Antioxidative effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability and gene expression in bovine oviduct epithelial cell and the developmental competence of bovine IVM/IVF embryos.

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) by RT-PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co-cultured with BOEC pre-treated with astaxanthin (500 µM) in the presence or absence of sodium nitroprusside (SNP, 1000 µM) for 24 h. Cell viability in BOEC treated with SNP (50-2000 µM) lowered, while astaxanthin addition (50-500 µM) increased it in a dose-dependent manner. Cell viability in astaxanthin plus SNP (1000 µM) gradually recovered according to the increase in astaxanthin additions (100-500 mM). The LPO in astaxanthin group (50-500 µM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 µM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT-PCR. Bcl-2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase-3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under co-culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 µM astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.

Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development.

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H(2)O(2)) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 µM). The sperm were treated with melatonin in the presence or absence of H(2)O(2) for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 µM) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H(2)O(2) groups were lower than H(2)O(2) only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.

Nuclear transfer using clonal lines of porcine fetal fibroblasts with different sizes and population doubling rates.

The aim of the present study was to examine the development of pig embryos produced by somatic cell nuclear transfer (NT) using the clonal lines of fetal fibroblasts with different population doublings (PD) per day and sizes. Clonal lines were established by plating fetal fibroblasts from a Day 35 pig fetus into 96-well clusters, one cell to each well. Four clonal lines (L1-L4) were selected for NT according to their PD per day (1.1 +/- 0.2 to 0.8 +/- 0.2) and mean cell size (15.1 +/- 2.0 to 20.1 +/- 2.9). Donor cells were transferred into enucleated oocytes, fused and activated simultaneously with electrical stimuli (two pulses of 125 V mm(-1) for 30 micros) and cultured for 6 days. The proportion of embryos that developed to the blastocyst stage in the L3 (19.6%) and L4 (25.3%) lines, which had a lower PD per day and larger cell size, were significantly higher (P < 0.05) than that of the L2 line (10.6%), which had a higher PD per day and the smallest cell size. The proportion of embryos developing to the blastocyst stage in the L1 line (17.3%), which had the highest PD per day and smaller cell size, was significantly lower (P < 0.05) than that of the L4 line. These results suggest that clonal lines with larger sized cell populations in mean and lower PD per day have a greater in vitro developmental potential following NT.

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos.

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

Morphologic and histologic comparisons between in vivo and nuclear transfer derived porcine embryos.

Nuclear transfer (NT) is an inefficient but invaluable tool of the biotechnology industry. This study looked at abnormalities associated with peri-implantation NT porcine embryos. Four experimental groups were examined: nonpregnant animals, in vivo pregnant animals, NT recipients, and manipulation control embryos (MC). Embryos (Day 10, 12, or 14) were evaluated for embryonic disc diameter, gross morphology, nucleoli density, and mitotic figure index. Day 12 (P < or = 0.03) and Day 14 (P < or = 0.01) NT embryos had increased numbers of nucleoli, and Day 14 NT embryos had an increased (P < or = 0.03) mitotic index compared to in vivo and MC embryos. In vivo produced Day 14 embryos had increased (P < or = 0.01) disk diameters when compared to other embryos except for MC Day 14, which also showed increases (P < or = 0.01) in disk diameter except when compared to in vivo produced Day 12 and Day 14 embryos. In vivo produced Day 12 had greater (P < or = 0.03) disk diameters when compared to NT and MC embryos except for MC Day 14, and in vivo produced Day 14 embryos, which had a significantly increased (P < or = 0.01) disk diameter. In vivo produced Day 14 embryos were morphologically more advanced (P < or = 0.01) than Day 14 NT and MC counterparts. NT embryos develop at a slower rate than their in vivo produced counterparts. The increase in nucleoli and mitotic index of NT embryos suggest the cell cycle may be affected or the NT embryos are employing other means to compensate for slow development. The techniques used during NT also appear to compromise embryo development.

Effects of maturational age of recipient oocytes and activation conditions on the development of porcine fetal fibroblast nuclear transfer embryos.

