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Keywords:

  • DEAD-box protein;
  • rck/p54 RNA helicase;
  • oocyte maturation;
  • spermatogenesis;
  • embryogenesis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals. Developmental Dynamics 233:1149–1156, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

DEAD-box (D-E-A-D is the single letter code of Asp-Glu-Ala-Asp) RNA helicases are present in almost all organisms and play important roles in RNA metabolism (Rocak and Linder, 2004). It was earlier reported that DEAD-box RNA helicases are involved in mRNA export (Liang et al., 1996; Snay-Hodge et al., 1998), pre-mRNA splicing (Staley and Guthrie, 1994), translation initiation (Chuang et al., 1997; Pestova et al., 2001), RNA decay (Coller et al., 2001), and ribosomal biogenesis (Daugeron and Linder, 1998). These effects on various processes of gene expression suggest that DEAD-box RNA helicases are involved in a variety of cellular functions.

The RCK gene was cloned through the study of the t(11;14)(q23;q32) chromosome translocation in the human B-cell lymphoma cell line RC-K8 (Akao et al., 1992), and its transcripts were found to be expressed ubiquitously in mouse and human tissues (Akao et al., 1995). The protein product (rck/p54) of this gene exhibits ATP-dependent RNA-unwinding activity (Akao et al., 2003) and participates in mRNA masking (Minshall et al., 2001) and 5′–3′ RNA decay (Cougot et al., 2004). The rck/p54 protein is highly homologous to its yeast, Caenorhabditis elegans, Drosophila orthologs, indicating its conserved functions in diverse organism (Navarro et al., 2001). Recent findings show that rck/p54 orthologs play significant roles in gametogenesis and early embryogenesis in eukaryotes. In yeast, ste13, the rck/p54 ortholog, is essential for sexual reproduction (Maekawa et al., 1994). CGH-1, the C. elegans ortholog, is required for gametogenesis and protection from physiological germline apoptosis. Xp54, the Xenopus ortholog; clam p47, the clam ortholog; and Me13B, Drosophila ortholog, are an integral component of stored (maternal) mRNP particles in oocytes (Ladomery et al., 1997; Minshall et al., 2001; Nakamura et al., 2001). In mammals, rck/p54 expression has been examined in mouse oocytes and embryos at the mRNA level (Paynton, 1998); however, no detailed studies have been carried out on gametogenesis and early embryogenesis.

Therefore, in the current study, we examined rck/p54 expression in the mouse ovary and testis by using anti-rck/p54 antibody raised in our laboratory. We further investigated rck/p54 expression in gametes and the effect of its overexpression on early embryogenesis. Our results suggest that rck/p54 plays important roles in both female and male germ cells, and in early mammalian embryogenesis, as it does in other eukaryotes.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

rck/p54 Is Involved in Oogenesis and Spermatogenesis

To investigate rck/p54 protein expression, we generated rabbit polyclonal anti-rck/p54 antibody against an N-terminus portion of the human rck/p54 protein. The antiserum was purified by antigen-coupled affinity column, and its specificity was confirmed by enzyme-linked immunosorbent assay (ELISA; data not shown). Western blot analysis showed this antibody to react specifically with 54-kDa rck/p54 protein and that to be expressed in both the ovary and testis (Fig. 1A).

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Figure 1. A,B: Western blot analysis of rck/p54 in tissues from ovary and testis (A) and localization of rck/p54 in the ovary studied by immunohistochemistry (B). A: Both ovary and testis express rck/p54 protein. (1 μg of protein/lane). B: Immunohistochemical study on rck/p54 in the ovary. Ovarian tissue was stained with anti-rck/p54 (c and e). a and b (merged image) is a negative control pair. d and f are merged images of rck/p54 (green) and F-actin (red) and nuclei (blue), respectively. Scale bar = 100 μm in a,c, 50 μm in e.

