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Structural Characterization and Expression Studies of Dby and Its Homologs in the Mouse

YUNMIN LI

YUN-FAI CHRIS LAU

MARTIN DYM

WAI-YEE CHAN^*,

From the ^* Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, California; and the Departments of Cell Biology and Pediatrics, Georgetown University, Washington, DC.

Correspondence to: Dr Wai-Yee Chan, Laboratory of Clinical Genomics, NICHD/NIH, Building 49, Room 2A08, 49 Convent Dr, MSC 4429, Bethesda, MD 20892-4429 (e-mail: chanwy{at}mail.nih.gov).

Received for publication November 13, 2005; accepted for publication April 3, 2006.

	Abstract

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In spite of recent evidence showing the importance of DBY (DEAD-box RNA helicase Y) in spermatogenesis in human, the biologic role of its homolog Dby (also known as Ddx3y) in the mouse is less clear. The present study aims at characterizing the molecular structure of Dby and comparing its expression with its X- and autosome-linked homologs in embryonic gonads and developing germ cells in mice. Molecular cloning by rapid amplification of 3'-cDNA ends showed that the Dby gene in the mouse gives rise to 2 transcripts that differ only in the length of the 3'-untranslated region as a consequence of the use of alternative polyadenylation signals. Measurement by quantitative real-time polymerase chain reaction showed that both transcripts were ubiquitously expressed and were present in male germ cells and Sertoli cells. They were more abundant in type A spermatogonia compared with pachytene spermatocytes and round spermatids. Expression of Dby in the embryonic gonad increased from day 10.5 and reached a peak at day 17.5. The expression level of Dby decreased after birth and remained low in adult male gonads. Although the level of expression of Dby was much lower than its X chromosome homolog, Ddx3 (also known as Ddx3x) in all samples examined, the pattern of expression of the 2 genes was comparable. In contrast, their autosomal homolog, D1Pas1(also known as PL10), was predominantly expressed in pachytene spermatocytes and round spermatids. This result is in accord with meiotic sex chromosome inactivation in that Dby and Ddx are replaced in pachytene spermatocytes by their autosomal retroposon. These observations indicate that unlike DBY in humans, the role of Dby in spermatogenesis is less obvious in the mouse and its biologic activity may be replaced by that of Ddx3 and D1Pas1.

     Key words: Ddx3, D1Pas1, 3'-UTR, gonad, spermatogonia, spermatocytes, spermatids

	Introductionb

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Similar to that in human, mouse Dby has a structural homolog, Ddx3 (also known as Ddx3x) on the X chromosome (Mazeyrat et al, 1998). A genomic Southern blot identified further an autosomal homolog D1Pas1on mouse chromosome 1 (Kingsmore et al, 1989). Murine D1Pas1, formerly designated PL10 (Leroy et al, 1989), was thought to be a retroposon of the X chromosome Ddx3 (Mazeyrat et al, 1989). D1Pas1 is probably involved in the initiation of mRNA translation, as it is able to complement the deletion of the homologous yeast gene DED1, which shares 53% of its amino acid composition with D1Pas1 and has been shown to bind mRNA to the ribosome (Chuang et al, 1997). To understand the role of Dby in reproduction, we studied the expression pattern of Dby and its X and autosome homologs in male germ cells, embryonic gonads, and different somatic tissues. Results support the previous belief that the Dby gene may not be required for mouse spermatogenesis.

	Materials and Methods

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Tissue Preparation

Embryos were collected from pregnant mice at embryonic days 10.5 (E10.5), E11.5, E12.5, E13.5, E15.5, and E17.5, with the first day of vaginal plug identification defined as E0.5. Whole genital ridges (gonad and mesonephros) were dissected from the earlier embryos (E10.5, E11.5, and E12.5). In later embryos (E13.5, E15.5, and E17.5), mesonephros and gonads were separated. Sexing of the embryos was done by polymerase chain reaction (PCR) detection of Sry in DNA of the residual embryo (E10.5 and E11.5) or by morphology of the embryonic gonad (E12.5 to E17.5). Neonatal and adult mouse testes were collected at postnatal days 0, 3, 6, 12, 18, 24, 30, and 56. These procedures were performed at the Veterans Affairs Medical Center, University of California, San Francisco and were approved by the Institutional Animal Care and Use Committee.

