C. Cai et al. Figure 2. Khdc1 family members are ex- pressed in oocytes and early embryos. (A) RT-PCR for Khdc1a and Khdc1b expression in the indicated tissues. Gapdh expression is shown as a loading control. RT- indicates the negative control sample run without reverse transcriptase. (B) RT-PCR for Khdc1a and Khdc1b expression in isolated germinal vesi- cle (GV) and meiotic M-phase II (MII) oocytes and embryos. The stage of embryo is indi- cated by the cell number or by embryonic day. (C) RT-PCR for Khdc1a and Khdc1b in fetal (E12.5–16.5) and postnatal (Day 0 –16) testes (T) and ovaries (O). E8.5 indicates whole embryonic day 8.5 embryos. (D) In situ hybridization for Khdc1a and Khdc1b in adult ovary sections. Sense probes were used as controls. Solid insets are higher magniﬁcation preantral follicles. Preantral follicles contain growing oocytes surrounded by one or a few layers of granulosa cells. Dashed insets are higher-magniﬁcation antral follicles. Antral follicles represent the ﬁnal stage in follicle growth, contain transcriptionally and transla- tionally quiescent oocytes surrounded by multiple layers of granulosa cells, and contain a ﬂuid-ﬁlled space termed the antrum. Scale bar 240 m. GV, GVBD, and MII oocytes as well as embryos up to E3.5 (Figure 3E). Intriguingly, the KHDC1 proteins localized to structures resembling germinal granules in the cytoplasm. Germinal granules (a.k.a. polar granules) are large protein and RNA complexes that are maternally deposited and di- rect germ cell speciﬁcation in Drosophila, C. elegans, and Xenopus (Chuma et al., 2009). Evidence for maternal speciﬁ- cation of germ cells is lacking in mice, but some studies have identiﬁed structures resembling germinal granules in mouse oocytes (Swetloff et al., 2009). In addition to appearing in granules, the KHDC1 proteins were also enriched in the cortex and in the perinuclear area of GVBD oocytes, MII oocytes, and 1-cell stage embryos (see arrows in Figure 3E, subpanels b, c, d, and e). KHDC1 Proteins Have Polynucleotide Binding and Oligomerization Activity The KH domain is a nucleotide binding domain, and most KH domain proteins associate with single-stranded RNA or DNA (Ryder et al., 2004; Galarneau and Richard, 2005). For example, Quaking I protein (QKI) regulates splicing and translation of mRNAs by binding to its target sequence NACUAAY-N1–20-UAAY. The presence of an atypical KH domain in the KHDC1 proteins suggests that they might be RNA-binding proteins. To test this possibility, we analyzed whether these proteins can bind to homopolymer RNA- agarose. We puriﬁed GST fused KHDC1A and KHDC1B and tested their binding activity using poly-U as a target. As shown in Figure 4A, GST-KHDC1A and GST-KHDC1B have poly-U binding activity, however, as a control, GST itself could not bind to poly-U. This demonstrates that KHDC1A and KHDC1B possess nucleotide binding activity and are likely to bind endogenous mRNA targets. KH domains are typically found in multiple copies, two in fragile X mental retardation protein (FMRP) (Siomi et al., 1993), three in hnRNP K and other members of the Poly rC binding protein family (Choi et al., 2009), and up to 14 in members of the vigilin family. These domains cooperate with each other 3140 and regulate the afﬁnity and speciﬁcity for target RNAs (Chmiel et al., 2006). Other KH domain proteins such as mem- bers of the STAR family contain only one KH domain, and it is proposed that multimerization facilitates their RNA-binding activity (Chen et al., 1997). To test whether KHDC1A can form multimers, HA-tagged KHDC1A was cotransfected with or without Flag-tagged KHDC1A. Immunoprecipitations using antibodies directed against the Flag tag resulted in the precip- itation of Flag-KHDC1A and coprecipitation of HA-KHDC1A, indicating that KHDC1A can form multimers (Figure 4B, lanes 1 and 2). Similar experiments proved that KHDC1B can also form multimers (Figure 4B, lanes 5 and 6). Furthermore, KHDC1A and KHDC1B can interact with each other (Figure 4B, lanes 3 and 4). KHDC1 Proteins Interact with the Cytoplasmic Polyadenylation Machinery The later stages of oocyte maturation and early embryonic development occur in the absence of transcription and con- sequently rely on the activities of a large number of RNA- binding proteins involved in translational regulation. The activity of many of these factors was initially characterized in Xenopus, making this a valuable system for testing the role of putative translational regulators (Brown, 2004). To test the activity of KHDC1A and KHDC1B, we ectopically expressed these proteins in Xenopus embryos. RNAs encoding for mouse Khdc1a or Khdc1b were injected into one cell of a two-cell embryo. By stage 6.5, embryos injected with Khdc1a exhibited small and strongly pigmented cells near the injec- tion site (Figure 5A). By stage 8.5, these effected cells had died and the embryos failed to gastrulate (Figure 5A and data not shown). This phenotype resembles the induction of apoptosis achieved by overexpressing Bix3 (Trindade et al., 2003) and would be consistent with the fact that overexpres- sion of KHDC1A has been shown to induce apoptosis in tissue culture cells (Rajpal et al., 2003). In contrast to KHDC1A, ectopic expression of KHDC1B resulted in large blastomeres at the injection site (Figure 5A). This large-cell Molecular Biology of the Cell