JEZ 788 250 Y.THE TSUNODA JOURNAL ANDOF Y. KATO EXPERIMENTAL ZOOLOGY 278:250–254 (1997) Full-Term Development After Transfer of Nuclei From 4-Cell and Compacted Morula Stage Embryos to Enucleated Oocytes in the Mouse Y. TSUNODA* AND Y. KATO Laboratory of Animal Reproduction. College of Agriculture and Research Institute for Animal Developmental Biotechnology. Kinki University, Nara, 631, Japan ABSTRACT The developmental ability of enucleated mouse oocytes reconstituted between the nucleus from 4-cell mouse embryos at different cell stages and recipient cytoplasms at different conditions, and the developmental ability of oocytes receiving nuclei from compacted morulae were examined. The highest development was observed with the nucleus at the G1 stage fused with cytoplasm at the M stage. Although the enucleated oocytes receiving a nucleus from 4-cell embryos did not develop after transfer to a recipient female, young were obtained after renuclear transfer to enucleated fertilized eggs. Live mice were also obtained from the nucleus of compacted morula. J. Exp. Zool. 278:250–254, 1997. © 1997 Wiley-Liss, Inc. Since the first report of a nuclear transfer sheep by Willadsen (’86), nuclear transfer has been done in farm animals by fusing enucleated oocytes at the second metaphase with a blastomere of donor preimplantation embryos. The developmental ability of nuclear transferred eggs is influenced by the cell cycle of the donor nucleus and the donation of the recipient cytoplasm. The nuclei fused with recently ovulated or mature oocytes show premature chromosome condensation (PCC) with dispersal of individual duplicate chromosomes, reform pronucleus-like nucleus after parthenogenetic activation, duplicate chromosomal DNA and division to the 2-cell stage (Tsunoda et al, ’89; Collas et al., ’92; Campbell et al., ’93). Only the G 1 nuclei are suitable for maintaining correct policy after nuclear transfer (Campbell et al., ’93). Nuclei fused with preactivated oocytes did not show PCC, but in the nuclei at the G1 stage, not G2 stage, DNA replicated, and in the nuclei at the S stage DNA duplication continued (Campbell et al., ’93; Barnes et al., ’93; Takano et al., ’96). In this case, a nucleus at any cell stage can be used as the donor nucleus. The developmental ability of nuclear transfer to activated oocytes was higher than that after transfer to nonactivated oocytes (Kono et al., ’94). After serial nuclear transfer in cattle, more than 40 blastocysts were produced from one cattle embryo (Takano et al., unpublished observation) and identical calves were produced (Bondioli et al., ’90). In contrast to the success in cattle, the success© 1997 WILEY-LISS, INC. ful production of offspring by nuclear transfer is limited in the mouse. Cheong et al. (’93) obtained young after transfer of enucleated oocytes fused with nuclei from 2-, 4-, and 8-cell stage embryos. They found that the developmental ability was higher in enucleated oocytes receiving nuclei at an early stage of the cell cycle than at the middle or late stage. However, the effect of the condition of recipient cytoplasm in connection with the stage of cell cycle of donor nucleus on the development of reconstituted oocytes has not been reported. Although a nucleus of a blastomere of morula (Rossant and Vijh, ’80) and the inner cell mass cell of blastocysts (Gardner, ’68; Kato and Tsunoda, ’95) can produce chimeric mice after aggregation with precompacted embryos or after injection to blastocysts, live mice have not been obtained after nuclear transfer beyond the 8-cell stage. In the present study we examined: (1) the developmental ability of oocytes reconstituted between the nucleus from 4-cell mouse embryos at different cell stages and recipient cytoplasms at different conditions, and (2) the developmental ability of oocytes receiving nuclei from compacted morulae. *Correspondence to: Dr. Y. Tsunoda, Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara, 631, Japan. Received 23 October 1996; Revision accepted 8 January 1997 NUCLEAR TRANSFER OF MOUSE EMBRYOS MATERIALS AND METHODS Donor embryos Two-cell stage embryos were obtained from superovulated F 1(C57BL/6 × CBA) female mice mated with F1 males 45 hours after injection of human chorionic gonadotrophin (hCG). The embryos were stored