Isolation of Testicular Cells

Germ cells were isolated by the STAPUT procedure (Dym et al, 1995). Six-day-old BALB/c mouse testes were used for isolation of type A spermatogonia. For the isolation of pachytene spermatocytes and round spermatids, testes from 60-day-old animals were used. Purity of germ cells was routinely higher than 95% for type A spermatogonia and higher than 90% for pachytene spermatocytes and round spermatids. Sertoli cells were isolated from neonatal (6-day-old) BALB/c mice as previously described (Dirami et al, 2001). MA-10 cells were used as Leydig cells. All germ cells and Sertoli cells were prepared at Georgetown University. The protocol was approved by the Georgetown University Animal Care and Use Committee.

RNA Preparation and cDNA Synthesis

Total RNA was isolated from mouse testicular cells and embryonic and postnatal gonads using TRIZOL reagent (Invitrogen, Gaithersburg, Md) and further purified by RNeasy Protect Mini Kit (Qiagen, Valencia, Calif) according to the manufacturer's protocol. The quality and quantity of the RNA was assessed with a 2100 Bioanalyzer (Agilent, Palo Alto, Calif). RNA of adult mouse tissues was purchased from Ambion (Austin, Tex). First strand cDNA was synthesized from RNA samples using Superscript II reverse transcriptase (Invitrogen, Gaithersburg, Md) according to the manufacturer's protocol.

Cloning of Mouse Dby Transcript Variant cDNAs

To obtain the transcript variant of Dby, the method of anchored PCR, rapid amplification of 3'-cDNA ends (3'-RACE), was performed using the GeneRacer Kit (Invitrogen, Gaithersburg, Md). Anchored PCR was performed using a gene-specific primer (5'-CTTACTCGTTACACTCGTCCTACTCC-3') and nested gene-specific primer (5'-CGTCCTACTCCAGTGCAAAAACATGCTT-3'). After amplification, the PCR products were cloned into pGEM-T (Promega, Madison, Wis) by TA-cloning according to the manufacturer's protocol. The sequence of the cloned cDNA was determined by PCR sequencing using BigDye Primer Cycle Sequencing Kits (Applied Biosystems, Foster City, Calif). DNA sequences were analyzed using DNASIS software v2.5 (MiraiBio, Alameda, Calif). Sequences used for alignment other than those reported here were extracted from public databases from the National Center for Biotechnology Information using BLAST searches.

Quantitative Real-Time PCR

cDNA was used as a template for quantitative PCR (QPCR) of Dby, Ddx3, and D1Pas1 mRNA levels. Gene-specific primers and Taqman probes for Dby, Ddx3, and D1Pas1 were designed using Primer Express software (Applied Biosystems, Foster City, Calif) according to the manufacturer's instructions. The sequences of the Taqman probes and primers are shown in the Table. Dby-L mRNA level was determined using the Dby-L primers and TaqMan probe, which were specific for the nonoverlapping region of the 3'-untranslated region of Dby-L. Since the sequence of Dby-S overlapped with that of Dby-L, the total mRNA levels of Dby-L and Dby-S, denoted as levels ofDby, were obtained with Dby primers and a TaqMan probe designed within the coding sequence. The amount of Dby-S mRNA was obtained by subtracting the total Dby mRNA level from the Dby-L mRNA level. 18S ribosomal RNA (rRNA) was used as the internal control of the reaction and was quantitated using the TaqMan rRNA control reagents (Applied Biosystems, Foster City, Calif). Standard procedures for the operation of the Prism 7900 HTS Sequence Detection System (Applied Biosystems, Foster City, Calif) were followed. Thermal cycler conditions consisted of 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. CT determinations were performed with the instrument for each reaction using default parameters. The CT values for Dby, Ddx3, and D1Pas1 were normalized to that of rRNA in each sample. QPCR was conducted in triplicate. The significance of the difference between expression levels was calculated using the Student's t test.

