Viability of blastocysts produced by aggregation of two half-embryos in the mouse

Although methods for production of chimeras from early cleavage stages have been well established, little research has been directed toward production of genetically identical chimeric offspring. This study was designed to examine survival of blastocysts produced by aggregation of two halved eight-cell stage embryos from two different mouse strains. Four blastomeres of an eight-cell embryo from a pigmented strain were aggregated with four blastomeres of an eight-cell embryo from a nonpigmented strain. Aggregates were cultured for 48 h and transferred as blastocysts to synchronized recipients of three treatment groups. Viability was determined by examining the number of offspring produced relative to the number of blastocysts transferred. Thirty-nine pups were born from 375 transferred blastocysts (10%), with 16 pups displaying coat-color chimerism. Both nonmanipulated eight-cell embryos cultured for 48 h (P < 0.05) and chimeric blastocysts (P < 0.001) displayed lower embryo survival after transfer to recipients than noncultured, nonmanipulated blastocysts used as controls. Viability of chimeric blastocysts was also lower than that of nonmanipulated embryos cultured for the same period and transferred to the same recipients (P < 0.001). Although posttransfer survival of chimeric blastocysts was low, the birth of morphologically normal offspring demonstrated that production of chimeras from half embryos was compatible with survival. Improvements in this procedure may be useful for production of tenetically identical chimeras from outbred populations, such as those commonly found in domestic livestock species.     

Birth of mice from vitrified/warmed 2-cell embryos transported at a cold temperature

Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24, 48 and 72 h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24 h, the rates were markedly decreased to low levels after 48 h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young.     

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Cold storage of mouse embryos of different stages of development

Experiments were designed to evaluate the survival rates of preimplantation mouse embryos of different stages of development in cold culture at 4 degrees C. Several developmental stages, from one-cell to the blastocyst, were stored at 4 degrees C from 1 to 8 d. Viability following cold culture was determined by blastocyst expansion during culture in Whitten's medium at 37 degrees C. Blastocyst formation of nonstored controls ranged from 93 to 100% for all developmental stages tested. Only 3% of one-cell embryos survived 1 d and none survived 2 days at 4 degrees C. Survival improved using two-cell embryos, with 84, 69 and 15% forming expanded blastocysts following storage for 1, 2 and 3 d, respectively. Eighty five and 38% of eight-cell embryos formed expanded blastocysts following cold storage for 3 and 4 d, respectively. Survival rates for cold stored morulae and blastocysts remained above 75% for 6 d but decreased significantly to 30 and 36%, respectively, when stored for 8 d. A large percentage of blastocysts were observed to collapse when placed in cold storage from 1 to 8 d but almost all expanded when placed in culture at 37 degrees C. This study showed that one-cell embryos were particularly sensitive to cold storage compared to later-stage mouse embryos. Cold storage survival increased with increasing age of the embryo; morula and blastocyst survival rate was similar.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.     

Differential expression of alternatively spliced transcripts of HLA-G in human preimplantation embryos and inner cell masses

It has been reported that preimplantation human embryos secrete HLA-G, and the levels may be predictive of their ability to implant. However, it is not known which of the membrane-bound (HLA-G 1-4) and soluble (HLA-G 5-6) alternatively spliced forms are present, nor the developmental stage at which they appear. Therefore, we have investigated HLA-G mRNA isoform expression on single embryos at the two-, four-, six-, and eight-cell, morula, and blastocyst stages. The percentage of embryos expressing each HLA-G isoform mRNA increased with developmental stage, but contrary to expectation, HLA-G5 mRNA was not detected in single two- to eight-cell embryos and was only expressed by 20% of morulae and blastocysts. Similarly, soluble HLA-G6 mRNA was not detected until the blastocyst stage and then in only one-third of embryos. In contrast, labeling with MEM G/9 Ab (specific for HLA-G1 and -G5) was observed in 15 of 20 two- to eight-cell embryos and 5 of 5 blastocysts. This disparity between mRNA and protein may be due to HLA-G protein remaining from maternal oocyte stores produced before embryonic genome activation and brings into question the measurement of soluble HLA-G for clinical evaluation of embryo quality. Although HLA-G is expressed in the preimplantation embryo, later it is primarily expressed in the invasive trophoblast of the placenta rather than the fetus. Therefore, we have investigated whether down-regulation of HLA-G first occurs in the inner cell mass (precursor fetal cells) of the blastocyst and, in support of this concept, have shown the absence HLA-G1 and -G5 protein and mRNA.     

