Figure 1. Alignment of KHDC1A and KHDC1B proteins. Overall the proteins are 65% identical (black-boxed amino acids). Identity is highest in the N-terminal region containing the KH domain (arrow). three times and incubated in diluted secondary antibody (Cy5 conjugated anti-rabbit IgG and FITC conjugated anti-mouse IgG) (Jackson Immunore- search Laboratories, West Grove, PA) at room temperature for 1 h. Samples were then washed as above and mounted in VECTASHIELD Mounting Medium (Vector labs, Burlingame, CA). Images were collected with the TCS SP2 confocal microscope (Leica, Bannockburn, IL) or DM6000 ﬂuorescence microscope (Leica) and analyzed with Volocity software (Perkin Elmer-Cetus, Waltham, MA). RESULTS The Khdc1 Gene Family Encodes for Putative RNA Binding Proteins Transcripts for Khdc1a (KH domain containing 1A) were detected in a transcriptional proﬁling screen for genes expressed in puriﬁed primordial germ cells (PGCs) (Molyneaux et al., 2004). Using q-RT-PCR, we conﬁrmed that expression of Khdc1a is elevated in E10.5 and E12.5 PGCs relative to expression in the somatic cells (Supplemental Figure 1). Searching GenBank revealed that Khdc1a is a member of the small KH domain containing 1 family originally de- scribed by Pierre et al. (2007). There are three members in this family, Khdc1a (gene ID: 368204), Khdc1b (gene ID: 98582), and Khdc1c (gene ID: 433278). All three genes are adjacent to each other on mouse Chromosome 1A4. In silico analysis identiﬁed 40 expressed sequence tugs (ESTs) for Khdc1a in cDNA derived from spleen, ovary, testis, fertilized eggs, and early embryos. In contrast, ESTs for Khdc1b were restricted to the ovary, oocytes, and early stage embryos. Khdc1c is very similar in sequence to Khdc1a making expression difﬁcult to detect unambigu- ously. In fact, UniGENE has listed mRNAs for Khdc1c under the Khdc1a reference. It is currently unclear whether Khdc1c is expressed or is a psuedogene, so we have con- ﬁned our analysis to Khdc1a and Khdc1b. Both Khdc1a and Khdc1b have three exons. The ORF (open reading frame) of mouse Khdc1a is 501 base pairs long, encoding a 166-aa protein. The ORF of mouse Khdc1b is 381 base pairs long, encoding a 126-aa protein. As shown in Figure 1, both KHDC1 proteins contain a N-terminal domain with similarity to the K homology (KH) nucleotide binding motif; however, an invariant GXXG loop shown to be re- quired for the RNA binding activity of the KH domain is changed (Lewis et al., 2000). Compared with KHDC1B, KHDC1A has a unique hydrophobic C-terminal extension predicted by InterProScan analysis to be a transmembrane helix motif. Vol. 21, September 15, 2010 KHDC1B Interacts with CPEB Khdc1 mRNAs Are Enriched in Oocytes and Early Stage Embryos In silico analysis suggested that Khdc1a and Khdc1b are ex- pressed in a limited subset of tissues. To conﬁrm this, we used RT-PCR to examine Khdc1a and 1b expression in a panel of adult mouse tissues (Figure 2A). Khdc1a was highly expressed in ovaries along with weak expression in several other tissues such as brain, lung, and testis. In contrast, Khdc1b was only observed in ovaries. There was no Khdc1b detected in the other tissues examined including testes. Next, we examined the expression of Khdc1 mRNAs in oocytes and early stage embryos. As shown in Figure 2B, Khdc1a and Khdc1b have similar expression patterns. The mRNAs of both genes are enriched in germinal vesicle (GV) and metaphase II (MII) stage oocytes along with one-cell and two-cell stage embryos. The mRNA levels of both genes were decreased in 4 – 8 cell stage embryos and could not be detected in blastocysts. To see when Khdc1 expression in ovaries initiates, we performed a developmental time series (Figure 2C). Weak Khdc1a expression could be detected in the fetal gonads. Expression of Khdc1a increased dramatically in postnatal day 2 ovaries. Additionally, Khdc1b expression initiated at postnatal day 2 and increased as the animals matured. This expression pattern indicates that Khdc1a and Khdc1b are likely to play a role in oocyte maturation or early embryonic development. To further conﬁrm expression of Khdc1a and Khdc1b in growing oocytes, we performed in situ hybridization on cryosections of mouse ovaries. Consistent with the RT-PCR results and previous observations (Pierre et al., 2007), the signal was enriched in oocytes (Figure 2D). The solid inset and spaced inset show a high magniﬁcation view of pre- antral and antral follicles, respectively, highlighting the strong expression of Khdc1 genes in later stage follicles. KHDC1 Proteins Are Expressed in Oocytes and Early Embryos Two antibodies were used to examine the distribution of KHDC1 proteins (Figure 3). First, a rabbit polyclonal antibody (pan-KHDC1) raised against full-length KHDC1A (Rajpal et al., 2003) was tested and found to recognize both KHDC1A and 1B expressed in 293T cells (Figure 3A). Second, a rabbit polyclonal antibody speciﬁc for KHDC1A ( KHDC1A) was generated using a peptide (RDHPLRDRDLELHP) corresponding to 103 aa to 116 aa of KHDC1A, a region not exhibiting high sequence similarity to KHDC1B (Figure 1). Using the isoform speciﬁc antibody, we demonstrated that KHDC1A is highly expressed in the ovary but is also moderately expressed in other tissues including the liver and testis (Figure 3B). There appear to be two isoforms of KHDC1A expressed in the testis, the expected size 19-kDa form and a lower-molecular-weight isoform that might be the product of tissue speciﬁc processing. This lower- molecular-weight form was also detected in mature oocytes and early embryos (Figure 3C). Protein levels decline in E3.5 (blastocyst) and E4.5 embryos. Immunoﬂuorescence using pan-KHDC1 of mouse ovary sections revealed that the KHDC1 proteins are enriched in oocytes (Figure 3D). KHDC1 proteins can be detected in oocytes within growing and antral follicles (Figure 3D). In most cases, the KHDC1 proteins were cyto- plasmic, but in 14% of follicles the proteins were located within the nucleus of oocytes (Figure 3D). Additionally, KHDC1B accu mulated in nuclei when overexpressed in HeLa cells (Figure 7A). Whole mount immunostaining of oocytes and early em- bryos revealed that the KHDC1 proteins are expressed in 3139