We examined the developmental ability of enucleated eggs receiving embryonic nuclei and male primordial germ cells (PGCs) in the mouse. Reconstituted eggs developed into the blastocyst stage only when an earlier 2-cell nucleus was transplanted (36 %) but very rarely if the donor nucleus was derived from a later 2-cell, 8-cell, or inner cell mass of a blastocyst (0–3%). 54–100%, 11–67 %, 6–43 % and 6–20 % of enucleated eggs receiving male PGCs developed to 2-cell, 4-cell, 8-cell and blastocyst stage, respectively, in culture. The overall success rate when taking into account the total number of attempts at introducing germ cells was actually 0-6 %. Live fetuses were not obtained after transfer of reconstituted eggs to recipients, although implantation sites were observed.

The developmental ability of reconstituted eggs in relation to embryonic genome activation and genomic imprinting is discussed.

Nuclei from mouse follicle cells (Tarkowski and Balakier, 1980) or thymocytes (Czolowska et al. 1984), when fused with eggs at the second metaphase, show premature chromosome condensation with dispersal of individual chromosomes, and they develop along a pronucleus-like pathway after parthenogenetic activation. These processes are considered to be prerequisites to nuclear reprogramming. In sheep (Willadsen, 1986), cattle (Prather et al. 1987) and rabbit (Stice and Robl, 1988), fused blastomeres from 8-cell embryos to enucleated halves of unfertilized eggs developed into blastocysts like pronuclear eggs and to normal young after transfer to recipients. However, in the mouse experiments on nuclear transplantation, eggs at the pronuclear stage have been mainly used as recipient cytoplasm. Although a low proportion of enucleated zygotes fused with nuclei from 2-cell embryos developed into blastocysts, none of them fused with nuclei from 4-cell or 8-cell embryos developed into blastocysts (McGrath and Solter, 1984; Tsunoda et al. 1987a; Howlett et al. 1987). The nuclei from 4-cell and 8-cell embryos transplanted into enucleated 2-cell embryos developed into blastocysts in culture, and to full term after transfer to recipients (Tsunoda et al. 1987a). However, from the facts that compaction of such reconstituted eggs occurred earlier than in the normal embryos (Tsunoda et al. 1987a) and that blastocysts cultured from eggs with nuclei from 8-cell embryos were smaller and had fewer nuclei (Robl et al. 1986), it appears that nuclei transplanted into enucleated 2-cell embryos did not fully reprogram into the normal nuclei found in 2-cell embryos.

Recently, we reported that mouse eggs at the second metaphase, from which chromosomes had been removed under a fluorescence microscope, could be used as cytoplasm for transplantation of nuclei from inner cell mass (Tsunoda et al. 1988a). However, the rate of development of those reconstituted eggs into blastocysts was quite low (5 %).

The study of nuclear transplantation in salamanders (Lesimple et al. 1987) demonstrated that nuclei from primordial germ cells (PGCs) were totipotent since adult animals were developed from reconstituted eggs with PGCs. Similar experiments were performed with PGCs in Rana pipiens and normal tadpoles were obtained from reconstituted eggs (Smith, 1965). On the other hand, there have been no such tests of developmental ability in mammals.

The present study was undertaken in order to compare the developmental ability of enucleated eggs fused with male PGCs and embryonic nuclei in the mouse.

Donor embryonic cells and primordial germ cells (PGCs) were obtained from albino CD-I females mated with the same strain of males. 2-cell embryos were obtained 39-54 h after hCG injection from superovulated females. 8-cell embryos and blastocysts were obtained 2.5 and 3.5 days after natural mating (day 0.5 is the day of vaginal plug). A single donor inner cell mass cell was obtained from blastocysts by immunosurgery according to Solter and Knowles (1975) and our previous report (Tsunoda et al. 1988a). PGCs were isolated from the genital ridges of fetal male mice from 12.5-17.5 days after coitum according to the procedures of Brinster and Harstad (1977), Felici and McLaren (1983) and Hogan et al. (1986). Morphological differences in the genital ridges of male and female embryos can first be detected at 12.5 days p.c. In the male, mitotic proliferation slows down at 13.0 days p.c., and male germ cells become arrested in the Gi stage of the cell cycle (Monk and McLaren, 1981). A single germ cell was collected by a small bore pipette under a phase-contrast microscope (x200). Almost 100% of PGCs collected in such fashion were positive for staining with alkaline phosphatase. The viability of PGCs, determined by the dye exclusion test (Felici and McLaren, 1983), was 86% (818/953).

