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Gamete Biology |
Laboratory of Reproductive Biology,3 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Maebashi Institute of Animal Science,4 Livestock Improvement Association JAPAN, Inc., Maebashi 371-0121, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gametogenesis, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During oocyte growth and follicular development, oocytes accumulate maternal-effect factors necessary for early embryogenesis, which occurs in the absence of de novo transcription of either parental genome [8]. Six such maternal-effect genes have been characterized using knockout mouse models [9–14]. Maternal antigen that embryos required (MATER) was first identified as an ooplasm-specific protein [15] encoded by a single-copy gene that is transcribed in growing oocytes. Homozygous null Mater males and heterozygous females have normal fertility, although homozygous females are sterile. Although folliculogenesis, ovulation, fertilization, and the first cleavage appear normal, early embryos lacking MATER are unable to progress beyond the two-cell stage [9]. Heat-shock factor-1 (Hsf1) was also identified as a maternal-effect gene. Embryos lacking HSF1 are blocked mainly at the one-cell stage and show ultrastructural abnormality in nuclei at the two-cell stage [16]. Recently, oocyte-specific gene, Zar1 (zygote arrest 1) and Npm2 (nucleoplasmin 2) have been identified using subtractive hybridization [13, 14]. Homozygous null Zar1 females are sterile because the embryos from the female are arrested at the one- to two-cell stage. Zar1 is detected after resumption of meiosis, persists in one-cell embryos and rapidly disappears at the two-cell stage, suggesting a critical role in the oocyte-to-embryo transition [14]. Npm2 knockout females have fertility defects because of reduced cleavage to the two-cell stage. In Npm2 null oocytes and zygotes, absence of coalesced nucleolar structures and loss of heterochromatin and deacetylated histone H3 are observed, suggesting that Npm2 is critical for nuclear and nucleolar organization and embryonic development [13]. Another germ cell-specific gene, Gasz has been identified using in silico subtraction and the genomic database [17]. Based on its unique structure that includes ankyrin repeats, a sterile-
motif and a putative leucine zipper domain and its cytoplasmic localization, the protein is suggested to function as an important cytoplasmic signal transducer in both male and female germ cells. Still other genes are transcribed only in oocytes, but it is unclear whether the transcripts are translated [18].
We previously identified a novel gene (c-1; herein called Oogenesin) that is expressed in two-cell mouse embryos [19]. In this study, Oogenesin mRNA expression was greater in two-cell blocked embryos than in normal developing two-cell embryos in which the Oogenesin mRNA is normally degraded. The reason for the higher expression in two-cell blocked embryos is unclear, although this has also been observed for other genes such as Tcl1 [19, 20]. Here we report the sequence of this gene and its stage- and tissue-specific expression, mRNA localization and the protein expression and subcellular localization in oocytes and preimplantation embryos. On the basis of its expression pattern, cellular localization and deduced protein structures, we postulate that the Oogenesin gene product (OOGENESIN) functions as an important factor in zygotic transcription of preimplantation embryos as well as folliculogenesis and oogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fragment of amplified cDNA obtained from differential display [19] was cloned using a TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Cloned DNA was sequenced with an ABI PRISM 377 automatic sequencer using an ABI DNA sequencing kit (Dye Terminator Cycle Sequencing FS Ready Reaction Kit; Applied Biosystems, Foster City, CA). DNA sequences were analyzed with the software package AutoAssembler (Applied Biosystems). The DNA sequences were compared with the sequences in the GenBank, EMBL, and dbEST databases using the BLAST program. Based on the cDNA sequence of expressed sequence tags (ESTs) identified, gene-specific PCR primers were designed and the targeted genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using a Marathon-Ready cDNA kit (cDNA of mouse 17-day embryo; Clontech, Palo Alto, CA) and an Advantage 2 PCR Enzyme System (Clontech). The complete sequence of the gene was constructed using the EST database and by 5'-RACE (rapid amplification of cDNA ends). The GenBank accession number of the Oogenesin cDNA is AB050008.
RNA Isolation and cDNA Synthesis from Somatic Tissues
Ovaries, testis, liver, kidney, spleen, heart, lung, and brain were obtained from 8-wk-old female ICR mice. Total RNA was extracted with ISOGEN (Nippon Gene Inc., Tokyo, Japan) from each somatic tissue. After treatment with Amplification Grade DNase I (Life Technologies, Inc., Gaithersburg, MD), reverse transcription was performed on approximately 1 µg of isolated RNAs in 20-µl reaction solution using M-MLV reverse transcriptase (Superscript II; Life Technologies).