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Next, we investigated the cellular localization of rck/p54 in the ovary and testis of the mice by immunohistochemistry. In the ovary, the rck/p54 protein was specifically localized in the cytoplasm of developing oocytes from the early phase to the mature state (Fig. 1B). In contrast, in the testis, the protein was expressed in the germinal epithelial cells lining the seminiferous tubules (Fig. 2A). rck/p54 protein formed cytoplasmic particles in spermatogonia and primary spermatocytes (Fig. 2A-e), however, its expression significantly decreased in the spermatids, and then the remaining rck/p54 was concentrated in the heads of the spermatozoa (Fig. 2A-F). Interestingly, the expression gradually decreased as the spermatozoa matured along the epididymal duct (Fig. 2B). To confirm this result, we performed Western blot analysis of the testis and matured spermatozoa derived from the tail region of the epididymis (Fig. 2C). The 54-kDa band corresponding to intact rck/p54 in the matured spermatozoa showed significantly lower density than that in the testis; however, an immunoreactive protein of approximately 30 kDa, which was a minor form in the testis, was evidently expressed in the mature spermatozoa. This 30-kD protein was presumed to be partially proteolyzed rck/p54 protein. These data indicate that the rck/p54 protein plays important roles in both oogenesis and spermatogenesis.

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Figure 2. Immunohistochemistry for rck/p54 in the mouse testis. A: Testicular tissue is immunoreactive with anti-rck/p54 antibody (c and e). d and f are merged images of rck/p54 (green) and nuclei (blue) staining. a and b (merged image) is a negative control pair. An enlargement of the boxed area of f shows that rck/p54 is concentrated in the head of spermatozoa. B: Immunohistochemistry for rck/p54 in epididymis and Western blot analysis of rck/p54 in the sperm derived from the tail region of the epididymis. Longitudinal three panels: head region, body region, tail region. Horizontal three panels: the middle panels are for rck/p54 staining, the upper ones are negative controls (N.C.), and the lower ones are stained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) for visualization of nuclei. C: The amount of 54-kDa rck/p54 is lower in sperm than in testis, but the expression of the putatively proteolyzed rck/p54 protein (arrowhead) shows the opposite. Five micrograms of protein/lane was examined. Scale bar = 100 μm in a–d, 25 μm in e,f, 100 μm in B.

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rck/p54 in Early Embryogenesis

Because oocytes strongly expressed rck/p54, we next investigated whether rck/p54 is involved in early embryogenesis. We examined the expression levels of RCK mRNA and protein in embryos of three different stages, i.e., pronucleus, morula, and blastocytes. Morula- and blastocyte-stage embryos expressed RCK mRNA at the same level, which was less than that of the pronucleus-stage embryos (Fig. 3A). Western blot analysis showed that the protein level also decreased in the morula- and blastocyte-stage embryos compared with that in the pronucleus stage (Fig. 3B). Similarly, the immunocytochemical examination showed that rck/p54 protein was present in the pronucleus stage, but significantly decreased in the morula and blastocyte stage (Fig. 3C). rck/p54 was detectable mainly in the cytoplasm; however, the protein was barely detectable in the cytoplasm of the inner cell mass of the blastocyte (Fig. 3C-B). These results indicate spatiotemporal control of rck/p54 during embryogenesis.

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Figure 3. RCK mRNA and protein (rck/p54) expression in early embryogenesis. A: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RCK by using a LightCycler. Elongation factor-1 alpha (EF-1α) was used as an internal control. Each melting curve is shown as an inset. P, pronuclear embryos; M, morulae; B, blastocytes. B: Western blot analysis of rck/p54. Proteins extracted from pronuclear embryos and morulae (25 of each) and from 20 embryos at the blastocyte stage were analyzed. β-Actin was used as an internal control. C: Immunocytochemistry for rck/p54 during early embryogenesis. Images of anti-rck/p54 staining (green) and nuclear staining (orange) were merged.