Unfertilized eggs were obtained from superovulated Ft (C57BL/6JxCBA) females 15–17 h after hCG injection. The cumulus cells were removed by treatment with hyaluronidase (300i.u. ml-1) in M2 (Fulton and Whittingham, 1978), washed three times and used for cytoplasmic recipient.

Nuclear transplantation

Before nuclear transplantation (McGrath and Solter, 1983), the zonae pellucidae of all the eggs and embryos were cut 10–20 % along the length with a fine glass needle. The method for cutting the zonae, setting the holding and enucleation pipette, and pretreatment with cytochalasin B and colcemid are described in our previous reports (Tsunoda et al. 1986; Tsunda et al. 1988b).

Removal of chromosomes from recipient eggs

Prior to removal of the chromosomes at the second metaphase, the eggs were pretreated with Hoechst 33342 at the concentration of l μg ml-1 for 5 min at 37°C. The chromosomes were mechanically removed within 15 seconds under a fluorescence microscope (Tsunoda et al. 1988a) before nuclear transplantation and parthenogenetic activation. By this method, all of the chromosomes from recipient eggs were completely removed.

Developmental ability of embryonic cell nuclei in enucleated eggs

Nuclei from 2-cell and 8-cell embryos with a small volume of cytoplasm or a single inner cell mass cell were introduced with inactivated Sendai virus (HVJ, 2500 haemagglutinating activity unit) into the perivitelline space of enucleated eggs at the second metaphase. At 5–15 minutes after injection, they were activated with 7 % ethanol for 7 min at room temperature (Cuthbertson, 1983), and were examined for pronuclear-like formations 5–6 h after activation. The eggs with a pronucleus were cultured for 5 days in M16 (Whittingham, 1971).

Developmental ability of male primordial germ cells (PGCs) in enucleated eggs

Single male primordial germ cells were fused with enucleated eggs at the second metaphase by means of HVJ. Within 15min after injection, the eggs were activated with ethanol, and the pronuclear-like formations were examined 5 to 6 hours after activation. The eggs with a nucleus were cultured up to 5 days in M16. After 1 to 2 days culture, some eggs developed into the 2- to 4-cell stage, and were transferred to the oviducts of day 1 pseudopregnant CD-I strain females. The recipients were killed on days 10.5–12.5 in order to examine the implantation sites, or were allowed to go to term. In some recipients, control embryos freshly obtained from F! females were transferred into the same recipients, in order to monitor the success of pregnancy.

Developmental ability of embryonic nucleus in enucleated eggs

Table 1 shows the developmental ability of karyoplasts from 2-cell and 8-cell embryos, and from single inner cell mass cells fused with enucleated eggs at the second metaphase stage. The proportion of karyoplasts fused with recipient cytoplasm was quite high (92–100%), but the fusion rate was not calculated due to the small size when inner cell mass cells were used.

Table 1.

In vitro development of enucleated mouse eggs receiving embryonic nuclei

In vitro development of enucleated mouse eggs receiving embryonic nuclei
In vitro development of enucleated mouse eggs receiving embryonic nuclei

The proportion of eggs fused with karyoplasts that showed pronuclear-like formations 5–6 h after nuclear transplantation and parthenogenetic activation was high (94–100%). In vitro development of reconstituted eggs was different according to the stage of the donor nucleus used for nuclear transplantation. When the nucleus from 2-cell embryos was used, 55–80% of reconstituted eggs developed into the 2-cell stage. Early 2-cell (39–41 h after hCG) nuclei could support development into blastocysts (36%), but middle (44–46h) and late (50–54 h) stages of 2-cell nuclei did not support development. When the reconstituted eggs were not parthenogenetically activated, 44% (12/27) developed into the 2-cell stage and none of them developed to blastocysts. Of the 36 enucleated eggs receiving nuclei from 8-cell embryos, only 6% developed into the 2-cell stage and none of them developed into blastocysts.

Of 111 eggs receiving an inner cell mass nucleus, 80 (72 %) showed a pronuclear-like formation. Of 80 eggs with a nucleus, 18 (23%) developed into the 2-cell stage, with each blastomere having one nucleus, 1 day after activation. However, only 2 of them (3%) developed into blastocysts 5 days after culture.

Developmental ability of male primordial germ cells in enucleated eggs

Table 2 shows the in vitro development of enucleated eggs receiving male primordial germ cells (PGCs). The proportion of eggs fused was not determined for the same reason as for the transfer of inner cell mass cells. The proportion of eggs that had a nucleus 5–6 h after activation was low (20–34%) compared with that of eggs receiving inner cell mass cells (Table 1). All of them had one nucleus (Fig. 1). The proportion of eggs receiving PGCs on days 12.5 and 13.5 that developed into the 2-cell stage (54 and 67 %, Fig. 2) was significantly (P<0·01) lower than that of eggs receiving PGCs at days 14.5 to 17.5 (87–100%). 11 and 27% of reconstituted eggs with PGCs on days 12.5 and 13.5 developed into the 4-cell stage. These are also significantly lower than with eggs receiving PGCs at later fetal stages (40–67%, Fig. 3). 6–44% of reconstituted eggs developed into the 8-cell stage (Figs 4, 5), but only a low proportion of eggs (0–20 %) developed into blastocysts (Fig. 6).