	Results

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Molecular Characterization of the Dby Transcript Variants

Dby contained 3767 bp including a 1977-bp open reading frame (ORF) identical to that published previously (GenBank NM_012008). Analysis of this sequence using the BLAST program against the mouse genome database showed that the Dby gene consisted of 15 coding exons (Figure 1A), 2 exons less than found for the human homolog DBY (Foresta et al, 2000). The ORF encoded a putative polypeptide with 88% identity to DBY. This is the only transcript identified to date (Mazeyrat et al, 1998). In humans 2 transcript variants of DBY, which have alternative polyadenylation sites, were initially identified (Foresta et al, 2000). A more recent study found at least 5 different RNA species on a Northern blot of testicular tissue (Ditton et al, 2004). To determine if any transcript variants of Dby existed in the mouse, a 3'-RACE experiment was performed. Two transcripts of the mouse Dby were identified that differed only in their 3'-untranslated region (3'-UTR) due to alternative polyadenylation signals (Figure 1B). The sequence of the longer transcript variant Dby-L was identical to the Dby published previously (GenBank NM_012008), whereas the Dby-S had a shorter 3'-UTR with a different polyadenylation sequence. Both Dby transcripts encoded the same protein containing the conserved motifs of DEAD-box RNA helicases, indicating that they likely shared a common ATP-dependent helicase function with other DEAD-box family members (Figure 1C).

View larger version (7K): [in this window] [in a new window]   Figure 1. Mouse Dby gene. (A) Schematic representation of the intron/exon structure. Exons are represented by solid boxes; untranslated regions (UTRs) are represented by open boxes. (B) Dby transcripts. The positions of start and stop codons and poly(A) tails are indicated, with numbering of nucleotides according to the cDNA sequence in GenBank (NM_012008). The translated region is represented by solid bars; UTRs are represented by open bars. (C) Conserved motifs of Dby protein, common to the DEAD-box family. Amino acid numbering is according to GenBank NM_012008.

View larger version (14K): [in this window] [in a new window]   Figure 2. Expression of Dby variants, Ddx3, and D1Pas1 in testicular germ and somatic cells of mouse. (A) Dby-L and Dby-S. (B) Dby (total), Ddx3, and D1Pas1. The Dby, Ddx3, and D1Pas1 mRNA levels, measured by quantitative polymerase chain reaction, were normalized with 18S ribosomal RNA and were shown as fold induction (Y-axis) compared with that of Dby-L in type A spermatogonia. Sg, type A spermatogonia; Sc, pachytene spermatocytes; Sd, round spermatids; Sertoli, Sertoli cells; Leydig, Leydig cells. Values are means ± SD of triplicates.

Expression of Dby and Its X and Autosomal Homologs in Nontesticular Tissues

Expression of Dby transcript variants was also studied in different mouse tissues including brain, heart, kidney, lung, liver, spleen, thymus, testis, ovary, and embryo (mixed male and female embryos). As shown in Figure 3A, the brain and heart were the major sites of Dby-L expression. Dby-L was found in all tissues examined with the exception of ovary, which does not have the Y chromosome and served as a negative control. Tissue distribution of Dby-S was similar to that of Dby-L, except that its expression level was significantly lower (P < .05) and it was absent in the thymus and ovary.

View larger version (16K): [in this window] [in a new window]   Figure 3. Expression of Dby variants, Dbx3, and D1Pas1 in nontesticular tissues of adult mouse. (A) Dby-L and Dby-S. (B) Dby (total), Dbx3, and D1Pas1. The Dby, Dbx3, and D1Pas1 mRNA levels, measured by quantitative polymerase chain reaction, were normalized with 18S ribosomal RNA and were shown as fold induction (Y-axis) compared with that of Dby-L in type A spermatogonia. Values are means ± SD of triplicates.