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Effect of Excessive Expression of rck/p54 in the Early Embryo

Our findings on rck/p54 expression in early embryogenesis indicate that rck/p54 might need to be reduced to a lower steady-state level after fertilization. To study the influence of rck/p54 overexpression on early embryogenesis, we microinjected a CMV-based RCK expression vector into the pronuclei of pronucleus-stage embryos and cultured them for 105 hr. Nontreated and vector-injected embryos were used as controls. A CMV-based RCK expression vector was shown to be transcribed into its mRNA in COS-7 cells (data not shown). All embryos appeared to develop normally until the four- to eight-cell stage at 69 hr (Fig. 4A). By 105 hr, all of the nontreated and vector-injected embryos had developed to the blastocyte stage; however, approximately 16% (8 of 49) of the RCK-injected embryos still remained at the morula stage (Fig. 4A,B). Although up-regulation of rck/p54 was not confirmed for our technical reasons, this frequency is reasonable in consideration of the efficiency of introduction of foreign genes by microinjection, i.e., 25–30% (Brinster et al., 1985). To prove this event to be a specific for RCK, we examined the effect of RCK-deletion mutant (1–189 amino acids) on early embryogenesis; however, we did not find the significant differences between wild and mutant (Table 1). Considering the characteristics of rck/p54 with a high affinity to RNAs and its nonspecific binding with RNAs (Matsui et al., 2004), this result might be inevitable. Actually, overexpression of the deletion-mutant affected the growth as well as the wild-type in HeLa and COS-7 cells (data not shown). Based on such situation, we further examined the fertility in generating RCK-transgenic mice, because it can be assumed that the developmental delay induced abnormal effect on fertility (Fig. 5). Although there was no significant difference in the pregnancy rate, the birth rate of RCK experiment was significantly lower than those of the others. These data, thus, suggest that overexpression of rck/p54 may cause the developmental delay, resulting in abnormal fertility.

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Figure 4. Effect of rck/p54 overexpression on early embryogenesis. rck/p54 expression vector DNAs or vector DNAs were microinjected into pronuclei. A: The cleavage of each type of embryo in vitro. Morula embryos were observed only in rck/p54 overexpressed embryos at 105 hr (arrowhead). B: Evaluation of rck/p54 overexpression in early embryogenesis. The eggs that could develop past the four-cell stage were examined in this experiment (nontreatment eggs, 20; vector DNA-injected eggs, 46; rck/p54-injected eggs, 49).

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Table 1. In Vitro Development of Wild and Mutant RCK-Injected Pronuclear Stage Zygotes
DNANo. of embryos culturedNo. of embryos developed to
2-cell4-cell8-cellMorulaBlastocyte (%)
wild-RCK5050 (100)48 (96.0)45 (90.0)41 (82.0)37 (74.0)
mutant-RCK5050 (100)49 (98.0)48 (96.0)46 (92.0)39 (78.0)
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Figure 5. Effect of rck/p54 on generating RCK transgenic mice. Two kinds of control transgenic mice (Exp. 1 and Exp. 2) were also generated. Exp. 1 and Exp. 2 were performed before and after the RCK experiment, respectively. The pregnancy rate of RCK experiment is slightly lower than those of other two experiments; however, there is statistically no significance between them. In contrast, the birth rate of RCK transgenic mice is significantly lower than those of controls.

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In the current study, we demonstrated the expression of rck/p54, a DEAD-box RNA helicase, in mouse testis, ovary, and embryos. It was earlier reported that some members of DEAD-box RNA helicases are present in germ cells and functionally expressed during development, indicating that these proteins may actively participate in gametogenesis, embryogenesis, and development (Rocak et al., 2004). Based on one of these previous reports, one DEAD-box helicase seemed to function in either male or female germ cells of mice, but not in both. For example, ERH (embryonic RNA helicase) is expressed only in oocytes (Sowden et al., 1995); whereas PL10, P68, Mvh (mouse homologue of Vasa), and GRTH/Ddx25 (gonadotropin-regulated testicular RNA helicase) were detected mainly in the male germ cells (Leroy et al., 1989; Lemaire and Heinlein, 1993; Fujiwara et al., 1994; Tang et al., 1999). Mvh and GRTH/Ddx25 were well examined by using mice deficient in these helicases (Tanaka et al., 2000; Tsai-Morris et al., 2004). Male mutant mice homogeneous for either gene were infertile, but the female mice were fertile and did not show abnormality in their reproductive system. In the present study, we showed that rck/p54 is highly expressed in both female and male germ cells. In the female germ cells, rck/p54 was localized in all developing oocytes and remained in the pronuclear zygotes after fertilization. In contrast, in the male germ cells, rck/p54 was expressed in a granular pattern in spermatogonia and primary spermatocytes, and then the level decreased in the spermatids. Thus, rck/p54 and its orthologs may play important roles in common processes in both female and male germ cells.

