Blastomeres from somatic cell nuclear transfer embryos are not allocated randomly in chimeric blastocysts

A marker has been developed to allow detection of blastomeres that originate from embryos produced by nuclear transfer (NT) of genetically engineered fetal fibroblasts. A plasmid (phEFnGFP) was constructed with a G418 resistance cassette for selection in fibroblasts and a nuclear localized green fluorescent protein (nGFP) expression cassette that expresses in every cell of day-6, -7, and -8 bovine embryos. This construct was utilized to follow the blastomere distribution in aggregation chimeras produced from fertilized embryos (in vitro produced, IVP) or parthenotes and NT embryos. Fluorescent and nonfluorescent NT embryos were aggregated early on day 4 and evaluated on day 8. Nuclei of blastomeres that carried the transgene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluorescence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the inner cell mass (ICM) or trophectoderm in NT<>NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres in the trophectoderm when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was significantly less (p < 0.05) when aggregated with parthenotes as compared to aggregation with IVP embryos. In NT<>NT aggregates, no chimeric embryos were produced that had an ICM composed of blastomeres from a single origin. However, in NT<>Parthenote aggregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 0% to 83% of the ICM that expressed nGFP. Similarly, in NT<>IVP aggregates 50% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We conclude that production of divaricated chimeras from NT origin is feasible. Other applications of this technology are discussed.     

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.     

Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period.

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period. Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: 1) single pronuclear (haploid) embryos and 2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1-cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that 1) the cell doubling time of the diploid parthenogenones was 12.25 +/- 0.34 h, and was not significantly different from the value obtained for the control group (12.74 +/- 1.17 h), and that 2) the cell doubling time of the haploid parthenogenones (15.25 +/- 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>)

In nonhuman primates (NHPs), there have so far been few reports about nuclear transfer (NT), especially using adult somatic cells. The objective of this study was to determine the developmental competence of NT embryos derived from various somatic cells embryonic stem (ES), amniotic epithelial, cumulus, or fetal fibroblast cells] and the nuclear transfer method, such as electro fusion or piezo microinjection, activation with chemical reagent [ionomycine/6-dimethylaminopurine (DMAP), calcium ionophore A23187/DMAP, or cycloheximide (CHX)] and reprogramming time (1, 2, or 4 h; in this study, the duration from injection or fusion to activation was defined as the reprogramming time). Our results showed that a 1-h reprogramming and activation with ionomycin/DMAP are suitable for NT in monkeys. Developing cleaved embryos up to the six-cell stage was similar among all experiments. However, beyond the eight-cell stage, developmental rates were higher in NT embryos reconstructed with fetal fibroblast cells and amniotic epithelial cells, and we were able to produce NT blastocysts from these cells. Interestingly, electro fusion is sufficient for amniotic epithelial cells and piezo microinjection is better suited for fetal fibroblast cells to produce NT blastocysts, thus suggesting that the best method for somatic cell NT may be different between cell types.     

Application of the zona-free manipulation technique to porcine somatic nuclear transfer

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.     

Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.     

Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells

Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.     

Development of cloned pig embryos by nuclear transfer following different activation treatments

This study compared the effects of activation treatments on the development and ploidy of nuclear transferred (NT) pig embryos. After in vitro maturation of oocytes collected from the slaughterhouse, oocytes were enucleated and reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 kV/cm, 20 musec). Oocytes were pulsed thrice electrically with 1.4 kV/cm for 20 musec and NT eggs were divided into three treatment groups: Group 1 (no further treatment), Group 2 (10 mug/mL cycloheximide [CHX], 3 hr), and Group 3 (1.9 mM 6-dimethylaminopurine [DMAP], 3 hr). All the eggs were cultured in sets of 30 in 60 muL drops of NCSU-23 supplemented with 4 mg/mL fatty acid free BSA, and compared for the rates of development and ploidy. The rates of cleavage, development, and total cell number of parthenotes in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Cleavage rates of NT embryos in Group 1 were significantly (P < 0.05) lower than those in Groups 2 and 3 (73% vs. 81% and 82%, respectively). Development into blastocyst stage and total cell number of NT embryos in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Although the embryos in Group 3 had higher development, approximately 58% of NT embryos evaluated were abnormal ploidy (6% haploidy, 9% polyploidy, and 42% mixoploid). The results suggested that although DMAP enhanced development and higher cell number, due attention should be paid to abnormal ploidy in pig NT embryos.     

