1 Department of Animal Science, Tokyo University of Agriculture, Tokyo, Japan and 2 Department of Animal Breeding and Reproduction, Utsunomiya University, Tochigi, Japan
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Abstract |
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Key words: androgenetic embryos/development/imprinting/mouse
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Introduction |
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To date, various procedures have been reported for producing uniparental mouse embryos. It has been shown (Edwards and Sirlin, 1959) that the treatment of mouse oocytes with colchicine at fertilization induced abnormal segregation of chromosomes; consequently, it led to gynogenesis and androgenesis, though the degree of heteroploidy was imperfectly controlled. On the other hand, the pronuclear exchange procedure using micromanipulation techniques has been the most widely used for many analyses (Barton et al., 1984
; McGrath and Solter, 1984
; Surani et al., 1986
; Kaufman et al., 1989
; Latham and Solter, 1991
; Mann and Stewart, 1991
; Hagemann and First, 1992
). Using these methods, the ploidy of uniparental embryos can be controlled perfectly, but there are also some disadvantages; it is not always clear which pronucleus is female or male (McGrath and Solter, 1984
; Kaufman et al., 1989
), and newly synthesized products by maternal and paternal genomes may be contained in egg cytoplasm and/or pronuclear components. These possibilities could affect the nature of uniparental embryo development. Actually, serial nuclear transfer into fertilized cytoplasm greatly improved developmental ability of cloned embryos (Kwon and Kono, 1996
; Kwon et al., 1997
). Previously we reported that diploid androgenetic embryos can also be produced by in-vitro fertilization of mouse enucleated oocytes and that these embryos develop to the blastocyst stage (Kono et al., 1993
). However, their post-implantation development has not been examined. On the other hand, several reports on in-vitro development of androgenetic embryos so far imply that culture media containing low sodium chloride, such as Whittin's medium (Whittin and Bigger, 1968
) and CZB medium (Chatot et al., 1989
), more strongly support in-vitro development than do media containing high sodium chloride, such as M16 medium (Whittingham, 1971
) (McGrath and Solter, 1984
; Surani et al., 1986
; Latham and Solter, 1991
; Mann and Stewart, 1991
; Hagemann and First, 1992
). However, precise experiments on the post-implantation development of androgenetic embryos are lacking.
In this study, to establish an efficient and reliable procedure for producing mouse androgenetic embryos, we conducted experiments to evaluate the developmental ability of androgenetic mouse embryos in vitro and in vivo, which were produced by in-vitro fertilization of enucleated oocytes and cultured using M16 and CZB media characterized by high and low sodium chloride, respectively. Further, to manifest the precise feature of mouse androgenetic development, we analysed the phenotype and the karyotype of androgenetic embryos after implantation.
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Materials and methods |
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In-vitro fertilization
Spermatozoa were collected from the cauda epididymis of mature B6CBF1 male mice and preincubated in T6 medium (Quinn et al., 1982) for 2 h in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 37°C in which the sperm concentration was adjusted to 1x106/ml (Kono et al., 1993
) to induce bispermic fertilization at high incidence.
In-vitro culture and embryo transfer
Diploid androgenetic embryos, which contained two male pronuclei due to bispermic fertilization, were cultured for 4 days with M16 and CZB media supplemented with 4 mg/ml bovine serum albumin, which were respectively modified with 0.1 mmol/l EDTA and 5.56 mmol/l glucose (Whittingham, 1971; Chatot et al., 1989
). Control biparental embryos produced by in-vitro fertilization of nonmanipulated oocytes were also cultured in vitro under the same conditions. Resultant blastocysts were transferred to the uterine horns of female mice on day 3 of pseudopregnancy. Their developmental ability was assessed at 92 h after insemination and on day 9.5 of gestation, and the assessment compared M16 and CZB media by
2 analysis.
Cell counting
For assessment of the quality of the androgenetic embryos, the total cell number at cavitation (blastocoel formation) and 92 h after culture (the time when the embryos were transferred) was counted after staining with 1 µg/ml Hoechst 33342. Cavitation was observed at 2 h intervals. Comparisons between the cell number of androgenones in M16 and CZB media were made using the t-test. A P-value of < 0.05 was considered to be significant.