at 4°C for 7 to 8 hours in M2 (Fulton and Whittingham, ’78) supplemented with 10% fetal calf serum (FCS). Then they were cultured in M16 medium (Whittingham, ’71) supplemented with nocodazole (3 µg/ml) for 14 to 17 hours at 37°C in 5% CO2 and 95% air atmosphere (Kato and Tsunoda, ’92). The treated embryos were washed several times with M2 and M16 and cultured in M16 for 1.5 hours. Some of them were incubated with 5 µg/ml aphidicolin for 1 hour to suppress the DNA synthesis (Spindle et al., ’85). Most 2-cell embryos divided into the 4-cell stage during the treatment. The nuclei of such embryos should be considered at the G1 stage. The other 2-cell embryos were culture for 1.5 to 2.0 hours (0.5 to 1.0 hours after cleavage, G1 to S stage), 3.5 to 4.0 hours (early S stage), 6.5 hours (middle S stage) and 12.5 hours (late S to G2 stage) in M16 before nuclear transfer. The nucleus with a small volume of cytoplasm (karyoplast) from 4-cell embryos was used for nuclear transfer. Morula stage embryos were obtained after a 24hour culture in M16 of 8-cell embryos, recovered from F1 females mated with F 1 males, 65 hours after hCG injection. They were treated with M16 supplemented with nocodazole for 6 hours and their zonae pellucidae were removed with 0.5% pronase. Zona-free embryos were pipetted into single blastomeres in trypsin-EDTA solution. After further culture for 30 minutes, divided blastomeres were immediately used for nuclear transfer. To inhibit DNA synthesis in a divided blastomere, aphidicolin was added in some cases to the media for all procedures from pronase treatment to single blastomere culture. Recipient oocytes Recipient oocytes were collected from superovulated F1 females 16 to 19 hours after the injection of hCG, and denuded by the treatment with hyaluronidase (300µ/ml) in M2 and washed three times with M2. Observation of oocytes under an inverted microscope with Nomarski optics revealed a small swelling around the metaphase chromosomes. The zonae pellucidae around the swelling was cut by using Narishige micromanipulators (Narishige, Japan) and the chromosomes 251 were removed as previously reported (Tsunoda et al., ’86; Tsunoda and Kato, ’95). The enucleated oocytes were immediately used for recipient oocytes, or after parthenogenetic activation. For activation, enucleated oocytes were transferred to Zimmerman’s cell fusion medium (Wolfe and Kraemer, ’92) in a chamber comprised of two wire electrodes mounted 1 mm apart on a glass slide (BTX, U.S.A.). Two DC pulses of 50 V/mm for 50 µseconds at 2-second intervals were given three times at 20-minute intervals. After electrical stimulation, the oocytes were cultured in M16 for 2.5 to 3.5, 5.5 to 6.5 or 10 to 11 hours before nuclear transfer. In some experiments, oocytes with chromosomes were activated, cultured in M16 containing cytochalasin B (CB, 5 µg/ml) for 6.5 hours and used as the recipient cytoplasm at the pronuclear stage. The fertilized eggs in vitro (Tsunoda and Chang, ’75) were also used at a pronuclear stage 6.5 to 10 hours after insemination. The pronuclei of activated or fertilized eggs were removed before nuclear transfer. Nuclear transfer A single karyoplast from a 4-cell embryo or blastomere from compact morulae was fused with an enucleated oocyte by using inactivated Sendai virus (HVJ; Tsunoda et al., ’86). The unfused karyoplast or blastomere pairs were further induced to fuse by electrical stimulation with DC pulses of 50V/mm for 50 µseconds. All oocytes used before activation were electrically stimulated three times at the same conditions for preactivation. When the nucleus at the G1 stage of the cell cycle was used, aphidicolin was added to all media used until fusion. The total aphidicolin treatment period was less than 3 hours. In a preliminary experiment, we observed that this treatment had no effect on the developmental ability of 2-cell mouse embryos in vitro. Some eggs that developed to the morula or blastocyst stage were transferred to the oviducts of day 1 pseudopregnant CD-1 strain females and killed on day 18.5 to examine the fetuses. Renuclear transfer into enucleated zygotes or 2-cell embryos When the reconstituted oocytes developed to pronuclear stage, the pronucleus was fused again with in vitro fertilized (F1 females and males) enucleated