Table 2.

In vitro development of enucleated mouse eggs receiving male primordial germ cells

In vitro development of enucleated mouse eggs receiving male primordial germ cells
In vitro development of enucleated mouse eggs receiving male primordial germ cells
Fig. 1.

An intact ovulated egg fused with a single inner cell mass cell and examined 5 hrs after fusion. Two sets of metaphase chromosomes from recipient egg (up) and from fused cell (down) can be seen.

Fig. 1.

An intact ovulated egg fused with a single inner cell mass cell and examined 5 hrs after fusion. Two sets of metaphase chromosomes from recipient egg (up) and from fused cell (down) can be seen.

Fig. 2.

An enucleated egg at the second metaphase stage fused with a single inner cell mass cell and examined 5 hrs after fusion. The chromosomes of reconstituted egg dispersed in the cytoplasm.

Fig. 2.

An enucleated egg at the second metaphase stage fused with a single inner cell mass cell and examined 5 hrs after fusion. The chromosomes of reconstituted egg dispersed in the cytoplasm.

Figs 3 to 6.

These figures show the in vitro development of enucleated eggs receiving a male primordial germ cell.

Figs 3 to 6.

These figures show the in vitro development of enucleated eggs receiving a male primordial germ cell.

59 eggs that received day 15.5 PGCs and developed into the 2- to 4-cell stage were transferred to the oviducts of day 1 pseudopregnant recipients, but none of the 11 recipients produced young, although 6 (10%) implantation sites were observed. 38 reconstituted 2cell eggs were transferred to recipients with control embryos from Fi females mated with CD-I strain males. 19 of 30 control eggs (63 %) developed into nonalbino young, but none of reconstituted eggs developed to term. 57 reconstituted 2-cell eggs were transferred into 7 recipients, which were examined on day 10.5-12.5. Three had 1-6 implantation sites (14 %), but none had fetuses.

In a preliminary study, when nuclei were introduced into oocytes at metaphase I, the donor chromosomes either mixed with host chromosomes (in intact oocytes) or scattered in enucleated oocytes as they underwent condensation. However, because nuclei introduced into newly ovulated eggs at metaphase II did not combine with host chromosomes (in intact eggs) and did not scatter in enucleated eggs, we decided to use newly ovulated enucleated eggs as recipient cytoplasm in preference to oocytes at metaphase I.

The developmental ability of enucleated eggs receiving embryonic nuclei was different according to the stages of the embryos used as nuclear donors (Table 1). 36% of reconstituted eggs with nuclei from 2-cell embryos that were recovered 39–41 h after hCG injection developed into blastocysts, but few or no eggs receiving nuclei from later 2-cell (44–54 h after hCG) embryos developed into blastocysts. This was so when 8-cell embryos or inner cell mass cells were transplanted. Howlett et al. (1987) reported that a high proportion of enucleated zygotes receiving nuclei from early 2-cell embryos (1–2 h after cleavage) developed into blastocysts, but that the development of zygotes receiving nuclei from late 2-cell embryos (12–18 h after cleavage) was quite low. On the other hand, a high proportion of single blastomeres from sheep (Willadsen, 1986), cattle (Prather et al. 1987) and rabbits (Stice and Robl, 1988) 8-cell embryos fused with the enucleated half of unfertilized eggs developed into the morula or the blastocyst stage, and live young were obtained after transfer of such reconstituted eggs to recipients. Several factors are considered for the inability of reconstituted eggs that received nuclei from 8cell embryos to develop in the mouse. First, in the present study, chromosomes at the second metaphase stage were removed under a fluorescence microscope after Hoechst staining. Although such treatment did not impair the viability of cytoplasm for zygotes when u.v. exposure time was shorter than 20–30 seconds (Tsunoda et al. 1988a), the sensitivity of the cytoplasm at the second metaphase stage to u.v. irradiation and Hoechst staining might differ from that of cytoplasm at the pronuclear stage. A more likely reason is the difference of time of embryonic genome activation among species. It occurs at the 2-cell stage in the mouse (Bolton et al. 1984), at the 8- to 16-cell stage in the sheep (Crosby et al. 1988) and cow (King et al. 1985) and at the 16-cell stage in the rabbit (Manes, 1973). It might be speculated that in enucleated eggs at the second metaphase, in which ‘maturation-promoting factor’ activity is high (Hashimoto and Kishimoto, 1988), receiving nuclei from embryos before embryonic genome activation with develop in vitro and in vivo. However, the enucleated eggs receiving nuclei from embryos after that may not develop. From the results of our previous study in which live young were obtained after transfer of enucleated 2-cell embryos receiving nuclei from 8-cell embryos (Tsunoda et al. 1987a) and after transfer of isolated blastomere of 8-cell embryos aggregated with parthenogenone (Tsunoda et al. 19876), it is clear that at least some of the nuclei of 8-cell mouse embryos have totipotency. This fact indicates that the conditions used in the present study were not adequate for conversion of the transplanted 8-cell nuclei into ‘fertilization’ ones.