RNA Isolation and cDNA Synthesis from Oocytes, Embryos, and Ovaries
Three- to five-week-old female Crj:CD-1(ICR) mice (Charles River Japan Inc., Tokyo, Japan) were superovulated with intraperitoneal injections of 5 IU eCG (Teikoku Hormone M.F.G. Co., Ltd., Tokyo, Japan) followed 48 h later by 5 IU hCG (Sankyo Zoki Co., Ltd., Tokyo, Japan). Ovulated oocytes (metaphase II-arrested oocytes) were freed from the surrounding cumulus cells and used for RNA isolation. Fertilized embryos were produced by in vitro fertilization (IVF) and in vitro culture as described previously [19]. Total RNA was isolated from each group of 50 oocytes or preimplantation embryos using an RNA isolation kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Reverse transcription was performed on RNA obtained from 50 oocytes or embryos in 20-µl reaction solution using M-MLV reverse transcriptase. Fetal ovaries were isolated from 12.5 days postcoitum (dpc) to 19.5 dpc embryos. Ovaries were also isolated from newborn and 2-wk-old ICR mice. RNAs were extracted using ISOGEN and reverse transcription was performed on approximately 1 µg of extracted RNAs as described above.
Reverse Transcription Polymerase Chain Reaction
To confirm the expression of Oogenesin, synthesized cDNA was subjected to PCR on a thermal cycler (PTC-100; MJ Research, Watertown, MA) using the following conditions: 94°C for 2 min and (94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min) x 35 cycles; the last cycle was followed by a 5-min extension at 72°C. The following sense and antisense primers were used for PCR (followed by the expected sizes of the PCR product): sense, 5'-CTTTTCACACTCACACTAGATCACAT-3'; antisense, 5'- GGGCCAGTCTCATAAAATGGTGC-3' (983 base pairs [bp]). The primers were designed to span an intro sequence (Fig. 1). Because the 1617-bp product was amplified in testis cDNA and genomic DNA using the same set of the primers, both products were sequenced and confirmed. For positive control, ß-actin was amplified using the following primers: sense, 5'-CGAGCGTGGCTACAGCTTCACC-3'; antisense, 5'-CCGATCCACACAGAGTACTTGCG-3' (444 bp). In two-cell stage embryos, RT-PCR was performed at each cell cycle stage (G1: 1 h; S, 4 h; early G2: 10 h, late G2: 21 h after cleavage). Embryos used for RT-PCR were collected as described previously [21].
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Northern Blot Analysis
Total cellular RNA was isolated by the alkaline guanidine phenol chloroform method from ovary, testis, liver, kidney, spleen, heart, lung, and brain. Five µg total RNA from these tissues was denatured and size fractionated on 1% agarose gels containing 2.2 M formaldehyde and transferred to a GeneScreen nylon membrane (NEN Lifescience Products, Boston, MA). A 1.0-kb fragment in the open reading frame (ORF) was used as a probe. Hybridization was performed using a 32P-labeled probe at 65°C in SSC-Denhardt-SDS buffer. The membrane was washed for 10 min each in 2x SSC and in 2x SSC and 0.1% SDS, then washed in 0.1x SSC and 0.1% SDS twice, and autoradiographed. The same membrane was stained with 2% methylene blue to locate 28S and 18S rRNAs and verify equal loading per lane before Northern blotting (data not shown).
In Situ Hybridization
Eight-week-old mouse ovaries were fixed by incubation in 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin. Paraffin-embedded ovaries were cut into 5-µm sections. In situ hybridization to ovarian cross-sections was performed as described previously [22]. Briefly, sense and antisense RNA probes were transcribed in vitro with T3 and T7 RNA polymerase from linearized pBluescript II vectors (Stratagene) containing approximately 1.0 kb of Oogenesin open reading frame cDNAs in the presence of digoxygenin-11-UTP (Roche Diagnostics, Basel, Switzerland). One hundred ng of labeled transcripts (1.0 kb) were hybridized after hydrolysis to tissue sections at 42°C. Digoxigenin was detected with alkaline phosphatase-labeled anti-DIG Fab fragments and 4-nitro blue tetrazolium chloride/5-bromo-4 chloro-3 indolyl phosphate (Roche Diagnostics). Sections were counterstained with safranin-O and mounted by coverslips with glycerol gelatin.