cytoplasm by HVJ. Some reconstituted oocytes forming a pronucleus were cultured to 2cell stage. In half of the oocytes, one nucleus of 252 Y. TSUNODA AND Y. KATO each reconstituted eggs at 2-cell stage was fused with the enucleated blastomere of an in vitro fertilized 2-cell stage embryo by HJV and the enucleated remaining blastomere of the 2-cell embryo was left without a nucleus. In the remaining oocytes, both nuclei of the reconstituted eggs at 2-cell stage were fused with each enucleated blastomere of fertilized 2-cell embryos. The 2-cell embryos with fused blastomeres were discarded. The renuclear transferred eggs were cultured for 3 or 4 days in vitro and some eggs developed to morula or blastocyst stage were transferred to the oviducts of day 1 pregnant CD-1 strain females mated with the same strain males, which were subsequently allowed to go to term. stage was used as a donor. Most of the oocytes that had been activated 10 to 11 hours before nuclear transfer became fragmented shortly after nuclear transfer. Forty-three morulae and blastocysts obtained in various groups were transferred to eight recipient mice but none of them developed to young. To inhibit the fragmentation after nuclear transfer, a parthenogenetic or fertilized egg at the pronuclear stage was fused with a nucleus at various cell stages shortly after removal of the pronucleus. However, none of them developed to morula or blastocyst stages (0/119 for activated and 0/54 for fertilized eggs, data not shown in Table 1). RESULTS Renuclear transfer of nucleus from 4-cell embryos The effect of the combination of cell cycle stage of donor nucleus and cytoplasm on the development of the enucleated oocyte receiving a nucleus from a 4-cell embryo was examined. As shown in Table 1, the developmental ability of the nuclear transferred embryo was influenced by the combination of the cell cycle stage of the donor nucleus and condition of cytoplasm of enucleated oocyte. The proportion of the nonactivated oocytes (0 hour) receiving the G 1 nucleus (0 hour) developed to morula and blastocyst stage was significantly (P<0.05) by χ2 test) higher than those obtained in the other groups (23% vs. 0% to 10%). In the oocytes nonactivated or activated 2.5 to 3.5 hours before nuclear transfer, the developmental ability after nuclear transfer was low regardless of the cell cycle of the donor nucleus, expect the G 1 stage. In the oocytes activated 5.5 to 6.5 and 10 to 11 hours before nuclear transfer, 2 to 10% developed to morula and blastocyst stages, respectively, except when the nucleus at the G2/S As shown in Table 2, 25% to 62% and 14% to 43% of renuclear transferred embryos developed to morula and blastocyst stages, respectively. After transfer to recipient, 8% to 36% of the embryos developed to young in all groups except one. Live young were obtained even from the donor embryos without aphidicolin treatment to suppress DNA synthesis after cleavage to 4-cell stage. Nuclear transfer of compacted morulae The proportion of oocytes developed to 2-cell stage after nuclear transfer without aphidicolin treatment (74/199, 37%) was significantly (P<0.001) lower than that obtained with aphidicolin treatment (92/162, 57%). As shown in Table 3, 18% to 26% and 5% to 18% developed to morula and blastocyst stages, respectively. After transfer to recipients, two normal live young (one male and one female) were obtained. TABLE 1. Developmental ability of enucleated oocyte receiving a nucleus from a 4-cell embryo in vitro Cytoplasm (h after activation) 0 2.5~3.5 5.5~6.5 10~11 1 Nucleus (h after division)1 0 71/88 20/88 37/40 0/40 57/68 7/68 9/66 1/66 1.5~2.0 (81) (23) (93) (0) (84) (10) (14) (2) 12/32 0/32 14/54 2/54 52/79 7/79 6/49 3/49 (38) (0) (26) (4) (66) (9) (12) (6) 3.5~4.0 4/29 0/29 2/44 0/44 23/59 1/59 12/49 4/49 (14) (0) (5) (0) (39) (2) (24) (8) 6.5 12.5 4/42 (10) 0/42 (0) 0/41 (0) 0/41 (0) 20/61 (33) 4/61 (7) 9/56 (16) 4/56 (7) 0/21 (0) 0/21 (0) 1/24 (4) 0/24 (0) 0/32 (0) 0/32 (0) 0/27 (0) 0/27 (0) Upper figures indicate no. of 2-cell embryos/fused (%) and lower figures indicate no. of morulae and blastocytes/fused (%) NUCLEAR TRANSFER OF MOUSE EMBRYOS 253 TABLE 2. Developmental ability of renuclear transferred embryo receiving a nucleus from a 4-cell embryo in vivo With or without