In C. elegans, the protein level of CGH-1, the ortholog of rck/p54 in this worm, decreased in its protein level during embryogenesis (Navarro et al., 2001). In our present study, rck/p54 expression also declined after the pronucleus stage and was barely detectable in the inner cell mass of the blastocyst during early embryogenesis. Therefore, rck/p54 may be controlled spatiotemporally and may need to be reduced to a lower steady-state in early embryogenesis. Our results on RCK overexpression in the embryos support this speculation. It has been reported that rck/p54 protein participates in RNA decay (Cougot et al., 2004) and works negatively to translation efficiency by mRNA masking (Minshall et al., 2001). Collectively, rck/p54 is thought to be a translational repressor. Importantly, the expression of rck/p54 is found to be regulated in gametogenesis and in early embryogenesis. This finding indicated that rck/p54 may execute its roles in the cells in which gene expression is strictly regulated or repressed. Moreover, we were able to show a pivotal role of rck/p54 in embryogenesis by examining the generation of RCK transgenic mice.

The rck/p54 protein represents a highly conserved protein in eukaryotes, as evidenced by its various orthologs, the expression of which has been well examined in gametogenesis and early embryogenesis. Among the studies on rck/p54 orthologs, good similarities between mouse (present study) and C. elegans (Navarro et al., 2001) was observed in terms of the expression pattern. In the female mouse gonad, rck/p54 was detectable in the cytoplasm of developing oocytes, and CGH-1 was also detected in the cytoplasm throughout oogenesis. In contrast, in the male mouse gonad, rck/p54 was localized in a granular pattern in spermatocytes, which cells undergo meiosis. CGH-1 was also detectable in male germ cells that were entering meiosis, in which it was colocalized with P granules (granules present specifically in germline cells and containing protein and mRNA). In early embryogenesis, similarity was also observed. For example, rck/p54 remained present in the pronucleus stage in the mouse embryo, and its level significantly decreased after the stage; and CGH-1 expression also decreased as development progressed. These results suggest that rck/p54 plays conserved and essential roles in germ line cells and during early embryogenesis.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Animals

C57BL/6 strain mice maintained in the animal facility of Gifu International Institute of Biotechnology were used in the present study, and all experiments were performed in conformation with the animal experimental guidelines of Gifu International Institute of Biotechnology. Killed mice were immediately immersed in liquid nitrogen for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting or frozen in TISSU MOUNT (Shiraimatsukiki, Osaka, Japan) for immunohistochemistry.

Antibodies

Affinity purified rabbit polyclonal anti-rck/p54 antibody was developed in our laboratory. Recognition of rck/p54 was confirmed by performing an enzyme-linked immunosorbent assay (data not shown) and Western blotting (Fig. 1A). Mouse monoclonal anti–β-actin antibody was purchased from SIGMA (St. Louis, MO). Alexa 488–conjugated goat anti-rabbit IgG antibody was obtained from Invitrogen (Carlsbad, CA); and fluorescein isothiocyanate (FITC) -conjugated goat anti-rabbit antibody, from Jackson Immuno Research (West Grove, PA). Horseradish peroxidase–conjugated sheep anti-mouse Ig and donkey anti-rabbit Ig antibodies were purchased from Amersham Biosciences (Piscataway, NJ).

Immunohistochemistry

Eight-week-old female and male mice were used for the immunohistochemical study. Frozen sections of 7-μm thickness were prepared and analyzed as previously described by Matsumoto et al. (2002). Briefly, the sections were first fixed with 4% paraformaldehyde and preincubated for 1 hr at room temperature in a blocking solution containing 1% bovine serum albumin (BSA) and 50% Block Ace (Dainihon-Seiyaku, Osaka, Japan) in phosphate-buffered saline (PBS). The sections were then incubated with anti-rck/p54 antibody at 4°C overnight. After having been washed with 0.1% Tween 20 in PBS (PBST), they were next reacted with the Alexa-488 conjugated secondary antibody at room temperature for 1 hr. As negative controls, sections were reacted with the secondary antibody only. After washing with PBST, F-actin staining was performed with Alexa 546–phalloidin (Invitrogen), and nuclear staining with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI; Invitrogen). The stained sections were observed under a fluorescence microscope, an Olympus BX-51 (Olympus, Tokyo, Japan).

