Mol. Hum. Reprod. Advance Access originally published online on April 1, 2005
Molecular Human Reproduction 2005 11(5):325-333; doi:10.1093/molehr/gah166
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Temporary developmental arrest after storage of fertilized mouse oocytes at 4°C: effects on embryonic development, maternal mRNA processing and cell cycle
1Division of Basic Molecular Science and Molecular Medicine, School of Medicine, 2Center of Genetic Engineering for Human Diseases and 3Department of Molecular and Developmental Science, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
4 To whom correspondence should be addressed at: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan. Email: sakurai{at}is.icc.u-tokai.ac.jp
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Abstract |
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The aim of this study was to examine whether fertilized mouse oocytes can survive after short-term incubation (for 6–48 h) at 4°C. When fertilized oocytes of ICR and C57BL/6 (B6) strain were incubated at 4°C and returned to normal culture conditions (37°C), development of these 4°C-treated embryos for up to 12 h (for ICR) to blastocyst stage did not differ from that of untreated oocytes. Even 4°C-treated embryos for 48 h developed to blastocysts at relatively good rates (33.3% for ICR and 50.8% for B6). The in vivo development of 4°C-treated embryos for 12, 24 and 36 h to fetal stage was similar to that of untreated ones. BrdU labelling assay revealed temporary cessation of DNA replication in 4°C-treated fertilized oocytes. Post-fertilization events including cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene and elevated mRNA expression of zygotic gene activation-related genes were temporarily suppressed in 4°C-treated embryos. These findings indicate that 4°C-treatment of fertilized murine oocytes results in temporary cessation of molecular events. We also show that 4°C-treated fertilized oocytes for 12 h can be used for preparation of transgenic mice.
Key words: cell cycle/fertilized oocyte/low temperature preservation/maternal RNA/4°C-storage
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Introduction |
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Since the first reports by Whittingham et al. (1972)
and Wilmut (1972) that murine preimplantation embryos could be successfully cryopreserved, many attempts have been made to improve the preservation of mouse embryos below 0°C. Many types of mammalian embryos from those of non-domestic animals such as rodents and those of domestic animals have been successfully cryopreserved, but the rate of survival of frozen-thawed embryos is usually lower than that of freshly isolated embryos (Dobrinsky, 2002; Leibo and Songsasen, 2002).
Long-term preservation of embryos is performed by immersing the embryos in appropriate cryoprotectants and subsequently storing them in liquid nitrogen. Methods used to maintain embryos in a metabolically inhibited but viable state include cooling at refrigerated temperatures (short-term storage), which is clearly simpler, more convenient and inexpensive than cryopreservation of embryos. Success with storage of preimplantation embryos at temperatures from 0 to 7°C has been reported for rabbits (Chang, 1947, 1948), cows (BonDurant et al., 1982; Lindner et al., 1983), sheep (Harper and Rowson, 1963) and mice (Sherman and Lin, 1959; Kasai et al., 1983; Kasai, 1986; Nakamura and Tsunoda, 1986; Herr and Wright, 1988; Miyoshi et al., 1992; Nakamura and Tsunoda, 1992; Wiggins et al., 1999; Tsuchiya et al., 2001). However, the rate of success depends on the species, stage of preimplantation embryo and methods used in each laboratory. For example, Lindner et al. (1983) found that survival of bovine blastocysts stored at 4°C for 48 h was similar to that of non-stored blastocysts. Hughes and Anderson (1982) maintained viability of rabbit embryos at 4°C for up to 15 days. Generally, embryo viability decreases more rapidly with increasing length of storage when embryos are stored at low temperatures (0–10°C), although the reason for this remains unclear. Otoi et al. (1999) reported a relationship between decrease in developmental ability of bovine embryos stored at 4°C and increase in number of necrotic cells. The mechanism underlying thermal shock of embryos during storage at low temperature above 0°C has recently been progressively clarified, but the effects of cooling to temperatures above 0°C on molecular events of preimplantation embryos are still unclear.
We hypothesized that exposure of embryos to low temperatures, which makes embryos metabolically inactive, may temporarily extend or discontinue several molecular events associated with fertilization, DNA replication, zygotic gene activation (ZGA), close association of female and male pronuclei, genome reprogramming, and inheritance and expression of maternal RNAs and proteins. Since the above mentioned molecular events proceed rapidly in preimplantation embryos, low temperature treatment would be helpful for study of the molecular mechanisms of preimplantation embryogenesis itself. In this study, we examined whether temporary 4°C-treatment affects development of fertilized mouse oocytes and expression of cell cycle-related molecules and maternal RNA. In addition, we performed pronuclear microinjection of DNA into low temperature-treated fertilized oocytes, with the expectation of increase in efficiency of introduction of exogenous DNA into the mouse genome.