aphidicolin Stage of renuclear transfer No. of eggs fused – 1-cell 2-cell half1 2-cell both2 1-cell 2-cell half1 2-cell both2 20 33 21 57 28 33 + 1 2 Developed to (%) morula 10 13 13 25 7 18 blastocyst (50) (39) (62) (44) (25) (55) 3 8 9 14 4 11 No. of recipients (15) (24) (43) (25) (14) (33) 6 5 7 17 2 5 No. of live young/ transferred (%) 1/6 1/13 4/11 4/25 0/6 2/15 (17) (8) (36) (16) (0) (13) A nucleus from a reconstituted 2-cell egg was fused with a blastomere of enucleated 2-cell embryos. Two nuclei from reconstituted 2-cell eggs were fused with both blastomeres of enucleated 2-cell embryos. DISCUSSION to the nuclear membrane since parthenogenetic or fertilized zygotes, whose pronuclei were removed immediately before nuclear transfer, fused with a nucleus at the S stage, did not develop. Effect of the combination of cell cycle between donor nucleus and recipient cytoplasm For the normal development of nuclear transferred embryos, the nuclear information must change from the advanced stage to fertilization. For the recipient cytoplasm, unfertilized oocytes at the second metaphase stage have been mainly used (Campbell et al., ’96). In the present study, the highest development of nuclear transferred oocytes was obtained when the nucleus at the G1 stage was fused with the cytoplasm at the M stage as suggested by Cheong et al. (’93). The nuclear membrane break down (NMBD) and premature chromosome condensation (PCC) of donor nucleus occurred soon after fusion. However, 2% to 10% of oocytes at the S stage which received a nucleus at any stage except late S/G2 developed to morulae or blastocysts. In these cases, neither NMBD nor PCC were observed. Since the developmental process of nuclear transferred embryos was not different, the process of NMBD and PCC is not necessary for the conversion of nucleus to the condition at fertilization. A cytoplasmic reprogramming factor(s) besides maturation promoting factor (MPF) is possibly present in the cytoplasm of unfertilized oocytes. Such a factor(s) might be located at or bind Full-term development of nuclear transferred oocytes receiving a nucleus from 4-cell and compacted morula stage embryos Cheong et al., (’93) reported that 22% of enucleated oocytes at the M stage fused with a nucleus from early 4-cell embryos developed to young. The present study was not in agreement with their report. The most likely explanation for the discrepancy is the difference in the method used to fuse the donor blastomere with cytoplasm of recipient oocytes, HVJ in the present study and electrical stimulation in the report of Cheong et al. (’93). When the pronuclei or nucleus(ei) originating from the 4-cell embryos were fused again with enucleated cytoplasm of fertilized zygotes or 2-cell embryos, 8% to 36% of the morulae and blastocysts developed to young. Tsunoda and Shioda (’88) reported that the developmental potential of reconstituted parthenogenetic eggs which received pronuclei of fertilized eggs was lower than that of reconstituted fertilized eggs. Unknown factors that enhance the developmental ability of reconstituted eggs might be present in the cytoplasm of fertilized eggs. TABLE 3. Developmental ability of renuclear transferred embryos receiving a nucleus from compacted morulae in vivo With or without aphidicolin Type of1 renuclear transfer – both half both half + 1 Developed to (%) No. of eggs fused morula 38 22 46 22 7 (18) 4 (18) 12 (26) 4 (18) blastocyst 2 1 8 4 (5) (5) (17) (18) A nucleus from a reconstituted 2-cell egg was fused with a balstomere of enucleated 2-cell embryos. No. of recipients 6 1 5 2 No. of live young/ transferred (%) 0/9 0/2 2/8 0/2 (0) (0) (25) (0) 254 Y. TSUNODA AND Y. KATO Although the sample size was small, the fact that live young were obtained after renuclear transfer of compacted morulae indicates that developmental totipotency of nucleus on the mouse germ line is maintained at least up to the morula stage. The cells used in the present study are considered to be at the blastocyst stage since individual cells separated at the compacted morula stage were used immediately after the next cell division. ACKNOWLEDGMENTS We thank Dr. K. Tanaka, Institute for Molecular and Cellular Biology, Osaka University, for providing HVJ. 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