Western Immunoblotting
Immunoblotting was performed using an affinity-purified polyclonal antibody raised against the specific peptide sequence (LGFLLERVGDTLKTLELDSC) in the putative protein. Fifty oocytes were collected 15 h after hCG injection. Fifty embryos at each developmental stage (one-, two-, and four-cell stage and morula/blastocyst stage) were collected 5, 22, 45, and 96 h after IVF, respectively. Each sample was subjected to 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). The membranes were first incubated for 2 h in PBS-Tween (PBS-T; 136 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4·12H2O, 1.47 mM KH2PO4, 0.1% Tween-20) containing 10% fetal calf serum (FCS). The blocked membranes were then incubated for 2 h with antibody diluted 1:100 in PBS-T containing 5% FCS. After incubation, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Biosciences Corp.) (1:1000 dilution) for 1 h. The blot was detected using the Enhanced Chemiluminescence (ECL) detection system according to the instructions of the manufacturer (Amersham Biosciences Corp, Piscataway, NJ). The molecular weights of detected bands were determined with a model 4.0 ATTO densitograph (ATTO Inc., Tokyo, Japan). The same membranes were reprobed with antibody absorbed by peptides for negative control as described above.
Immunohistochemistry and Immunocytochemistry
Immunohistochemistry was performed with the same antibody used in immunoblotting. An avidin-biotinylated enzyme complex (ABC) staining kit (Vector Laboratories, Burlingame, CA) was used on the paraffin sections according to the protocol specified by the suppliers. Briefly, 2-wk-old mouse ovaries were fixed with 4% paraformaldehyde at 4°C overnight. Five-µm-thick sections were mounted onto polylysine-coated slides, blocked with 10% normal goat serum, reacted with 100 µl primary antibody diluted 1:100 in PBS, and incubated for 1 h. Control sections were treated with antibody absorbed by antigen peptides. Counterstaining was done with methyl green (Vector Laboratories). Ovulated oocytes and preimplantation embryos used for immunocytochemistry were recovered at 15 h (oocyte, n = 21), 18 h (early one-cell stage, n = 30), 24 h (middle one-cell stage, n = 51), 30 h (late one-cell stage, n = 51), 32 h (early two-cell stage, n = 121), 40 h (middle two-cell stage, n = 45), 48 h (late two-cell stage, n = 49), 54 h (four-cell stage, n = 31), 66 h (eight-cell stage, n = 24), 84 h (morula stage, n = 34) and 92 h (blastocyst stage, n = 28) after hCG injection. Embryos were fixed for 10–15 min at room temperature in 3.7% formaldehyde in PBS [23]. The fixed cells were blocked for 1 h in blocking buffer (10% normal goat serum, 0.1% Triton X-100 in PBS) and then incubated with antibody diluted 1:100 in PBS for 1 h. The cells were washed three times for 5 min each in PBS containing 10% normal goat serum, then incubated in Alexa 488 conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) at a dilution of 1:200 in PBS for 1 h and washed as described above. Observation of fluorescence was carried out with a confocal laser microscope (Carl Zeiss, Oberkochen, Germany) and Zeiss image analysis system. Nuclear localization of the protein was confirmed by Hoechst staining and transmission image. For negative control, we performed immunofluorescence experiment for OOGENESIN with peptide-absorbed primary antibody. All performances were carried out at room temperature.
Experimental Animals
All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of Kyoto University.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fragment of Oogenesin that we first sequenced was used for a BLAST homology search of the mouse EST database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). We found six mouse ESTs (AA413536, AU020451, AU018055, AA690259, AA546733, and AU015289), which are homologous to the Oogenesin fragment. These fragments were used for a further homology search (EST walk) and another five mouse ESTs (AA681175, AA549437, AA823887, AA681501, and AA549248) were found. All of these ESTs were derived from mouse embryos. Using these ESTs, we reconstructed a part of Oogenesin, the length of which was 980 bp. After reconstruction, we used the 5'-RACE reaction to determine the full length of Oogenesin and determined another 406-bp sequence. During the process of determining the full length of Oogenesin, a sequence from mouse BAC library of chromosome 12 that contains the Oogenesin gene was published (AC079644). Using these data, we finally determined the full length of the Oogenesin cDNA (Fig. 1). Recently, however, several homologous sequences on chromosome 4 have been published (AL627131, AL626771, and AC118466). A homology search using Ensembl Genome Data Resources (Sanger Institute, http://www.ensembl.org/Mus_musculus/) indicated that Oogenesin is really located on chromosome 4. In addition, genomic Southern blot analysis in our preliminary experiments revealed that the Oogenesin is a single-copy gene (data not shown).