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Mice and culture media
ICR and C57BL/6J (hereafter termed B6) mice (CLEA Japan Inc., Tokyo, Japan) were used in this study. These mice were kept on a 12 h light/12 h dark schedule (lights on from 7:00 to 19:00 h) and allowed food and water ad libitum. Experiments were carried out in accordance with The Guide for Care and Use of Laboratory Animals of Tokai University.
All reagents were obtained from Sigma-Aldrich Japan Co. Ltd (Tokyo, Japan) unless otherwise stated. A culture medium, modified Tyrode's medium (mTM) (Fraser, 1993; Table I), was used for IVF in mice. Potassium modified simplex optimized medium (KSOM) was used to cultivate fertilized mouse oocytes (Lawitts and Biggers, 1993). For storage of fertilized oocytes at 4°C, we used a modified KSOM [hereafter termed ‘Hepes-KSOM’, which contained 20 mM Hepes and a reduced amount (2.5 mM) of NaHCO3 and was adjusted to pH 7.4] and KSOM (Lawitts and Biggers, 1993). The compositions of these media are shown in Table I.
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Isolation of fertilized oocytes
Fertilized oocytes of ICR or B6 mice were prepared by a standard method of IVF (Fraser, 1993
). Briefly, spermatozoa were obtained from the cauda epididymides of 5–10-week-old males. They were suspended in 500 µl of mTM covered with paraffin oil (#26137-85; Nakalai Tesque Co. Ltd, Tokyo, Japan) on a 35 mm culture dish (#1008; Nippon Becton Dickinson Co. Ltd, Tokyo, Japan) and incubated for 1.5 h. To collect ovulated oocytes, 5–10-week-old females were ovulation inducted by injection of 5 IU of eCG (Serotropin; Teikokuzouki Co. Ltd, Tokyo, Japan) followed by 5 IU of HCG (Gonatropin; Teikokuzouki Co. Ltd, Tokyo, Japan) 47 h later. Oocyte–cumulus masses were taken from the excised oviducts into 300 µl of mTM (covered with paraffin oil) at 14–15 h after HCG administration. Spermatozoa at a final concentration of 300 cells/µl were added to the mTM containing the oocyte–cumulus masses of the same strain. Dishes were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 6–9 h. After oocytes were washed three times in mTM, they were checked for the presence of both pronuclei and the second polar body, which provides evidence of successful fertilization of oocytes. Insemination time was in this study considered the time of fertilization. For example, fertilized oocytes at F-6 h correspond to oocytes incubated for 6 h after insemination.
In vitro culture and 4°C-storage of fertilized oocytes
Each of 20 fertilized oocytes from F-6 to F-9 h was transferred to 30 µl of KSOM covered with paraffin oil and incubated under normal conditions (at 37°C in a humidified atmosphere of 5% CO2 in air) until experiments were carried out. As a control, some of the fertilized oocytes were continuously cultivated at 37°C. For 4°C-treatment, each of 20 fertilized oocytes was first transferred into 30 µl of Hepes-KSOM covered with paraffin oil on a culture dish and left for 10 min at room temperature (24°C). They were then cooled to 4°C (rate of decrease in temperature, 5°C/min) by directly transferring the dish to a general laboratory refrigerator (MPR-213F; SANYO Co. Ltd, Japan) and left for adequate periods of time. Under these conditions, pH was estimated to be 7.4 for Hepes-KSOM. Just before return to normal conditions at 37°C, the cooled dishes were left for 10 min at room temperature. After that, the 4°C-treated fertilized oocytes were picked up, washed three times in fresh KSOM and finally transferred into 30 µl of KSOM covered with paraffin oil on culture dishes for further cultivation at 37°C in 5% CO2 atmosphere. The in vitro development of the fertilized oocytes was inspected with a stereoscopic microscope (SZX12; Olympus, Tokyo, Japan) or an inverted microscope (IX70; Olympus, Japan).
Estimation of cell number in blastocysts
Cell numbers of trophectoderm and inner cell mass (ICM) of blastocysts, which had developed from the control or 4°C-treated fertilized oocytes in Hepes-KSOM, were determined by the method of Handyside and Hunter (1984) with some modifications. Briefly, the zona pellucida of blastocysts was first removed with acid Tyrode's solution. These blastocysts were then transferred into 50% rabbit anti-mouse serum in Hepes-KSOM for 15 min at room temperature. After washing for three times in Hepes-KSOM, they were transferred to 15% guinea pig complement serum in Hepes-KSOM, 20 µg/ml Hoechst 33342 and 10 µg/ml propidium iodide, and incubated in the dark for 60 min at room temperature. After washing three times in Hepes-KSOM, they were fixed in 4% formalin containing 10 µg/ml Hoechst 33342 and 1 µg/ml propidium iodide in the dark for 20 min at room temperature. The fixed blastocysts were sealed with a cover slide on a glass slide and observed under an Olympus IX70 inverted fluorescence microscope with an appropriate filter set (U-MWU2; Olympus, Japan). Cells exhibiting pink and blue fluorescence were judged to be trophectoderm and ICM, respectively. All blastocysts were photographed and the number of cells of each type was recorded.