Developmental Onset of the Expression of Dby and Its X and Autosome Homologs

The expression of Dby, Ddx3, and D1Pas1 in the developing male gonad from E10.5 to postnatal day 56 was examined (Figure 4). The expression level of Dby showed a 3-fold increase from E10.5 to E17.5, and then decreased slowly after birth. The Ddx3 expression level was 5- to 10-fold more than that of Dby in embryonic male gonads at all ages examined. Ddx3 expression remained relatively stable throughout all embryonic ages and decreased after birth, although it was still significantly higher (P < .001) than that of Dby up to postnatal day 56. The expression level of Ddx3 at postnatal day 56 was about 6-fold lower than that in the embryonic male gonads. D1Pas1 expression was first observed at postnatal day 12 and continued to increase. It peaked at postnatal day 24 and then decreased afterwards. This pattern of expression coincides with the timing of the appearance of meiotic spermatocytes (Bellve et al, 1977).

View larger version (14K): [in this window] [in a new window]   Figure 4. Expression of Dby (total), Dbx3, and D1Pas1 in embryonic and postnatal male gonadal tissues of mouse. The Dby, Dbx3, and D1Pas1 mRNA levels, measured by quantitative polymerase chain reaction, were normalized with 18S ribosomal RNA and were shown as fold induction (Y-axis) compared with that of Dby in embryo day 10.5 male gonad. Values are means ± SD of triplicates.

	Discussion

Top Abstract Introductionb Materials and Methods Results Discussion References

Sequence alignment revealed significant homology among Dby, Ddx3, and D1Pas1xs. When the coding sequences were compared, Dby showed 90% and 84% identity with Ddx3 at the amino acid and nucleic acid level, respectively, while Dby and D1Pas1 shared 87% and 80% homology at the amino acid and nucleic acid levels, respectively. Significant stretches of absolute identity and conserved substitutions were distributed over the entire alignment when the 3 gene products were compared (Figure 5). The proteins encoded by the 3 genes share the highly conserved RNA helicase motif of the DEAD-box family. The differences among the 3 proteins are predominantly located at the N- and C-termini. These observations suggest that these 3 proteins might all have RNA helicase function but might be involved in different pathways. The germ cell–specific expression of mouse D1Pas1 was similar to the short form of human DBY (Foresta et al, 2000) and the DBY protein detected by immunostaining (Ditton et al, 2004). This implies that the spermatogenesis-specific function of DBY in humans is replaced by D1Pas1, but not Dby, in the mouse.

View larger version (46K): [in this window] [in a new window]   Figure 5. Amino acid alignment of mouse Dby with Ddx3 and D1Pas1. Gaps were introduced for maximal homology. The homologous amino acid residues are shaded. The consensus motifs I–VI in proteins identified as DEAD box RNA helicases are indicated by asterisks above the sequences.

D1Pas1 maps to mouse chromosome 1 (Kingsmore et al, 1989) and is thought to be a retroposon of the X chromosome homolog Ddx3 (Mazeyrat et al, 1998). Blasting of D1Pas1 against the human genome revealed a sequence with 80% homology in chromosome 4. This sequence is 84% homologous to Ddx3 and is probably the same as one of the pseudogenes reported by Kim and colleagues (2001). It is not known whether the human sequence is expressed. Interestingly, both D1Pas1 and the human sequence are intronless (results not shown), a property common to retroposons (Sedlacek et al, 1999). It is known that the X and Y chromosomes are transcriptionally silenced during meiosis in pachytene spermatocytes because of sex chromosome meiotic inactivation (Handel, 2004). In fact, Fernandez-Capetillo and coworkers (2003) used microarrays to show that the expression of X-linked genes and 3 Y-linked genes, including Dby, were suppressed in histone H2AX-deficient spermatocytes which do not initiate meiotic sex chromosome inactivation. Autosomal transposons of a number of X-linked genes including Cetn1 (Hart et al, 1999), Cstf2t (Dass et al, 2001), G6pd^x (Hendriksen et al, 1997), Pdha2 (Dahl et al, 1990; Takakubo and Dahl, 1992), Pgk2 (Boer et al, 1987; McCarrey et al, 1992), Zfa (Ashworth et al, 1990; Erikson et al, 1993), which are silenced during male meiotic prophase are expressed only in spermatocytes (Handel, 2004). The present study showed that D1Pas1 is predominantly expressed in pachytene spermatocytes. This observation confirms the previous report that the D1Pas1 protein is expressed predominantly in the nuclei of germ cells undergoing meiosis (Session et al, 2001). Thus, the pattern of differential expression of Dby and Ddx and their autosomal homolog D1Pas1 is similar to the other X and Y genes and their autosomal retrogenes and is in accord with the meiotic sex chromosome inactivation model (Handel, 2004).