This study was conducted to evaluate the nuclear remodeling patterns and the developmental potential of porcine fetal fibroblast nuclear transfer embryos (NTs) following the maturational age of recipient oocytes and activation conditions. Donor cells were transferred into the enucleated oocytes that were matured for 36 or 44h. Electrofused embryos were cultured in PZM-3 for 6 days without activation treatment (EF group). Some of these embryos were additionally activated by electric stimulus (ES; EF+ES group) or a combination of ES and DMAP (EF+ES+D group) before culture. The reconstituted embryos were fixed 2.5h after fusion to evaluate the nuclear remodeling patterns. The nuclear remodeling pattern of NTs reconstituted with 44 h-matured recipients showed a tendency to form a pronucleus-like structure, while that of NTs reconstituted with 36 h-matured recipients showed a tendency to undergo a premature chromosome condensation (PCC) and form one set of chromatin clump. In EF+ES+D group, blastocyst development was significantly increased regardless of maturational age of recipient oocytes (P<0.05). The result indicates that additional activation treatment is necessary to induce the activation of embryos reconstituted with 36 h-matured recipients, and treatment with the combination of electrical stimuli and DMAP could enhance the blastocyst formation rate of porcine NTs reconstituted with both 36 h- and 44 h-matured recipient oocytes.

Effects of plasmin on sperm-oocyte interactions during in vitro fertilization in the pig.

The present study was undertaken to examine the effect of plasmin on sperm viability and sperm-oocyte interaction during in vitro fertilization in the pig. Porcine sperm, which were washed in Dulbecco's PBS were re-suspended and incubated in fertilization medium (mTBM; modified Tris-buffered medium) containing 0, 0.1, 1.0, 10.0 or 100.0ng/mL of plasmin. Sperm viability was not affected by plasmin treatment. Addition of plasmin in doses ranging from 0.1 to 100.0ng/mL for 2, 4 or 6h to washed boar sperm resulted in enhancement of acrosome reaction (AR) compared with untreated cells. The concentration of 0.1ng/mL plasmin (95+/-18 sperm/oocyte) had no effect on sperm binding, whereas 1.0ng/mL (123+/-21 sperm/oocyte), 10.0ng/mL (124+/-16 sperm/oocyte) and 100.0ng/mL (124+/-15 sperm/oocyte) of plasmin increased sperm binding compared with the control (83+/-15 sperm/oocyte). The zona pellucida solubility (zona dissolution time) was less in medium with 1.0ng/mL (123+/-24s), 10.0ng/mL (99+/-15s) or 100.0ng/mL (95+/-19s) plasmin compared with control (176+/-27s). When pig oocytes and sperm were co-incubated in various concentrations of plasmin for 6h, the penetration rate was greater in medium with 1.0ng/mL plasmin (77.5+/-3.1%) compared with the control. However, there were no differences in the polyspermic rates and mean number of sperm (MNS)/oocyte between the groups treated with plasmin and control. These results suggest that plasmin might play a role in events related to fertilization.

Effect of a fertilization-promoting peptide on the fertilizing ability and glycosidase activity in vitro of frozen-thawed spermatozoa in the pig.

This study has evaluated the effect of fertilization-promoting peptide (FPP) on the fertilizing ability and glycosidase activity in vitro of frozen-thawed boar spermatozoa. Use of chlortetracycline (CTC) fluorescence analysis, as well as various glycosidase analyses and the oocyte penetration test showed that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. There were significantly (P < 0.05) more acrosome-reacted and penetrated in medium with 100 nM FPP than with 0, 50, 200 or 400 nM. The beta-N-acetylglucosaminidase (beta-GlcNAcase) activity was at least two-fold higher than other glycosidase regardless of FPP concentrations. In the same glycosidase, there were no differences in medium with different concentrations of FPP. The percentages of spermatozoa that reached acrosome reaction were affected by different periods (0, 1, 2, 3 or 4 h) of spermatozoa preincubation and were higher in medium with than without FPP. Penetration rates were decreased with preincubation periods of spermatozoa when oocytes were inseminated with spermatozoa preincubated in medium with and without FPP for the different periods. These rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 or 24 h. The activities of alpha-fucosidase, alpha-mannosidase, beta-galactosidase and beta-GlcNAcase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may have a positive role in promoting sperm function and glycosidase activity in the pig.

