Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs

In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD-1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.     

Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion method

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.     

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.     

Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse

A new and reliable pronuclear transplantation procedure for the mouse egg has been developed by McGrath and Solter ('83). To overcome the technical difficulties of such a procedure, especially in uniformly preparing enucleation pipettes and in reducing damages during micromanipulation, we have examined the effect of cutting the zona pellucida of the eggs. By making a slit in the zona of an egg, the time for pipetting and exchange of pronuclei between eggs was shortened because the sharp tip of the pipette was not necessary. Although the proportion of pregnant recipients and young obtained after transfer of pronuclear transplanted eggs cultured for 1 day or 3 days was quite low, it was significantly increased (70% for pregnancy rate and 32% for the young) following transfer of eggs cultured for 4 days. These values were comparable with those after transfer of unoperated eggs cultured to morulae and blastocysts.     

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.     

Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: Effect of media and electrical fusion parameters

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro-produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 μsec. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle. © 1993 Wiley-Liss, Inc.     

Factors affecting electrofusion of 2-cell mouse embryos

Parameters of electrofusion of 2-cell mouse embryos were optimized for application as a model for nuclear transplantation. There was considerable lysis of embryos with M(2) as the medium for fusion; however, 100% fusion (n = 58) was obtained with a single 0.31-kv / cm, 1280-musec pulse. With mannitol and sucrose solutions as the medium, a wide range of field strengths (0.31-1.41 kv / cm for 0.26 M sucrose solution and 0.31 to 2.04 kv / cm for 0.3 M mannitol solution) and durations of the electrical pulse (10-1280 musec) resulted in high rates of fusion (often 100%). Likewise, osmolarity of sucrose and mannitol solutions did not affect the rate of fusion using a 0.47-kv / cm pulse. With a field strength of 2.04 kv / cm, the proportion of embryos that fused in mannitol solution increased (P<0.05) and the proportion that were lysed decreased (P<0.05) as osmolarity increased. Both fused (162 642 , 25%) and control embryos (32 72 , 44%) continued to develop in culture for 48 h, after which they began to compact. Fused embryos were only at the 4-cell stage by this time, while control embryos were at the 8-cell stage. Optimal pulse durations are plotted for field strengths between 0.31 and 1.41 kv / cm with 0.26 M sucrose as fusion medium.     

Production of bovine tetrapolid embryos by electrofusion and their developmental capability in vitro

Optimal conditions of electrofusion for blastomeres of two-cell bovine embryos to produce tetraploid embryos were investigated. The high fusion rate (73–95%), viability, and develop mental capacity were obtained under a field strength of 1.0 kV/cm with direct current pulses of 10 or 25 μsec duration applied twice. Cytological study showed that 78.6% (11/14 embryos) of embryos exposed to electrofusion had tetraploid chromosome sets and the others were diploid or hexaploid. The tetraploid embryos had the capability to develop up to morulae stage in vitro.     

Cloned piglets born after nuclear transplantation of embryonic blastomeres into porcine oocytes matured in vivo

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.     

Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro

The present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus-free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 μsec. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 μsec, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 μsec, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation. It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.     

Effect of aging of recipient oocytes on the development of bovine nuclear transfer embryos in vitro

The present study was undertaken to examine the effect of the length of in vitro maturation of oocytes on the efficiency of enucleation, parthenogenetic activation and blastomere fusion by electric stimulus. In vitro development of reconstituted oocytes receiving a blastomere from 8 to 16-cell bovine embryos fertilized in vitro was investigated to assess the effect of aging of the oocytes. The proportion of oocytes with a first polar body at 22 to 24 hours after maturation was high (80%) compared with those obtained at 16 to 18, 28 to 30 or 42 to 44 hours (50 to 75%). The success rate of enucleation significantly decreased with aging (88, 85, 74 and 55%). The activation rate significantly increased with the length of maturation in vitro (P<0.01) (1 to 4, 24 to 41, 57 to 70 and 80 to 87%). The proportion of oocytes fused with a blastomere from 8- to 16-cell embryos was not dependent on the age of the oocytes (54 approximately 59%). The ability of the reconstituted oocytes to develop to the 2-cell and the 8- to 16-cell stage increased with the length of maturation of recipient oocytes. When oocytes enucleated and a blastomere at 22 to 24 hours were incubated further for 22 to 23 hours until electrofusion. The proportions of oocytes which developed to the 2-cell and the 8- to 16-cell stages (74 and 17%) were similar to those obtained at 42 to 44 hours after maturation. However, only 1 to 6% of reconstituted eggs receiving a blastomere from 8- to 16-cell embryos fertilized in vitro developed into a blastocyst in vitro.     

Stage-specific gene expression in asynchronous tetraploid mouse embryos formed by fusion of blastomeres and fertilized eggs

Asynchronous tetraploid mouse embryos were generated by electrofusion of fertilized eggs with blastomeres from different cleavage stages. The majority of the cytoplasm was always contributed by the egg. The best development was observed when eggs were fused with 2-cell blastomeres. Both genomes became active in fusion embryos (at least the genes for glucose phosphate isomerase did). Stage-specific protein synthesis seemed to be more adjusted to the developmental stage of the egg's than of the blastomere's genome, but at the 2-cell stage both contributed slightly differently to the protein patterns. Also, the time range of the first appearance of the stage-specific embryonic antigen SSEA-1 was wider in fusion embryos than in controls. It seems that the two genomes are not completely synchronized in these tetraploid embryos, a further indication that, in the mouse, the cytoplasm of fertilized eggs might not be compatible with older embryonic nuclei. Some results were presented at the 83. Jahresversammlung der Deutschen Zoologischen Gesellschaft in Frankfurt, 04.-09.06.1990 Correspondence to: U. Petzoldt     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Nuclear transplantation in early pig embryos

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of choline acetyltransferase activity in chick cranial neural crest cells in culture

The types of mouse parthenogenones obtained in a medium modified with respect to Ca²⁺ and/or Mg²⁺ ions were investigated in “spontaneously” activating eggs after culturing cumulus masses in vitro for 5 hr. The second meiotic division was affected in eggs cultured in medium lacking Ca²⁺ and Mg²⁺ or Ca²⁺ alone, resulting in suppression of second polar body extrusion in a high proportion of cases, giving rise to two pronuclear eggs or eggs that underwent immediate cleavage. Extrusion of the second polar body occurred normally when the cumulus mass was cultured in complete medium and, in a high proportion of eggs, when Mg²⁺ alone was lacking in the medium. The results are discussed with reference to the second meiotic division. The method provides an efficient way for obtaining a large number of different types of parthenogenetic embryos.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.