Embryo transfer
Ten 2-cell ICR embryos, which had developed from the control or fertilized oocytes 4°C-treated for 12, 24, or 36 h in Hepes-KSOM, were used for each experiment. To avoid transfer of embryos treated under the same conditions into both oviducts of a recipient female, embryos in each different treatment group were transferred to either the left or right oviduct of ICR surrogate females (10–15-week-old). For example, one oviduct underwent transfer of control embryos and the other oviduct embryos 4°C-treated for 12 h. A total of 12 recipients were used. A total of 12 oviducts (6 right and 6 left oviducts) underwent transfer in each experimental group. The surrogate females were killed on day 16–18 of gestation to determine number of implantation sites and live fetuses.
BrdU labelling and immunological detection
For detection of DNA replication in fertilized oocytes, we used BrdU labelling and subsequent immunostaining using anti-BrdU antibody (Spector et al., 1998) with some modifications. Briefly, fertilized oocytes were cultivated in KSOM containing 20 µM BrdU for 1 or 3 h at 37°C in a humidified atmosphere of 5% CO2 in air. Some fertilized oocytes were cultivated in Hepes-KSOM containing 20 µM BrdU for 12 h at 4°C. After cultivation with BrdU-containing media, fertilized oocytes were all fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.2) for 15 min at room temperature. After washing with PBS and 0.02% Tween 20 (PBT) several times, the oocytes were permeabilized in 1% Triton-X in PBS for 30 min at room temperature. They were then incubated in 2 M HCl in PBS for 60 min and then in 0.1 M Na2B4O7 in PBS (pH 8.5) for 15 min at room temperature to denature chromosomal DNA. They were next incubated with mouse anti-BrdU monoclonal immunoglobulin G (IgG) antibody (Boehringer Mannheim GmbH, Mannheim, Germany) diluted 50-fold with PBS and 10% goat serum for 1 h, and then with Alexa 546-labelled anti-mouse IgG goat polyclonal antibodies (Molecular Probes Inc., Eugene, OR, USA; 1:2000 in PBT) for 1 h in a dark box at room temperature. Fluorescence was observed using an Olympus IX70 inverted fluorescence microscope with an appropriate filter set (U-MWIG; Olympus, Japan).
RNA blot analysis of mouse embryos
Total RNAs were extracted from 25–30 preimplantation ICR embryos using an ISOGEN RNA extraction kit (Nippon Gene Co. Ltd, Tokyo, Japan) according to the manufacturer's protocol with some modifications. Briefly, embryos were mixed with 50 µl of ISOGEN in a 1.5 ml Eppendorf tube and vortexed thoroughly. Total RNA was mixed with 10 µg yeast tRNA (Roche Diagnostics GmbH, Mannheim, Germany) as a carrier prior to ethanol precipitation. These isolated RNA samples were next separated on 1% agarose gels in formaldehyde-MOPS buffer and then transferred to Magnagraph nylon transfer membrane (OSMONICS Inc., Minnetonka, MN, USA). A hybridization solution containing 50% formamide, 5 x saline sodium citrate (SSC), 5 x Denhardt's, 0.4% sodium dodecyl sulphate (SDS), 7.5% dextran sulfate and 200 µg/ml herring sperm DNA was used for both pre-hybridization and hybridization. The blot filters were hybridized with 32P-CTP-labelled riboprobe (1 x 106 cpm/ml) for antisense ß-catenin (Oh et al., 2000) and stage-specific embryonic clone-D (SSEC-D) (Rothstein et al., 1992; Sakurai et al., submitted. ... USA) at 56°C overnight. The filters were washed in 2 x SSC/0.1% SDS at room temperature, 0.5 x SSC/0.1% SDS, and then 0.1 x SSC/0.1% SDS at 65°C prior to exposure to Kodak BioMax film (EASTMAN Kodak, NY, USA). The same filters were later re-probed with 18S ribosomal RNA cDNA probe as a control.