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

In the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.     

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs : A cytophotometric study

Mouse one-cell embryos were taken 1, 1.5, 2, 3, 4, 6, 8, 10, 13 and 18 h after insemination. One-cell parthenogenones were induced by treatment of mouse eggs obtained 20 h after HCG injection with hyaluronidase and cultured for 0.5, 1, 3, 4.5, 6, 8, 10, 12 and 24 h. Some parthenogenones were pulse-labelled with tritiated thymidine, cut and autoradiographed. Both the embryos and parthenogenones were Feulgen-stained, and integrated relative optical absorption of either pronuclei or nuclei of polar bodies was measured with a cytophotometer. In some fertilized eggs and parthenogenones the DNA synthesis sets in 4–6 h after either insemination or parthenogenetic stimulus. Between the 8th and 13th hour after insemination the fraction of DNA synthesizing embryonic pronuclei remained at the level 30–40%. Most parthenogenones duplicated their DNA content between the 8th and 12th hour after hyaluronidase treatment. The DNA synthesis time in pronuclei of embryos was determined to be 3.5–4.0 h and that of pronuclei of parthenogenones approx. 4 h. The minimal time of the G2 phase was estimated to be 3–5 h. The first labelled pronuclei of parthenogenones were detected 6 h after stimulus. Male pronuclei started and ended DNA synthesis earlier than female pronuclei. Differences in the DNA content between pronuclei of the parthenogenones (when there are two in one parthenogenone) were observed beginning with the 10th h after hyaluronidase treatment.The DNA content in the nuclei of the second polar bodies (PB) of embryos increased slowly between the 8th and 22nd hour after insemination, up to an overall value of 1.4 C. That of the nuclei of the polar bodies of parthenogenones accompanied the synthesis of DNA in pronuclei to the 10th hour after hyaluronidase treatment, up to an overall value of 1.4 C.     

Comparison of the interferon-tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 70/5 (93%); NT 67/1 (99%)] and less (P < .05) for P blastocysts [65/27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day.     

Effects of sucrose treatment on the development of mouse nuclear transfer embryos with morula blastomeres as donors

In this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180+/-10 s, 130+/-10 s and 120+/-10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p>0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.     

Analysis of the fifth cell cycle of mouse development

The 5th cell cycle of mouse development was analyzed to determine the lengths of each cell cycle phase. The DNA content of Feulgen-stained blastomere nuclei was measured at various times throughout the cell cycle by microdensitometry. To achieve precise timing of the start of the 5th cell cycle, experiments utilized isolated 16-cell blastomeres and cell pairs obtained by in-vitro division of isolated 8-cell blastomeres. The following estimates were made for a mixed population of polar and apolar 16-cell blastomeres: G1, less than or equal to 2 h; S, 8-9 h; G2 + M, 2 h. No significant difference was found in the timing of DNA synthesis between polar and apolar cells or between cell pairs and whole embryos.     

Birth weight and birth rate of heavy calves conceived by transfer of in vitro or in vivo produced bovine embryos