The identified cDNA contains a 1149-bp-long open reading frame (ORF), which encodes a predicted protein of 326 amino acids with a leucine zipper domain. A search for common protein motifs in the deduced protein revealed a leucine zipper structure at amino acid positions 131–152 and a leucine-rich domain at positions 131–254 (Fig. 1). There are multiple, potential protein kinase C, casein kinase II, and tyrosine kinase phosphorylation sites along the OOGENESIN polypeptide chain.
Tissue- and Stage-Specific Expression of Oogenesin
We examined the expression of Oogenesin in various tissues, oocytes, and preimplantation embryos. RT-PCR analysis, using primers specific for Oogenesin and ß-actin (positive control) transcripts, were performed on total RNA from eight different tissues, oocytes, and preimplantation embryos until the blastocyst stage. Expression of the Oogenesin gene was detected in ovary, ovulated oocytes, and preimplantation embryos until the end of the two-cell stage (Fig. 2, A and C). Northern blot analysis revealed a single prominent transcript of approximately 1.7 kb only in ovarian RNA (Fig. 2B). The size of the mRNA was in agreement with the predicted transcript length (1485 bp cDNA plus 150–200 nt poly-A tail). To confirm the expression stage of Oogenesin, RT-PCR analysis was performed using various stages of fetal, newborn, and 2-wk ovaries. Oogenesin expression initiated at 15.5 dpc and continued to be expressed through the adult stage (Fig. 2D and data not shown).
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To determine the expression site of Oogenesin mRNA in the ovary, we performed in situ hybridization. Ovaries from 8-wk-old mice were fixed, sectioned, and hybridized with digoxigenin-labeled sense and anti-sense probe. Specific signals for Oogenesin mRNA were clearly detected in oocytes of antral and secondary follicles and faint signals were detected in oocytes of primordial and primary follicles (Fig. 3, A–C). Oogenesin is only detectable in oocytes because no signal was observed in cumulus cells isolated from ovulated oocytes by RT-PCR (Fig. 3D).
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Western Immunoblotting of OOGENESIN Protein in Oocytes and Early Cleavage Embryos
Whole extracts of oocytes and preimplantation embryos were analyzed by Western blot analysis to confirm the expression of OOGENESIN protein during development. The analysis using an affinity-purified rabbit polyclonal antibody raised against a synthetic peptide (LGFLLERVGDTLKTLELDSC) and revealed that the molecular size of OOGENESIN protein is approximately 46 kDa (Fig. 4), which is a little larger than that of the predicted size (37 kDa) on the basis of the Oogenesin cDNA. OOGENESIN is expressed predominantly in oocytes and one- to four-cell-stage embryos, whereas it is expressed only weakly in morula/blastocyst-stage embryos. Increasing expression was observed from oocytes to the one-cell stage and the expression decreased toward the four-cell stage. The same membrane stripped the first antibody and reprobed by peptide-absorbed antibody (negative control) showed no signal.
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Immunohistochemistry of OOGENESIN Protein in Mouse Ovary
Immunohistochemistry of ovary sections revealed that OOGENESIN already existed in ovarian oocytes. As shown in Figure 5, strong signals were detected in oocytes of primary, secondary, and antral follicles and weak signals were detected in oocytes of primordial follicles.