RT–PCR
We employed a method described by Iwamori et al. (2002) with some modifications. Briefly, total RNA was isolated from 15 fertilized oocytes or embryos by adding 25 µl of the cell lysis buffer from ‘Cells to cDNA kit II’ (Ambion, Inc., Austin, TX, USA) and EGFP RNA (at a final concentration of 1 pg/µl) as an internal control. The reverse-transcribed cDNAs were then prepared according to the manufacturer's instructions. RNA was synthesized in vitro using an RNA transcription kit (Stratagene) with a vector plasmid containing whole EGFP cDNA. For PCR of reverse-transcribed products derived from mouse retinoblastoma (Rb) mRNA, sense (5'-CAACCCCCCCAAACCACTGA-3') and antisense (5'-CCAGATGTAGGGGGTCAGGA-3') primers (Iwamori et al., 2002) were used. For detection of murine endogenous retrovirus-like (MuERL-V) mRNA, sense (5'-TTTCTCAAGGCCCACCAATAGT-3') and antisense (5'-GACACCTTTTTTAACTATGCGAGCT-3') primers (Kigami et al., 2003) were used. For detection of mouse eukaryotic initiation factor 1A (eIF-1A) mRNA, sense (5'-AAGAAGTCTGAAGGCCTATG-3') and antisense (5'-CAGAGAACTTGGAATGTAGC-3') primers (Davis et al., 1996) were used. For detection of EFGP mRNA, sense (5'-TCGCCACCATGGTGAGCAAG-3') and reverse (5'-ATGTTGCCGTCCTCCTTG-3') primers were used. The components of the reaction buffer were 10 mM Tris–Cl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) gelatin, 0.4 mM each of dATP, dTTP, dGTP and dCTP, 0.4 µM each of two PCR primers and 1.25 U of Taq DNA polymerase (Takara Shuzo Co. Ltd, Kyoto, Japan). Two microlitres of the reverse-transcribed cDNA containing solution was mixed with 23 µl of the reaction buffer. This mixture was estimated to yield PCR products from mRNAs of 0.5 oocytes or embryos. Forty (for Rb mRNA), 50 (for eIF-1A mRNA) or 30 cycles (for MuERL-V and EGFP mRNAs) of PCR were performed with cycle times of 30 s at 94°C, 1 min at 60°C and 1 min at 72°C, respectively. The RT–PCR products were electrophoresed in a 1% agarose gel/TAE buffer system, stained with ethidium bromide and photographed under UV illumination. In a preliminary test, numbers of PCR cycles required for detection of each mRNA were determined to obtain PCR products under exponential amplification phase through testing of several points of amplification cycles.
DNA microinjection
The pCAGGS-EGFP (hereafter termed pCX-EGFP) vector, contains cytomegalovirus enhancer, chicken ß-actin promoter, enhanced green fluorescent protein (EGFP) cDNA, and a portion of the second intron, third exon and 3' non-coding region of the rabbit ß-globin gene (Niwa et al., 1991; Okabe et al., 1997). pCX-EGFP was constructed with a strategy similar to that described by Okabe et al. (1997). Briefly, EGFP cDNAs were amplified by PCR of pEGFP-N1 (Clonetech Laboratories, Inc., Palo Alto, CA, USA) as a template with EGFP primers having an EcoRI site. Amplified EGFP cDNAs digested with EcoRI were cloned into the EcoRI site of the pCAGGS vector. For DNA microinjection, the 3.2 kb DNA fragment obtained after digestion of pCX-EGFP with SalI and BamHI was purified using a ‘Gel extraction kit’ (Qiagen K.K., Tokyo, Japan), dissolved in 10 mM Tris–Cl/0.2 mM EDTA (pH 7.4) (500 copies/pl) and stored at –80°C until use.
DNA microinjection was carried out using the method of Hogan et al. (1994) with some modifications. Briefly, fertilized ICR oocytes prepared by IVF as described above were divided into two groups. For one group, pronuclear microinjection of approximately 2 pl of the DNA fragment was performed. Some of these injected embryos were cultured in 30 µl of KSOM covered with paraffin oil at 37°C in 5% CO2 atmosphere up to morula/blastocyst stage (‘control’), while the other embryos were transferred to 30 µl of Hepes-KSOM, 4°C-treated for 12 h, and then returned to 30 µl of KSOM (covered with paraffin oil) at 37°C in 5% CO2 atmosphere (‘DNA injection and 4°C-treatment’). In the other group, fertilized oocytes were first 4°C-treated for 12 h and then cultured at 37°C for 1–2 h. They were next microinjected and then cultured (‘4°C-treatment and DNA injection’). Expression of EGFP fluorescence was observed at morula/blastocyst stage using an Olympus IX70 inverted fluorescence microscope with an appropriate filter set (U-MWIBA2; Olympus, Japan). Using the criteria of Kato et al. (1999), embryos exhibiting bright fluorescence throughout were judged to be embryos that would develop as transgenics (‘uniform-EGFP expression pattern’). Embryos exhibiting no fluorescence or spotted (patchy) fluorescence were designated non-transgenic embryos unable to be transgenic probably due to transient expression of exogenous DNA or lack of integration of the exogenous DNA (‘mosaic-EGFP expression pattern’).