Developmental potential of porcine nuclear transfer embryos derived from transgenic fetal fibroblasts infected with the gene for the green fluorescent protein: comparison of different fusion/activation conditions.

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3-5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 microsec (group A), or one pulse of 1.6 kV/cm for 30 microsec followed in 30 min by one pulse of 1.2 kV/cm for 30 microsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was higher (P < 0.05) than in group B (59.4% +/- 2.3%). The rates of cleavage (50.1% +/- 4.6% to 62.8% +/- 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% +/- 4.2%) was higher (P < 0.05) than in group C (58.9% +/- 2.7%). The cleavage rates were not different among control and treatment groups (58.1% +/- 8.1% to 73.6% +/- 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and group C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (70.9% +/- 0.2%). The cleavage rate of the control group (85.5% +/- 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% +/- 2.7% and 63.9% +/- 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% +/- 2.5% to 19.1% +/- 6.0%) and the mean cell number of blastocysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.

Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro.

The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells.

This study tested the effects of oocyte collection method, activation protocol and maturational age of recipient oocytes on the in vitro development of nuclear transfer embryos reconstructed with cultured cumulus cells. Cumulus cells synchronized in G0/G1 phase by serum-starvation culture were transferred into enucleated oocytes that were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and cycloheximide (CHXM), and cultured for 6 days. Oocyte collection methods, activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44-h-matured recipients. However, the development of embryos reconstituted with 33-h-matured recipients was significantly improved (P<0.05) by activation with the combination of electric pulse and CHXM. The present study shows that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage, and that their development can be improved by reconstruction with young oocyte cytoplasts following activation with a combination of electric pulse and CHXM.

Effect of superoxide dismutase(SOD) on pronucleus formation of porcine oocytes fertilized in vitro.

This study was undertaken to evaluate the effects of superoxide dismutase (SOD) on pronucleus formation in porcine oocytes fertilized in vitro by frozen-thawed spermatozoa. No differences were found in penetration rates when SOD was added to maturation or fertilization medium at any level tested in first and second experiments. Pronucleus formation rates were higher (P < 0.05) when SOD at 10 and 100 units was added to the maturation medium (46 and 53%, respectively) compared with the controls (26%). On the other hand, when the fertilization medium was supplemented with SOD at different concentrations (1, 10 and 100 units/ml), pronucleus formation rates (55, 52 and 50%) were significantly higher (P < 0.05) than in the control group. In third experiment, the oocytes were cultured in medium with (1 unit/ml) or without SOD for 8, 16, 24 and 32 h after insemination. The penetration rates had a tendency to increase as time of sperm-oocyte culture was prolonged. No significant differences, however, were observed in penetration rates between groups with and without SOD. On the other hand, the pronucleus formation rates were higher in medium with than without SOD at 8 (7 vs 0%), 16 (14 vs 3%), 24 (48 vs 16%; P < 0.01) and 32 h (49 vs 22%; P < 0.05). These findings demonstrate the advantage of culture with SOD on pronucleus formation in porcine oocytes penetrated by spermatozoa. However, SOD does not affect penetration rates and polyspermy.

Effect of cumulus cells and exposure period to ethanol on in vitro development of mouse diploid parthenogenones.

Cumulus-intact and cumulus-free mouse oocytes were exposed to 7% ethanol for 1, 4 and 7 min, and treated with cytochalasin-B. The activation rate and the proportion of diploid parthenogenones in all groups were not significantly different. After 96 hr in culture, a higher number of blastocysts was obtained when either cumulus-intact or cumulus-free oocytes were exposed for shorter times (1 and 4 min) to ethanol. The presence or absence of cumulus cells at activation had no effect on the percentage of blastocysts. However, at 1 and 4 min ethanol-exposure periods, the parthenogenones derived from cumulus-intact oocytes had a higher number of cells than ones derived from cumulus-free oocytes.

Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes.

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0-91.6%), and activation (95.2-96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos.