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Immunocytochemistry of Oocytes and Preimplantation Embryos
The pattern of nuclear localization of OOGENESIN protein was examined. In all early one-cell embryos, OOGENESIN protein exists only in cytoplasm and gradually moved to the nuclei at the middle one-cell stage. At the late one-cell stage, most of the embryos (43/51: 84.3%) exhibited greater signals in nuclei than in cytoplasm and almost the same pattern was maintained at the early two-cell stage (68/121: 56.2%). After the middle two-cell stage, most of the protein existed in cytoplasm and never existed in nuclei after the four-cell stage. The typical appearances of OOGENESIN localization in oocyte and preimplantation embryos are shown in Figure 6. The OOGENESIN protein sequestered in the cytoplasm of oocytes and early one-cell-stage embryos (Fig. 6, A and B); however, the protein started to translocate to the nuclei of middle one-cell-stage embryos (Fig. 6C) and accumulate in the nuclei of the late one-cell- and early two-cell-stage embryos (Fig. 6, D and E). At the middle and late two-cell stage, some embryos exhibited equal signals in nuclei and cytoplasm (Fig. 6F), others exhibited strong signals in cytoplasm (Fig. 6G). The protein did not accumulate in the nuclei after the four-cell stage (Fig. 6, H–J).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[2, 28, 29]. Oogenesin mRNA, which is detected during oogenesis, is not detected after the four-cell stage. OOGENESIN protein is first detected in growing oocytes, dramatically increases at the one-cell stage, remains present until the four-cell stage, and has almost disappeared by the morula/blastocyst stage. The nearly same pattern of gene expression has been reported in the Mater [9]. Expression of the Mater is observed in the growing oocyte and continues to the blastocyst stage. In homozygous null Mater female mice, folliculogenesis, ovulation, and fertilization occur normally, although early embryos lacking MATER are unable to progress beyond the two-cell stage [9]. This phenotype resembles the two-cell block, which occurs in cultured embryos [30, 31] and a striking decrease in transcriptional activity is observed in the early embryos lacking MATER [32]. In many mammalian species, the developmental block occurs at the time of zygotic genome activation [33], suggesting the relationship between early embryonic development and zygotic genome activation. The increased expression of Oogenesin in two-cell blocking embryos [19] may well be the result of suboptimal culture conditions in vitro, as seen previously for Tcl1 [20]. However, the protein expression pattern and the timing of nuclear localization of the protein indicate the possibility that the protein is involved in the development of mouse embryos via transcription regulation. Different product size amplified in testis cDNA indicates the possibility of alternative splicing of the gene and may suggest that the protein product detected only in oocytes functions in pre-mRNA splicing during embryogenesis as reported in Drosophila [34].
A maternal-effect gene, Zar1, is present in oocytes and one-cell embryos; however, a rapid disappearance was observed at the two-cell stage. Zar1 null embryos synthesize less transcription requiring complex (TRC), makers of embryonic genome activation [35], than control embryos and are predominantly arrested at the one-cell stage, suggesting that this maternal factor functions before embryonic genome activation and is required for the activation [14].
The OOGENESIN protein contains a leucine zipper domain (at amino acid positions 131–152). In this respect, it resembles other oocyte-specific genes, such as MATER [36] and GASZ [17], and may thus function as a transcriptional regulator. The presence of a leucine-rich domain (amino acid positions 131–254) as well as a leucine zipper domain, both of which are known to mediate protein-protein interactions [37, 38], suggests that there are other proteins in oocytes and/or embryos that interact with OOGENESIN. In the mouse, de novo zygotic transcription is first detected in the pronuclei of one-cell embryos [39]. However, the major zygotic genome activation occurs at the late two-cell stage [40, 41], and the maternal transcripts accumulated during oogenesis are degraded by the two-cell stage. In the present study, it is revealed that OOGENESIN localizes in nuclei of the late one-cell- to early two-cell-stage embryos. This time coincides with the timing of de novo zygotic transcription. In view of these results, OOGENESIN may have a role in zygotic genome activation and it is possible that the gene represents a novel maternal-effect gene in the mouse. A higher molecular weight band (58 kDa) observed at the four-cell stage may represent the ubiquitination of OOGENESIN protein before degradation or phosphorylation; however, the details remain to be determined. Transcription factors are often used for more than one function due to the economy of developmental systems. For example, an oocyte-specific transcription factor, FIG, which regulates the expression of the three zona pellucida genes (Zp1, Zp2, and Zp3), was also found to play a key regulatory role in primordial follicle formation [29]. Thus, OOGENESIN, like FIG, may function as both a transcriptional regulator and a key regulator of folliculogenesis and/or oogenesis. It should be possible to generate Oogenesin knockout mice because our preliminary experiment revealed that Oogenesin is only detectable in oocytes.
In summary, we have identified an oocyte-specific protein that contains a leucine zipper structure and a leucine-rich region and that localizes nuclei of early cleavage-stage embryos. Future identification of proteins that interact with OOGENESIN should provide insights into the mechanisms that regulate gene expression within female germ cells during oogenesis and/or embryogenesis. In addition, generation of Oogenesin knockout mice should help us to elucidate the physiological function of this novel protein.
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FOOTNOTES |
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2 Correspondence: Naojiro Minami, Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. FAX: 81 75 753 6329; naojiro{at}kais.kyoto-u.ac.jp
Received: 10 April 2003.
First decision: 8 May 2003.
Accepted: 10 July 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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