Statistical analysis
Percentages are presented as means ± standard deviation of results of separate experiments. They were compared using one-way analysis of variance (ANOVA) following arcsin transformation of proportions: the Tukey–Kramer test was performed if ANOVA revealed significant differences among compared values. Values of P<0.05 were considered to indicate significant differences. These analyses were conducted using StatView ver. 5 software (SAS Inc., Cary, NC, USA).
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Results |
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Strain difference in tolerance of low temperature
Fertilized mouse ICR or B6 oocytes from F-6 to F-8 h were incubated in Hepes-KSOM for 12, 24, 36 or 48 h at 4°C and then incubated at 37°C. As a control, some oocytes were continuously cultivated at 37°C. In vitro rates of development of the low temperature-treated embryos to blastocysts were recorded. The results are summarized in Table II. Treatment at 4°C for 12–48 h (for C57BL/6J) and 24–48 h (for ICR) resulted in decrease in in vitro rates of blastocyst development. For ICR, however, within a treatment period of 12 h, no significant difference was noted in the in vitro rates of blastocyst development between the 4°C-treated embryos and the normal controls. For C57BL/6J, no significant difference was noted in in vitro rates of development of 2-cell embryo to morula stage between the 4°C-treated embryos and the normal controls. These results suggest a strain difference in tolerance to low temperature. The abnormal 4°C-treated embryos exhibited developmental arrest or fragmentation, which was also noted in the controls.
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Effect of 4°C-treatment on cell number in blastocysts developing from 4°C-treated fertilized oocytes
In the previous experiment, we observed that the rate of development of fertilized oocytes treated at 4°C for 48 h to blastocyst stage was approximately 30–50%. To determine effects of low temperature treatment on preimplantation development, cell numbers of trophectoderm and ICM in blastocysts derived from ICR and C57BL/6J fertilized oocytes 4°C-treated from 0 to 48 h were determined. The results are summarized in Table III. Deleterious effects of 4°C-treatment on the trophectoderm cells were remarkable. However, within a treatment period of 12 h, no significant difference was noted in number of either cell type between the 4°C-treated embryos and normal controls for either strain. These results indicate that blastocysts developing from fertilized oocytes treated at 4°C for 12 h would not be qualitatively different from those from the control oocytes. Treatment at 4°C for 24–48 h would be deleterious to fertilized oocytes from each strain, since cell number of trophectoderm and ICM was decreased (Table III).
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In vivo rates of development of 4°C-treated fertilized oocytes
To examine in vivo rates of development of low temperature-treated embryos, 2-cell ICR embryos, which had developed from fertilized oocytes treated at 4°C for 12–36 h, were transferred to the oviducts of pseudopregnant ICR females. The rates of implantation and numbers of living fetuses at gestational days 16–18 were then recorded. The results are summarized in Table IV. Treatment at 4°C for 12–36 h yielded fetuses at a frequency comparable to the control (54.2–63.3 versus 60.8%). Thus, blastocysts developing from fertilized oocytes treated at 4°C for 12–36 h can develop to normal fetuses, although in vitro rates of development tended to be reduced when low temperature treatment was prolonged (Table II).
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Effects of 4°C-treatment on the morphology and in vitro rates of development of fertilized mouse oocytes to 2-cell stage
Morphology of pronuclei in fertilized ICR oocytes after storage at 4°C for 0 (control), 6, 12 18, 24 and 36 h was first examined by light microscopy. These oocytes were photographed just before return to normal culture conditions at 37°C. As shown in Figure 1, morphology and relative position of the two pronuclei were indistinguishable among oocytes treated for 6 (Figure 1B), 12 (Figure 1C), 18 (Figure 1D), 24 (Figure 1E) and 36 h (Figure 1F) and control oocytes from F-7 to F-8 h (Figure 1A).
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Next, these ICR oocytes treated at 4°C for 6–18 h were then returned to normal culture conditions and observed every 6 h. The results are shown in Figure 2. The control oocytes exhibited centering of the male and female pronuclei or close association of female and male pronuclei after 6 h in culture, and almost all of them divided to 2-cell stage after 12 h in culture. Oocytes treated at 4°C exhibited a similar pattern of development when they were returned to normal culture conditions (Figure 2). Almost all oocytes treated at 4°C for 6, 12 or 18 h exhibited centering of the male and female pronuclei or close association of female and male pronuclei 6 h after return to 37°C. In addition, these embryos divided to 2-cell stage after 12 h in culture. These findings suggest the occurrence of developmental arrest during low temperature storage.