Birth of mice after transplantation of early cell-cycle-stage embryonic nuclei into enucleated oocytes.

The present study was conducted to investigate the influence of cell cycle stage of the donor nucleus on chromatin structure and development of mouse embryonic nuclei transplanted into enucleated oocytes. Donor cell-cycle stage was controlled in order to examine, in addition, the developmental potential of nuclei from 2-, 4-, and 8-cell-stage embryos. The cell cycle stage of donor nuclei was classified as early, middle, or late. After nuclear transfer, electrofusion, and activation, early-stage transplants formed a single pronucleus-like structure, but middle-stage transplants formed very irregular types of structures and late-stage transplants extruded a polar body. A high proportion of development to the blastocyst stage (77.8%) and an increased cell number (62.1 cells) were obtained from the early 2-cell-stage transplants as opposed to the middle- (0%) and late-stage (20.8%, 37.0 cells) transplants (p < 0.001). With transplantation of early-stage nuclei, high proportions of development to the blastocyst stage and of offspring were obtained from nuclear transplant embryos with a nucleus from a 2-, 4-, or 8-cell-stage embryo. The results confirm that the donor cell-cycle stage critically affects the chromatin structure and development of nuclear transplant embryos. The results also demonstrate that the nuclei from 2-, 4-, and 8-cell-stage mouse embryos in the early stage of each cell cycle can be reprogrammed when transplanted into enucleated mature oocytes.

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes.

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.

Development of mouse embryonic nuclei transferred to enucleated oocytes and zygotes.

The present study was conducted to examine the development of nuclear transplant embryos produced by transplanting nuclei to either oocytes or zygotes in the mouse. Metaphase II oocytes and one-cell zygotes were enucleated and fused with transferred nuclei from late two-, four- and eight-cell stage embryos. Enucleation of metaphase oocytes was achieved using the interference microscope without staining. Fusion and oocyte activation were performed by means of electric fields. Similar development rates to the blastocyst stage were obtained from enucleated oocytes (28.0%) and zygotes (30.9%) reconstituted with nuclei from late two-cell embryos. Cleavage and blastocoele formation of reconstituted embryos occurred at around the same time as observed in the control embryos, with some exceptions. After transfer to recipient females, live young were obtained from both reconstituted oocytes (9.1%) and zygotes (11.5%) that received a nucleus from late two-cell embryos. The results indicate that enucleated zygotes as well as oocytes can support development to term of nuclei introduced from late two-cell embryos in which activation of the embryonic genome has occurred, which may be a result of the reprogramming of the donor nucleus.

Developmental capacity of reconstituted mouse embryos: influences of nucleus and cytoplasm sources.

This study was undertaken to examine the developmental capacity of reconstituted mouse embryos, and the influences of nucleus and cytoplasm on the development of these embryos following reciprocal pronuclear transplantation between in vitro 2-cell blocked and nonblocked embryos. Karyoplast containing pronuclei was transferred into the perivitelline space of the enucleated zygote and fused to cytoplasm with electrofusion. Maximum fusion rate was obtained when a field strength of 1.5 kV/cm was used. The fusion rates were high (86.2 +/- 3.2 to 90.6 +/- 2.0%) regardless of the strains of donor nucleus and recipient cytoplasm. Developmental rates of reconstituted embryos to the blastocyst stage, which were similar to that of the F1 (C57BL/6J x CBA) control were high when F1 embryos were used as the cytoplasm recipients (88.8 +/- 1.5 and 91.9 +/- 2.0%). When ICR embryos were used as the recipient cytoplasm, developmental rates were significantly reduced (71.5 +/- 2.9 and 54.1 +/- 3.2%), and affected by the source of nucleus. There were no significant differences in the cell number of embryos that developed to blastocysts and in the developmental rates to live young among the embryos reconstituted with different nuclei and cytoplasm, and the ICR control. The results of this study show that the development of reconstituted embryos is hardly affected by nuclear transplantation and electrofusion procedures. It is indicated that the recipient cytoplasm, rather than the donor nucleus, has the greater influence on the in vitro development of the reconstituted embryos to the blastocyst stage.

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.