Previous studies in humans showed that DBY is expressed mainly in spermatogonia while DBX is expressed mainly in spermatids. This led the investigators to conclude that DBY contributes mainly to the premeiotic spermatogonia phase while DBX functions in postmeiotic activities (Ditton et al, 2004). The fact that the expression level of D1Pas1 in the postmeiotic spermatids is significantly higher than that of Dbx and Dby makes the Dby/Ddx3/D1Pas1xs system of the mouse distinct from the DBY/DBX system of the human and suggests that D1Pas1 may not simply serve as the backup for Dbx and Dby during meiotic sex chromosome inactivation in the mouse.

Differential expression of Dby, Ddx3, and D1Pas1 in gonad and germ cell development is interesting. The role of D1Pas1 in spermatogenesis seems obvious since its expression is testis specific and peaks at the time when meiotic spermatocytes appear. On the other hand, the biologic function of Dby is less clear. Its expression in Sertoli cells is significantly higher (P < .05) than in germ cells. Sertoli cells are essential for supporting germ cell proliferation and differentiation (Griswold, 1998), and a number of Sertoli cell products are known to affect germ cell division, differentiation, and metabolism (Jegou, 1993; Eddy, 2002). It is tempting to speculate that Dby may contribute to Sertoli cell functions. However, since Sertoli cells used in the study were derived from 6-day-old animals, it is not known if Dby is present in more mature Sertoli cells. The expression of Dby in the embryonic gonads peaks at E17.5, at a time when gonocyte proliferation stops (de Rooij and Russell, 2000; Grootegoed et al, 2000). From that time on, the level of expression of Dby drops. Therefore, one possibility is that Dby plays a role in early gonocyte development. The major problem in assigning a function to Dby is that its expression pattern in embryonic gonads, germ cells, and somatic tissues is largely comparable to that of Ddx3, but its expression levels are significantly lower, usually by several fold. This is contrary to that in humans in which the expression level of DBY is higher than that of DBX in all tissues analyzed (Ditton et al, 2004). Thus, the biologic significance of Dby in the mouse is not obvious. It provides further evidence that Dby is not required at any stage of the mouse male germ line (Mazeyrat et al, 2001).

In conclusion, this study showed that in spite of structural and organizational similarities, the significance of the DBY/Dby genes in spermatogenesis in human and mouse is different. D1Pas1, instead of Dby, plays a role in spermatogenesis in the mouse. Unlike that of humans in which DBY is not expressed in spermatids, D1Pas1 expression remains high in these postmeiotic cells. There are distinct differences between the human DBY/DBX system and the mouse Dby/Ddx3/D1Pas1 system. Further studies are required to explore the physiologic functions of the Dby protein and its relationship with its X and autosomal homologs. This study affirms that gene function and expression data in mice cannot always be directly translated to humans.

	Footnotes

 
Supported in part by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health, and NIH grants HD38117 (Y.-F.C.L.) and HD33728 and HD36483 (M.D.).

DOI: 10.2164/jandrol.106.000471

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