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Temporary cessation of DNA synthesis in fertilized mouse oocytes under storage at 4°C
To assess the low temperature-induced developmental arrest of fertilized oocytes at the molecular level, we first monitored DNA synthesis in 4°C-treated fertilized ICR oocytes by measuring incorporation of BrdU. Since DNA replication has been observed in fertilized oocytes from F-9 to F-12 h in our in vitro culture conditions (unpublished observation), control oocytes from F-8 to F-9 h were incubated at 37°C for 1 or 3 h with KSOM containing 20 µM BrdU. As expected, distinct immunostaining for the presence of BrdU was noted in each nucleus (Figure 3A and B for 3 h incubation). However, no staining was observed when fertilized oocytes were incubated at 4°C for 12 h in Hepes-KSOM containing 20 µM BrdU (Figure 3C and D). When fertilized oocytes were incubated at 4°C for 12 h in Hepes-KSOM and then at 37°C for 1 or 3 h in KSOM containing 20 µM BrdU, incorporation of BrdU into embryo nuclei was confirmed (Figure 3E and F for 3 h incubation). These findings suggest that DNA synthesis temporarily ceases under storage at 4°C, but recovers immediately after return of oocytes to normal conditions (37°C).
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Cytoplasmic polyadenylation of maternal RNA during low temperature storage of fertilized oocytes
Recently, it was reported that (1) de novo independent poly(A) tail elongation (or cytoplasmic polyadenylation) or shortening of certain maternal RNAs occurs after fertilization in mouse oocytes and (2) the mechanism of regulation of length of poly(A) tail of these maternal RNAs may be controlled at the translational level in a stage-specific manner in fertilized oocytes (Oh et al., 2000
). In this study, we examined the effects of low temperature treatment on the cytoplasmic polyadenylation of maternal RNAs (ß-catenin and SSEC-D), all of which exhibited temporary post-fertilization extension and shortening of the poly(A) stretch (Oh et al., 2000; Sakurai et al., 2005), by Northern blot hybridization. The results are shown in Figure 4. As expected, the poly(A) stretch pattern of ß-catenin and SSEC-D RNAs (Oh et al., 2000; Sakurai et al., submitted) in the 4°C-treated oocytes was almost identical to that in the control oocytes from F-7 to F-8 h. For maternal ß-catenin mRNAs, the poly(A) tail extended in control embryos from F-12 h and low temperature-treated embryos from F-18 h (cultured at 37°C for 12 h after treatment at 4°C for 6 h). After that, poly(A) tail began to decrease in length in control embryos from F-18 h and 4°C-treated embryos from F-24 h (cultured at 37°C for 18 h after treatment at 4°C for 6 h). Finally, maternal ß-catenin mRNAs were degraded in control embryos from F-24 h and 4°C-treated embryos from F-30 h (cultured at 37°C for 24 h after treatment at 4°C for 6 h). For maternal SSEC-D mRNAs, poly(A) tail extended in embryos at stages from F-12 to F-18 h (control) and 4°C-treated embryos from F-18 to F-24 h (cultured at 37°C for 12–18 h after treatment at 4°C for 6 h). However, the poly(A) tail began to decrease in length in control embryos from F-24 h and 4°C-treated embryos from F-30 h (cultured at 37°C for 12–24 h after treatment at 4°C for 6 h). These findings together suggest that cytoplasmic polyadenylation of maternal RNAs is tightly associated with oocyte metabolism.
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Molecular dynamics of Rb mRNA during low temperature storage of fertilized oocytes
Iwamori et al. (2002)
demonstrated that shortening of the G1 phase, which is known to be characteristic of early mouse embryos, is due to degradation of Rb protein following a decrease in the amount of maternal Rb RNA. We used RT–PCR to examine whether the low temperature-treated embryos exhibited sustained expression of Rb RNA. Control oocytes from F-6 h exhibited a decreasing trend in the amount of maternal Rb mRNA by a stage corresponding to F-26 h (Figure 5). Oocytes that had been treated at 4°C for 12 h exhibited a similar decrease in the amount of Rb mRNA by a stage corresponding to F-38 h (only 12 h delay in disappearance of Rb mRNA) (Figure 5). This finding indicates that expression of cell cycle-related molecules is also affected by cooling.