Embryo sexing
Individual day 9.5 androgenetic embryos were sexed by polymerase chain reaction (PCR) using the X- and Y-specific sequences, respectively. DNA from the embryonic tissue was obtained by extraction with a buffer containing 1 mg/ml protease K. Amplification of sequences from Y chromosome-specific gene, Zfy, was carried out using a primer set, forward: 5'-GAC TAG ACA TGT CTT AAC ATC TGT CC-3' and reverse: 5'-CCT ATT GCA TGG ACA GCA GCT TAT G-3' (Kay et al., 1994), and the X chromosome specific-sequences in the Xist gene were also amplified using a primer set, forward: 5'-AGG ATA ATC CTT CAT TAT CGC GC-3' and reverse: 5'-AAA CGA GCA AAC ATG GCT GGA G-3' (Zuccotti and Monk, 1995
). PCR was carried out in a 50 µl reaction containing 10 mmol/l TrisHCl, 50 mmol/l KCl, 1.5 mmol/l Mg2+, 0.1 µmol primers, and 1.25 U of Taq polymerase (Takara, Tokyo, Japan). When amplifying X chromosome-specific sequences, the PCR conditions consisted of a 5 min denaturation at 95°C, followed by 30 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min, followed by a 7 min extension at 72°C. When amplifying Y chromosome-specific sequences, the PCR conditions consisted of a 5 min denaturation at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min, followed by a 10 min extension at 72°C.
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Results |
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The ability of diploid androgenetic embryos to develop to blastocysts in vitro was tested using M16 and CZB media, which are respectively characterized by high and low sodium chloride concentration. In the first three cleavages, M16 medium tended to support highly androgenetic development (Table I). However, during the process of compaction, the viability of the androgenetic embryos was greatly impaired when cultured with M16 medium (P < 0.01). Consequently, CZB medium increased the frequency of blastulation of the androgenetic embryos compared with the rate of the embryos cultured with M16 medium (56.6% versus 45.0%, P < 0.001). Such differences in the media were not found in control biparental embryos (96.4% versus 94.7%). Blastocoel formation occurred significantly earlier in embryos cultured with CZB medium, compared with the embryos cultured with M16 medium, in both androgenetic (82.4 h versus 84.5 h, P < 0.05) and control (78.2 h versus 84.0 h, P < 0.001) embryos, although the time-lag until cavitation in androgenetic embryos was not as long as that observed in control embryos (Table II
). The total number of cells at cavitation was quite similar between the androgenetic embryos in the two culture media (Table II
), independent of the time of cavitation (data not shown). More importantly, no apparent differences in the cell number between androgenetic and control biparental embryos were observed by this time. However, following cavitation, there was a massive difference in the cell number between androgenetic and control biparental embryos. The cell proliferation rate of androgenetic embryos after blastocoel formation tended to be accelerated in M16 medium, but such a tendency was more exclusive in control biparental embryos. These results showed that androgenetic embryos had a greatly reduced viability after cavitation.
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Discussion |
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Androgenetic mouse embryos can develop to the blastocyst stage, but a conspicuous developmental stagnation occurs after the 8-cell stage (Table I). A comparison of the development of androgenetic embryos cultured in two different media showed that the proportion of embryos that developed to the 8-cell stage was slightly higher when they were cultured in M16 medium, but, adversely, CZB medium significantly increased the survival of the androgenones after compaction. This difference was not found in control biparental embryos. An improved development after compaction in CZB medium has been observed in severely manipulated embryos (Kwon and Kono, 1996
; Kwon et al., 1997
), and it has been observed that cavitation occurs more rapidly in embryos cultured in CZB medium (Table II
). These results could be attributed to the difference in the concentration of sodium chloride between the media, which promotes greater egg metabolism at low concentration (Ho et al., 1994
).
The results from post-implantation development showed that the proportion of androgenetic blastocysts to develop to somite stage fetuses is greatly affected by the culture medium, M16 or CZB (Table III), though there was no significant difference in the quality of embryos at the blastocyst stage (Table II
). To obtain accurate data, a significant number of embryos developed in the two media were transferred to 10 and 15 recipient mice respectively. The implantation rate was considerably worse in both embryos compared with the rate for control biparental embryos (Table III
) and parthenogenetic embryos (Yoshimizu et al., 1998
). The embryos cultured with M16 medium were much superior to those cultured with CZB medium in the development to somite stage fetuses (28% versus 11%). The reason for this distinction is not clear, but the lower rate of cell division after cavitation in CZB medium may affect the proliferation and differentiation of inner cell mass cells only in androgenones, which may lead to a further loss of embryos, at least in part. A computation of the present results demonstrates that the efficiency for producing live androgenetic fetuses totalled 7.0% in M16 medium and 3.5% in CZB medium. This shows that an assessment of the developmental ability of embryos up to the blastocyst stage is not always indicative of the ability to develop to more advanced stages.