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.     

Development of normal mice after microinjection of round spermatids into oocytes stored at room temperature for one day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 hr still retained full developmental potential. In this study, we stored mouse COCs and denuded oocytes (DOs) at room temperature for 24 hr and activated these oocytes with 10 mM SrCl(2) or injected the oocytes with round spermatids. We found that DOs were better than COCs when stored at room temperature for 1 day and more normal oocytes were obtained when COCs were stored in more H-CZB medium at room temperature for 1 day. The rates of normal oocytes were significantly different after preservation with three schemes (90.01%, 55.81%, and 86.70%, P < 0.05). Our results also indicated that oocytes stored at room temperature for 1 day were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei [PN]) after microinjection of round spermatid nuclei, and that the existence of cumulus cells (CCs) during oocyte storage did not significantly influence the early cleavage but had a detrimental effect on later embryo development and full-term development. After fertilization, most embryos developed to two-cell stage after being cultured for 24 hr, and the development rates of four- to eight-cell embryos between two experiments were similar. However, the rates of morula/blastocyst formation were significantly different (47.44% and 26.27%, respectively, P < 0.05). The birth of four healthy pups from stored DOs indicated that the storage of DOs at room temperature for 1 day might become a practical procedure in mammalian reproduction.     

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

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.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

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.     

Full term development of normal mice after transfer of IVF embryos derived from oocytes stored at room temperature for 1 day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 degrees C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 microg/ml 17beta-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p<0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

The effect of supercooling on the developmental capacity of mouse embryos.

The effect of supercooled storage (at subzero temperatures without ice formation) on compacted mouse morulae and early blastocysts was studied. The embryos were equilibrated with one of three storage solutions containing 1, 3, or 6% each of methanol and glycerol and cooled to -2, -5, -10, or -15 degrees C and stored for up to 24 h to assess the effect of subzero storage at different temperatures and concentrations of the permeating cryoprotectants on embryo survival. Early blastocysts showed substantially greater survival than morulae and, in general, survival of embryos of either stage increased with the concentration of cryoprotectant, while the proportion of embryos surviving decreased with decreasing storage temperature and with increased duration of storage.     

Effects of microinjection-related treatments on the subsequent development of in vitro-produced bovine oocytes

The effects of gene injection-related handling on the subsequent development of in vitro-produced bovine oocytes were studied. In Experiment 1, centrifuged oocytes were stored in an injection chamber for 30 min on a warm (+39 degrees C) stage at 18, 22, 26 or 30 h post insemination. In Experiment 2, centrifuged oocytes were stored for 60, 90 or 120 min on a warm stage, while in Experiment 3 they were stored for 60 min on a warm or cool (+22 degrees C) stage. In Experiment 4, the centrifuged zygotes were injected with buffer either into the pronucleus or cytoplasm. Development to morulae and blastocysts at Day 7 was monitored. The results indicate that handling of oocytes either very early (18 h post insemination) or very late (30 h post insemination) significantly reduced development (P<0.05). The duration of the storage or the temperature during storage did not have any significant effect on the development of the embryos. Development (counted from the cleaved ova), however, was significantly lower (P<0.01) in pronucleus-injected than in cytoplasm-injected or control embryos (27.7, 45.5 and 44.0% morulae and blastocysts, respectively). The conclusion of this study is that the main reason for decreased development of pronucleus-injected, in vitro-produced bovine zygotes is the pronucleus-injection itself rather than injection-related handling or the overall damage caused by zygote piercing.