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Initiation of ZGA in low temperature-stored mouse fertilized oocytes
In fertilized mouse oocytes, transcription of the embryonic genome commences during the period from around 8–13 h post-fertilization to the early 2-cell stage, and is referred to as ‘ZGA’ (Schultz, 1993
; Matsumoto et al., 1994; Kigami et al., 2003). The murine endogenous retrovirus-like (MuERV-L) and eIF-1A (formerly termed eIF-4C) genes are now known to be ‘early ZGA genes’ (Davis et al., 1996; Kigami et al., 2003). De novo transcription of the MuERV-L gene can be rapidly activated from 8 to 10 h post-fertilization (Kigami et al., 2003). On the other hand, eIF-1A RNA exists in a fertilized oocyte, probably as maternal RNA (Davis et al., 1996). However, de novo transcription of the eIF-1A gene commences by 2-cell stage. Consequently, the amount of eIF-1A RNA in 2-cell embryos increases to about 3–4 times than in fertilized oocytes (Davis et al., 1996). To monitor initiation of ZGA in low temperature-stored mouse fertilized oocytes, expression of MuERV-L and eIF-1A mRNA was examined by RT–PCR using embryos corresponding to F-18–F-58 h (which correspond to F-6–F-46 h for intact embryos) after storage at 4°C for 12 h. The results are shown in Figure 5. Increasing tendency of the amount of MuERV-L and eIF-1A mRNAs was observed in the control (from F-15 to F-26 h) and low temperature-treated embryos (from F-27 to F-38 h; corresponding to control embryos from F-15 to F-26 h), which were at nearly the same developmental stages. This finding indicates that ZGA is also arrested during storage at 4°C.
Gene transfer to 4°C-treated oocytes in an attempt to increase efficiency of transgenic mouse production
We performed pronuclear microinjection of DNA into the low temperature-treated fertilized oocytes expecting an increase in efficiency of introduction of exogenous DNA into the mouse genome. Fertilized oocytes were microinjected with EGFP-expressing transgenes, and these embryos were then divided into two groups: one was continuously cultured at 37°C up to the blastocyst stage (control), and the other was subjected to 4°C for 12 h and then returned to 37°C (‘DNA injection and 4°C-treatment’). On the other hand, fertilized oocytes were first treated at 4°C for 12 h and then microinjected (‘4°C-treatment and DNA injection’). Inspection for EGFP fluorescence was performed at the morula/blastocyst stage. The results are summarized in Table V. The rate of survival of the DNA-injected embryos was approximately 90% in all groups. In a control experiment, 74.6% of the injected embryos developed to morula/blastocyst stage. The percentages of embryos exhibiting uniform-EGFP expression pattern and those exhibiting mosaic-EGFP expression pattern were 21.7 and 24.9%, respectively, and was basically in agreement with the results obtained by Kato et al. (1999). No significant difference was noted in the incidence of either uniform- or mosaic-EGFP expression pattern among the three groups, that is, the control, ‘DNA injection and 4°C-treatment’ and ‘4°C-treatment and DNA injection groups’. These findings suggest that low temperature treatment of fertilized oocytes is not tightly associated with enhanced integration of exogenous transgenes into host chromosomes.
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Discussion |
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There have been several reports on the effects of temporary storage at low temperature on the development of fertilized mouse oocytes. For example, Nakamura and Tsunoda (1986)
cultured fertilized oocytes derived from a hybrid between CD-1 and CBA mice in PB1 containing 20% FCS for 4 days at 4°C, and then returned them to normal conditions (37°C). Similarly, Herr and Wright (1988) cultured fertilized oocytes derived from a hybrid between Swiss Webster and C57BL/6J mice in modified Whitten's medium containing 10 mM Hepes for 1–3 days at 4°C, and then returned them to normal conditions (37°C). In both cases, the rates of development of the low temperature-treated embryos to blastocyst stage were less than 2%, when embryos were stored for 1 day at 4°C. Herr and Wright (1988) also found that no embryos stored for 2 days at 4°C survived after return to normal culture conditions. In contrast, we have shown here that the rate of development of 4°C-treated embryos to blastocyst stage was as high (86.7% for ICR) as that of control embryos (without treatment at 4°C), when embryos were first stored in 20 mM Hepes-KSOM (pH 7.4) at 4°C for 12 h and then cultured in KSOM at 37°C (Table II). Furthermore, even embryos treated at 4°C for 48 h developed to blastocyst stage at somewhat better rates (33.3% for ICR and 50.8% for C57BL/6J) than those reported previously. When in vitro development of 4°C-treated embryos to blastocyst stage was evaluated, ICR oocytes exhibited more tolerance to low temperature than C57BL/6J oocytes (Table II). On the other hand, when in vitro development of 4°C-treated embryos to morula stage was evaluated, C57BL/6J oocytes exhibited more tolerance to low temperature than ICR oocytes (Table II). These observations suggest that sensitivity to cold shock differs among mouse strains. It remains unclear why the findings presented by Nakamura and Tsunoda (1986) and Herr and Wright (1988) differ greatly from ours. Interestingly, Nakamura and Tsunoda (1986, 1992) suggested that as storage at 4°C is prolonged, deleterious effects on chromosomal function and cytoplasm of fertilized mouse oocytes increase. However, the molecular mechanism underlying this phenomenon is still unclear. Examination of conditions (e.g. selection of mouse strain and employment of improved media) suitable for low temperature preservation of mouse embryos will probably explain this discrepancy in findings. We believe that with performance of these examinations, the period of low temperature (above 0°C) preservation of mouse embryos will be further prolonged. The in vivo rate of development of ICR embryos stored at 4°C for 12–36 h was almost the same as that of control embryos (0 h) (Table IV), although in vitro rates of blastocyst development tend to decrease when low temperature treatment was prolonged (Table II). This suggests that normal blastocysts surviving after a short period of low temperature treatment can develop normally in vivo. In support of this hypothesis, we observed normal development of ICM even after storage at 4°C for 12–24 h (Table III). In this context, 4°C-treatment for several hours may be useful for selection of embryos exhibiting tolerance to cooling shock.