Although nuclear transplantation studies revealed that primordial germ cells have totipotency or pluripotency in salamanders (Lesimple et al. 1987) and Rana pipiens (Smith, 1965), such information in mammals is lacking. From the present study, enucleated eggs receiving male PGCs formed one nucleus when parthenogenetically activated 15 min after fusion, and reconstituted eggs with a nucleus developed into the 2cell to blastocyst stage more frequently than those with inner cell mass cells. However, live fetuses were not obtained after the transfer of reconstituted eggs at the 2- to 4-cell stage to recipients although implantation sites were observed. The in vitro developmental ability of reconstituted eggs receiving PGCs of days 12.5 and 13.5 was significantly low compared with that of eggs receiving PGCs of later stages. This observation was coincident with the results of Felici and McLaren (1983), who cultured PGCs in vitro. There are several reasons for the failure of the development of reconstituted eggs with PGCs into live fetuses after transfer to recipients. Recent studies on nuclear transplantation in the mouse have revealed that paternal and maternal genomes play complementary roles during embryogenesis, and that both are essential for development to term (see the review of Surani, 1986). The maternal and paternal genomes are considered to be programmed (imprinting) to function differently during development (Reik et al. 1987; Sapienza et al. 1987). It would be logical to consider that such imprinting would occur during gametogenesis; however, it could be at the premeiotic stage.

Another reason is that cytoplasm of parthenogenone might be not adequate for the normal development of transplanted PGCs. Mann and Lovell-Badge (1984) reported that a very high proportion of young was obtained from recipients after the transfer of enucleated parthenogenones receiving pronuclei from fertilized eggs. In contrast, Tsunoda and Shioda (1988) demonstrated that development of reconstituted eggs using parthenogenetic 1-cell eggs as recipients for donor pronuclei developed significantly poorly, compared to when 2-cell parthenogenetic embryos were used as recipient cells for nuclei from 2-cell-stage embryos. When the nuclei of 2-cell embryos developed from reconstituted eggs with PGCs were transplanted again to enucleated blastomeres of normal 2-cell embryos, 18 of 29 eggs (62 %) developed into blastocysts but 15 % of them developed into blastocysts when retransferred into enucleated 1-cell eggs (preliminary results).

The reconstituted eggs with PGCs frequently showed the abnormality before the compaction stage; and, as shown in Fig. 6, most of the blastocysts obtained from reconstituted eggs did not show a distinct inner cell mass. Although we observed preliminarily that the reconstituted eggs with PGCs synthesized the 68/70K proteins as well as control 2-cell embryos, the presence of such proteins would be not sufficient for normal development into blastocysts. Since we did not examine the normality of chromosomes of reconstituted eggs in the present study, chromosomal abnormality might be another reason for the low developmental ability in vitro and the failure to develop into fetuses. No marker had been employed to check that development was due to the donor nucleus, although the chromosomes of recipient egg could be completely removed in our system (Tsunoda et al. 1988a).

One of the purposes of studying nuclear transplantation is to attempt to obtain cloned offspring. The most feasible way to obtain clones in mammals is to repeat the nuclear transplantation at the preimplantation stages, before embryonic genome activation (Marx, 1988). Another possible way is to use the nucleus from a cell before the imprinting of genomes. Further studies are necessary to improve the development of reconstituted eggs with PGCs in vitro and to obtain young after transfer to recipients.

We thank Dr Anne McLaren, MRC Mammalian Development Unit, London, U.K.; Dr M. C. Chang, Worcester Foundation for Experimental Biology, Mass., MA., USA for their critical evaluation of this manuscript. This investigation was supported by a grant from Japanese Ministry of Agriculture, Forestry and Fisheries (Nuclear Transfer), to YT and a grant from the Ministry of Science and Technology (Development Biotechnology) to YT and TT.

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