On the other hand, there was no difference in the phenotype of the fetuses at day 9.5 of gestation. Androgenones were pre-rotated and head-fold stage embryos with relatively abundant trophoblastic cells (Figure 1A and B). It is well known that androgenetic embryos have well-developed extra-embryonic tissues, but an embryo proper cannot develop beyond the early somite stage (Barton et al., 1984
). Intriguingly, in this study, some androgenones were observed to have 20 or more somites, even though they had not rotated and had still-open neural tube, suggesting that such imbalanced development is a result of the onset of the imprinting effect. In addition, androgenetic embryos can promote the proliferation of trophoblastic and extra-embryonic mesodermal cells but not differentiate chorioallantoic placenta (Figure 1D
). In humans, androgenetic development has been known to cause CHM, and 10 to 20% of CHM lead to choriocarcinoma (Wake et al., 1984
). Although this is not the case in mice, we and others have shown grossly swollen chorion and abnormal development of placenta. This similarity between humans and mice may also be caused by some imprinted genes. Androgenetic embryos would repress maternally expressed genes. In both humans and mice, several maternally expressed genes are thought to act as tumour suppressor genes (e.g. p57KIP2, WT1 and Ipl); therefore, silencing maternal transcripts may give rise to malignant trophoblasts.
There are several studies that have reported androgenetic development in vitro (McGrath and Solter, 1984; Surani et al., 1986
; Latham and Solter, 1991
; Mann and Stewart, 1991
; Hagemann and First, 1992
; Latham and Sapienza, 1998
). The results implied that developmental ability in vitro was altered by mouse strain-dependent modification. Here, we showed that the development of androgenetic embryos both in vitro and in vivo was also affected by the sodium chloride concentration of the culture media. On the other hand, we can find only two reports in which successful post-implantation development of androgenones is described (Barton et al., 1984
; Kaufman et al., 1989
). In these reports the androgenetic embryos produced by pronuclear exchanges were transferred into oviducts at the 1- to 2-cell stage. In the present study, by contrast, embryos were cultured in vitro up to the blastocyst stage and then transferred to the uterine horns of recipients, showing that culturing androgenetic embryos causes no detrimental effect on post-implantation development, especially in M16 medium. The proportion of somite stage androgenetic embryos yielded was higher than those of previous reports. These results indicate that M16 medium is superior for the culturing of androgenetic embryos. There was no great difference in the developmental ability of androgenetic embryos when maternal genomic components were removed from the oocyte at the MII stage, although it is proposed that genomic interaction occurs at the pronuclear stage and it affects embryonic development and gene expression (Latham and Solter, 1991
; Reik et al., 1993
; Moore and Reik, 1996
; Latham and Sapienza, 1998
).
Sexing of the androgenetic embryos at 9.5 days of gestation showed intriguing evidence that the embryos containing XX chromosomes can develop to the somite stage. Dosage compensation for the X-linked gene is crucial in mammalian development. Mammalian XX female embryos require that only one of the two X chromosomes is inactivated to confine a single dosage of X-linked gene products. In the mouse trophoblastic lineage, the paternally derived X chromosome is preferentially inactivated, while in the epiblastic lineage, X chromosome inactivation (XCI) is induced at random, governed by counting mechanisms in biparental embryos. In the androgenetic embryos, theoretically, the expected XX:XY:YY ratio is 1:2:1 when they are constructed. It is thought that YY androgenones lack an ability to develop into blastocysts (Morris, 1968). Consequently, XX and XY androgenetic embryos are allowed to develop to the blastocyst stage (Kay et al., 1994
), but the ability of post-implantation development was thought to be quite different due to dosage abnormalities of X-linked gene products (Kay et al., 1994
; Latham, 1996
). It was reported that only XY mice androgenetic embryos are permitted to survive up to the egg cylinder stage, but XX embryos are not (Kaufman et al., 1989
). Therefore, the two models have been proposed: XX androgenones die before blastocyst stage because of complete inactivation of both X chromosomes (Latham, 1996
) and activation of both X chromosomes (Kay et al., 1994
). However, here we analysed 112 androgenetic embryos at day 9.5 of gestation and showed that both XX and XY androgenones could develop into somite stage embryos in the Mendelian row. This suggests that mouse XX androgenones do not have a problem with XCI that affects ongoing development. The reason for the difference between the previous report and our findings is not clear. The Xist gene, which is known as an imprinted gene in mice, is expressed exclusively from the paternal allele in the extra-embryonic tissues and is essential for XCI. Therefore, further studies on the Xist expression in mouse androgenetic embryos may shed light on the mechanisms of XCI and its imprinting.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Department of Animal Science, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
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References |
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Submitted on May 13, 1999; accepted on January 14, 2000.