Our findings suggest the possibility that transient low temperature treatment of fertilized oocytes will be useful for the analysis of molecular mechanisms underlying cold shock or cryo-injury in mammalian embryos. We found that fertilized mouse oocytes stored at 4°C exhibited temporary developmental arrest at several steps including movement of pronuclei (Figures 1 and 2), cleavage of fertilized oocytes (Figure 2), DNA replication (Figure 3), cytoplasmic polyadenylation of maternal RNAs (Figure 4), and initiation of the cell cycle and ZGA (Figure 5). We plan to investigate the state of energy metabolism (Gardner and Leese, 1988) and critical point of cryo-injury in fertilized oocytes during storage at 4°C.
Recently, we found that even at ZGA stage, several maternal RNAs were regulated by cytoplasmic polyadenylation (Sakurai et al., 2005). This finding suggested that ZGA may be associated with behaviour of maternal RNAs exhibiting post-fertilization cytoplasmic polyadenylation. Both of these events commence earlier in mouse preimplantation development (5–18 h post-fertilization for cytoplasmic polyadenylation and 13–24 h post-fertilization for ZGA) (Schultz, 1993; Davis et al., 1996; Oh et al., 2000; Iwamori et al., 2002; Sakurai et al., unpublished data) and rapidly transit to the next phase. In this study, low temperature storage of embryos synchronized embryos at defined stages, which was in turn useful for analysis of the behaviour of each embryo over an extended period (Figures 1–5). Thus, 4°C-treatment for several hours may also be useful for assessing molecular mechanisms linking ZGA and cytoplasmic polyadenylation.
Low temperature treatment of fertilized oocytes also appears to be applicable to the field of early embryo manipulation. In this study, we found that the outline of the pronucleus was clearly maintained even after storage at low temperature (Figures 1 and 2), indicating synchronization and temporary maintenance of pronucleate stage under this condition. Therefore, one such application might yield efficient production of transgenic mice. To test this possibility, we introduced exogenous DNA into pronuclei of fertilized oocytes stored at 4°C for 12 h using a standard DNA microinjection procedure, but no improvement of efficiency of transgenic mouse embryo creation was achieved (Table V). However, this finding suggests that the machinery related to transgene integration remains operative even in 4°C-treated embryos. This will allow more convenient performance of transgenic experiments. For example, microinjection generally has to be performed late afternoon of day 0 (probably from 15:00 to 19:00 h) when the outline of a pronucleus becomes clear. If the time of collection of IVF-mediated fertilized oocytes is assumed to be 18:00–19:00 h on day 0, it would be desirable to perform DNA injection the following morning (corresponding to day 1) using oocytes stored at 4°C overnight (about 15 h). This would also be convenient for transfer of DNA-injected oocytes to pseudopregnant recipient females, since transfer would then be finished late in the afternoon (probably by 17:00 h).
Another advantage of low temperature preservation of fertilized oocytes is its usefulness in the animal bioresource field. That mouse fertilized oocytes can be stored at 4°C for even 48 h with maintenance of potentiality to develop to normal blastocysts (Table II) is advantageous for convenient transfer of oocytes between laboratories as an alternative to transfer of an animal itself.
In conclusion, we have demonstrated here that fertilized mouse oocytes could be maintained for at least 12 h in storage at 4°C without decreasing their developmental ability. Notably, embryos 4°C-treated for 48 h developed to blastocysts at rates better than those reported previously. This method of low temperature storage of embryos will be useful for analysis of the mechanism of cryo-injury of fertilized oocytes and in the field of reproductive engineering. It will also be useful for temporary storage of preimplantation embryos prior to experiments, and for simple and convenient delivery of embryos from one laboratory to another.
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Acknowledgements |
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We would like to thank Dr Jun-ichi Miyazaki (Osaka University) for the gift of pCAGGS vector. This research was supported by a grant from the Ministry of Education, Science and Culture, Japan (T.S and M.K).
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Submitted on December 21, 2004; accepted on March 1, 2005.
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