.
Immunofluorescence staining and antibodies
Immunostaining of germ cells was performed as previously described with minor adaptations (Kawasaki et al., 1998; Strome and Wood, 1983). Gonads were extruded and fixed with cold methanol and cold acetone for anti-PH3, J67 and SP56 immunostaining, and with cold methanol and 3% paraformaldehyde for anti-phospho-CDK-1 immunostaining. Specimens were incubated with the following primary antibodies; rabbit polyclonal anti-PH3 antibody (Hendzel et al., 1997) diluted 1:200, rabbit polyclonal anti-phospho-CDK-1 antibody (Hachet et al., 2007) (Calbiochem; phospho-Tyr15-specific-CDK1-Hu) diluted 1:100, and undiluted cultured supernatant of mouse monoclonal antibodies J67 (Strome, 1986) and SP56 (Ward et al., 1986). Secondary antibodies used were goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology) diluted 1:200 for anti-PH3 and anti-phospho-CDK1, and goat anti-mouse IgG-FITC (Santa Cruz Biotechnology) diluted 1:100 for J67 and SP56. The specimens were counter stained with 0.5 mg/ml Hoechst 33342, and mounted in Sigma mounting medium (M1289).
Mounted specimens were observed under a fluorescence microscope (Axioskop 2 MOT, Zeiss). Images were taken using an Orca ERG digital camera (Hamamatsu) and processed with Openlab (Improvision) and Photoshop (Adobe) software.
Each bacterial clone was incubated in LB broth containing 100 mg/ml ampicillin for at least 16 hours at 37°C. The overnight culture was diluted 100-fold in LB broth containing ampicillin, incubated for 6 hours at 37°C, and spread onto an NGM plate with 0.2% lactose and 100 mg/ml ampicillin. The plate was incubated for 2 to 3 days at room temperature to induce dsRNA. Synchronized L1 worms were transferred to the RNAi plate and grown for 3 days at 20°C until adulthood. The adult worms were dissected and their extruded gonads were examined for nuclear and/or immunofluorescence staining. Soaking RNAi was performed as described previously (Maeda et al., 2001).
Quantitative real-time RT-PCR
Total RNA was prepared from synchronized populations of larval stages L1 to L4, young adult (YA) and gravid adult (Ad) wild-type N2 hermaphrodites, from adult N2 males, as well as from temperature-sensitive fem-1, fem-3 and glp-1 mutant adult hermaphrodites. Wild-type N2 hermaphrodite and male populations were grown at 20°C, and the temperature-sensitive mutant worms were grown at 25°C. Worms were collected in TRIzol (Invitrogen) and total RNA was extracted using a phase lock gel (MaXtract High Density, Qiagen). cDNA was synthesized using oligo-dT primer and M-MLV reverse transcriptase (Invitrogen). qPCR reactions were conducted using Power SYBR®Green PCR Master Mix (Applied Biosystems) in a 96-well plate with a 25 ml reaction volume. Primers for act-1, which served as the internal control, were 5′-CCA GGA ATT GCT GAT CGT ATG CAG AA-3′ and 5′-TGG AGA GGG AAG CGA GGA TAG A-3′ (GenBank accession no. NM_073418). Primers for cdc-25.2 were 5′-ACT AGA GAC ATT TGA GGA GGA-3′ and 5′-GAT GGC GTT CTT GAT GTG AC-3′ (GenBank accession no. NM_071045). The relative expression level of each gene was defined as the mRNA level for each gene that was averaged from triplicate experiments and normalized to that of act-1.
This work was supported by Forest Science & Technology Projects (No. S110708L0505604C) provided by Korea Forest Service, the second BK21 project to Y.H.S. We thank CureBio for support on this project. Worm strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.