The aim of this study was to evaluate the difference in birth weight and gestation length between Japanese Black calves obtained from transfer of bovine embryos produced in vitro (IVP) and those developed in vivo (IVD). An additional objective was to clarify the sire effect on birth weight and gestation length and to examine the birth rate of heavier calves. Two Japanese Black bulls breed at our experimental station were used as a semen source for production of IVP and IVD embryos. Thirty-eight Japanese Black heifers and cows of various genetic backgrounds were used as embryo donors for IVD embryos. Ovaries for IVP embryos were collected at random at a local slaughterhouse from Japanese Black cattle of various genetic backgrounds. IVP embryos were produced using co-culturing with cumulus cells in 5% CS+TCM 199. Both the IVD and IVP embryos were transferred non-surgically to Holstein recipients on day 7+/-1 of estrous cycle. In this study, the birth weights and gestation lengths of half-sib single calves for bull A and B were analyzed.The numbers of single calves born by transfer of IVP and IVD embryos for bull A and B were 133 and 121, 243 and 465, respectively. The birth weight of the IVP calves was significantly higher (P<0.01) than that of the IVD (bull A: 31.0+/-0.4 kg versus 27.2+/-0.4 kg and bull B: 29.9+/-0.6 kg versus 26.6+/-0.2 kg). Gestation length of the IVP calves for bull A was significantly longer (P<0.01) than that of the IVD (291.9+/-0.9 days versus 283.6+/-0.5 days). However, for bull B, there were no differences in gestation length between the IVP and IVD calves (285.9+/-0.7 days versus 286.2+/-0.3 days). These results clearly indicated that IVP calves had heavier birth weights than IVD calves but that the average gestation length of IVP calves was not always longer than that of IVD calves. Furthermore, the birth rate of heavier calves and the incidence of stillbirth and perinatal mortality up to 48 h post partum in IVP calves (bull A: 11.3%, bull B: 7.8%) were greater (P<0.05) than those in IVD calves from both bulls (bull A: 4.1%, bull B: 3.7%).     

Cell allocation and chromosomal complement of parthenogenetic and IVF bovine embryos

Considerable concerns exist regarding the quality of parthenogenetically activated embryos in terms of sufficient numbers of cells comprising the inner cell mass (ICM) and trophectoderm (TE) and the ploidy. Therefore, these two parameters were used to assess the quality of embryos derived from parthenogenetic activation by using calcium ionophore A23187 (CaI) followed by either 6‐dimethylaminopurine (6‐DMAP, 3.5 hr or 6.5 hr) or cycloheximide (CHX) plus cytochalasin D (CD). The conventional in vitro (IVF) produced embryos served as a control. Double staining of the parthenogenetic blastocysts showed that the total cell number (TC) of embryos from the 6‐DMAP 3.5 hr (87.0 ± 5.3) and CHX+CD (79.0 ± 6.1) groups was not different (P > 0.05), but was lower than that of control embryos (116.0 ± 5.8, P < 0.001). The mean ratios of inner cell mass (ICM) and trophectoderm (TE) cells in the 6‐DMAP 3.5 hr group (0.57 ± 0.04) and the control IVF group (0.50 ± 0.02) did not differ significantly. Both were higher than those of the CHX+CD group (0.36 ± 0.02; P < 0.05). Further analysis of chromosomal compositions of developing stage embryos at day four after IVF or parthenogenetic activation demonstrated that prolonged treatment with 6‐DMAP for 6.5 hr resulted in a significantly lower percentage of diploid embryos and a significantly higher percentage of abnormal ploidy embryos compared to treatment with 6‐DMAP for 3.5 hr or with CHX and IVF. In conclusion, parthenogenetic activation of bovine oocytes with CaI followed by 6‐DMAP for 3.5 hr could produce better quality embryos in terms of total cell numbers, the number of cells allocated to the ICM, and the ploidy of embryos. Mol. Reprod. Dev. 54:57–62, 1999. © 1999 Wiley‐Liss, Inc.     

Analysis of the third and fourth cell cycles of mouse early development

The third (4-cell) and fourth (8-cell) cell cycles of early mouse development have been analysed in populations of blastomeres synchronized to the preceding cleavage division. DNA content was measured microdensitometrically. The entry of blastomeres into these cell cycles showed considerable heterogeneity both within and between individual embryos. This heterogeneity was greater in the fourth than in the third cell cycle. The component phases of the third cell cycle were estimated as G1 = 1 h, S = 7 h, and G2 + M = 2-5 h, and those of the fourth cell cycle as G1 = 2 h, S = 7 h, and G2 + M = 1-3 h.     

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.