Western Blotting

Tissue samples were homogenized in chilled lysis buffer containing 10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% deoxycholic acid, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, and 1% Protease Inhibitor Cocktail (Sigma) and left standing for 30 min on ice. After centrifugation at 14,000 rpm for 20 min at 4°C, supernatants were collected as protein samples. Protein contents were measured with a DC Protein assay kit (Bio-Rad, Hercules, CA). Each protein sample was analyzed as previously described by Matsumoto et al. (2003).

Quantitative Real-Time RT-PCR

Total RNA was isolated by using an RNAqueous-4PCR kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA samples were reverse-transcribed by using Super Script II RNase H- reverse transcriptase (Invitrogen) and oligo(dT) primer (Invitrogen). Prepared cDNA samples were purified by use of a PCR Purification kit (QIAGEN, Hilden, Germany) and used for PCR.

Quantitative real-time PCR was performed on a LightCycler (Roche, Mannheim, Germany) using a LightCycler Fast Start DNA Master SYBR Green I kit (Roche) according to the manufacturer's instruction. Elongation factor-1α (EF-1α) was used as an internal control because of its stable expression in vivo (Matsumoto et al., 2002). Primers for RCK and EF-1α were as follow: RCK forward, 5′-CATTCCCAT-TGCTTTATCTGGTAG-3′; RCK reverse, 5′-ATCATCTGGACATGATCAACCTT-T-3′; EF-1α forward, 5′-CTCAGGTGATTATCCTGAACCATC-3′; EF-1α reverse, 5′-AACAGTTCTGAGAC-CGTTCTTCCA-3′. The PCR products were evaluated by inspection of their melting curve.

Preparation of Embryos and Immunocytochemistry

C57BL/6 mice were used as fertilized embryo donors. They were caused to superovulate by being injected sequentially with 5 IU of equine chorionic gonadotropin and 5 IU of human chorionic gonadotropin (hCG) given 48 hr apart. At 22 hr after the hCG injection, zygotes were collected from the oviducts of superovulated females that had been mated with male of the same strain. Before being used for an experiment, the zygotes were cultured in M16 medium at 37°C under 5% CO2 in air. Pronucleus-, morula-, and blastocyte-stage embryos were used for RT-PCR, Western blotting, and immunohistochemistry.

Each embryo or earlier stage cells were fixed for 1 hr in ice-cold 1% acetic acid prepared in 95% methanol and then equilibrated sequentially with ice-cold 100% ethanol and 70% ethanol, each for 1 hr. After having been washed with PBS, the embryos were blocked for 1 hr at room temperature with 5% normal goat serum and 1% BSA in PBS. The embryos were then incubated with anti-rck/p54 antibody at 4°C overnight. After washing with PBS, the embryos were reacted with the FITC-conjugated secondary antibody at room temperature for 1 hr. After additional washing with PBS, nuclear staining was performed with propidium iodide (Invitrogen). The stained embryos were observed under a LSM510 Zeiss confocal laser-scanning microscope (Carl Zeiss, Jena, Germany).

Generation of RCK Transgenic Mice

For construction of transgenic mice, human RCK transgene was cloned into KpnI and XbaI site of pCAGGS plasmid. To isolate the expression unit of RCK transgene, the plasmid was cleaved with SalI and BglII. The purified DNA fragments (1–2 pl at a concentration of 10 ng/ml) were injected into pronucleus of zygotes obtained from superovulated. The injected eggs were then transferred to the oviduct of pseudopregnant recipient mice 0.5 days post coitus (CD-1 strain).

To examine the role of RCK in early embryogenesis, the injected eggs were cultured in M16 medium. As controls, nontreated eggs, vector DNA fragment-injected eggs, and RCK-deletion mutant (1–189 amino acids)-injected eggs were used.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Y.C. was funded by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES