Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.     

Influence of insulin-like growth factor-I on cytoplasmic maturation of horse oocytes in vitro and organization of the first cell cycle following nuclear transfer and parthenogenesis

In vitro maturation of horse oocytes cultured with or without IGF-I supplementation and their first cell cycle organization were studied in reconstructed horse oocytes made by somatic cell nuclear transfer versus intact oocytes stimulated parthenogenetically. The rates of metaphase II oocytes (47% and 45%) and of reconstructed oocytes that developed to the two-cell (27% and 25%) and blastocyst stages (11% and 3%) were not different between the media, with or without IGF-I, respectively. However, significantly more parthenogenetic embryos exhibited two-cell development with IGF-I (P < 0.05). The results also demonstrated that the first cell cycle organization in the reconstructed oocytes involved two different ways of nuclear remodeling. The donor nucleus in the Type I embryo showed normal nuclear remodeling that resulted in normal embryonic development. In the Type II embryos, however, the donor nucleus formed a polyploid nucleus or the embryo fragmented. Addition of IGF-I to the maturation medium significantly increased the rate of normal Type I embryonic development from the reconstructed oocytes (45% vs. 28%, P < 0.05). Maturation-promoting factor (MPF; including cdc2 and cyclin B) and mitogen-activated protein kinase (MAPK; including ERK1 and ERK2) were present at the beginning of culture, just after the oocytes had been harvested from the ovaries. The quantities of cyclin B remained stable no matter how long a period of in vitro culture the oocytes underwent, whereas cdc2 showed a tendency to accumulate in the oocytes toward the end of the 30-h culture period. Addition of IGF-I to the medium may induce a bigger accumulation of MAPK in the cytoplasm of the horse oocyte, especially in the ERK2 component, which might, in turn, increase the chance of the reconstructed oocyte undergoing nuclear remodeling to form a Type I embryo following nuclear transfer.     

Development of hamster one-cell embryos recovered under different conditions to the blastocyst stage

One-cell stage embryos, recovered from superovulated golden hamsters (8 to 12 weeks of age) 12 hours after egg activation, were cultured in HECM-1 medium at 37 degrees C and 5% CO(2) in air. The culture conditions investigated were the time and temperature required for embro recovery, the pH shift of the washing medium, and the oxygen concentration of the gas phase during and after embryo recovery. Each condition was assessed by the developmental efficiency of the embryo as determined by morphological criteria. As the time required for embryo recovery was reduced, the developmental rates of the embryos were improved: 2.3% (3 128 ) 26.9% (35 130 ) at 5 and 3 minutes, respectively, as determined by the number of embryos developed to the blastocyst stage. No blastocysts were obtained when more than 10 minutes were required for embryo recovery. As the oxygen concentration was reduced from 40 to 20% or to 5%, rather high developmental rates were obtained even when the time required for embryo recovery was prolonged: 6.9% (9 130 ) and 21.7% (28 129 ) of the embryos developed to the blastocyst stage when they were recovered under 5% oxygen within 10 and 5 minutes, respectively. Neither the temperature during embryo recovery (37 degrees C and 25 degrees C) nor the pH shift (pH 7.22 to 7.52) of the washing medium used in embryo recovery procedures influenced the development of the embryos. These findings suggest that the developmental block in hamster embryos may involve oxidative stress, which may result from exposure to high oxygen concentration and light during the manipulation of oocytes and embryos.     

Cryopreservation of mouse embryos affects later embryonic development possibly through reduced expression of the glucose transporter GLUT1

In order to study the effects of cryopreservation on later embryonic development, two-cell mouse embryos were frozen, thawed, and then allowed to develop into blastocysts. The percentage of cryopreserved embryos which developed into blastocysts was significantly lower than that of fresh two-cell embryos. The amount of glucose incorporation in terms of ³H-2-deoxyglucose uptake in blastocysts developed in vivo, and in vitro from fresh or frozen-thawed two-cell embryos, was 473 ± 108, 105 ± 75, and 43.0 ± 28.3 fmol per embryo per hour, respectively. Quantification of glucose transporter GLUT1 in these embryos by Western blotting was reflective of the degree of glucose incorporation. The implantation rate of blastocysts developed in vitro from frozen-thawed two-cell embryos (22.0%) was significantly lower than that developed in vivo (41.1%). These data suggest that cryopreservation may have later consequences on embryonic development through a mechanism that involves altered GLUT1 expression. Mol. Reprod. Dev. 48:496–500, 1997.© 1997 Wiley-Liss, Inc.     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronous division of mouse two-cell embryos with nocodazole in vitro.

Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12.5-14.5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.     

Nuclear transfer of mouse follicular epithelial cells pretreated with spermine, protamine, or putrescine

The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 microg/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. J. Exp. Zool. 289:208-212, 2001.     

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.     

Similar effects of osmolarity, glucose, and phosphate on cleavage past the 2-cell stage in mouse embryos from outbred and F1 hybrid females

One-cell-stage embryos derived from most random-bred and inbred female mice exhibit an in vitro developmental block at the two-cell stage in classical embryo culture media. However, embryos derived from many F1 hybrids develop easily past the two-cell stage under the same conditions. This has given rise to the commonly accepted idea that there exist blocking and nonblocking types of female mice, with only the former being prone to a two-cell block. Recently, culture media have been improved to the point that even embryos prone to the two-cell block will develop past the block in vitro, making it possible to study its etiology. Here, we show that either increased osmolarity or increased glucose/phosphate levels induced the expected two-cell block in random-bred CF1 embryos and the two-cell block at increased osmolarities could be rescued by the organic osmolyte glycine. Surprisingly, one-cell embryos from B6D2F1 (BDF1) F1 hybrid females, considered to be nonblocking, also became blocked at the two-cell stage when osmolarity or glucose/phosphate levels were increased. They were also similarly rescued by glycine from the osmolarity-induced block. The most evident difference was that the purportedly nonblocking embryos became blocked at a higher threshold of osmolarity or glucose/phosphate level than those considered prone to this developmental block. Thus, both blocking and nonblocking embryos actually exhibit a similar two-cell block to development.     

Possible function of 17 beta-hydroxysteroid dehydrogenase in preimplantation hamster embryos

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) catalyzes the interconversion of estradiol-17 beta (E2) and estrone (E1). The present study is designed to investigate the following: (1) the developmental stage of hamster embryos at which 17 beta-HSD activity first becomes detectable, and (2) the E1----E2 and E2----E1 conversion rate in the preimplantation hamster embryo. Embryos obtained from superovulated hamsters on days 1-4 were cultured in medium containing 107 ng [3H]E1 or -E2/ml and the respective conversion product, [3H]E2 or -E1, was isolated and assayed. The results show that (1) E1----E2 conversion was active in all embryos at the rate of 0.57, 0.66, 0.54 and 0.48 fmol/embryo/hr for day 1 (one-cell), 2 (two-cell), 3 (eight-cell) and 4 (blastocyst), respectively, and (2) E2----E1 conversion was not detectable in hamster embryos. In long-term blastocyst culture, E2----E1 conversion becomes detectable at 25 hours and increases sharply from 25 to 47 hours. These results suggest that (1) 17 beta-HSD may function mainly to convert E1 into E2 in preimplantation hamster embryos and (2) E2----E1 conversion may become active only during and after implantation.     

Effects of the removal of cytoplasm on the development of early cloned bovine embryos

Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

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.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Influence of genetic background and media components on the development of mouse embryos in vitro

One-cell embryos from some inbred and random-bred mice, but not those derived from certain F1 hybrids, suffer from a block during in vitro development known as the two-cell block. This two-cell block can be overcome by removing glucose or inorganic phosphate from the culture system or by altering the ratio of other medium components such as sodium, potassium, or bicarbonate. This issue is made more complex by the fact that the rate of development is different for each strain of mouse and this rate of development is invariably slowed under in vitro culture conditions. This study investigated the role of glucose and inorganic phosphate, individually or in combination, in relation to the two-cell block, and rate of development in vitro of two random-bred strains (CF-1 and CD-1) and an F2 hybrid derived from a nonblocking F1 hybrid cross (C57B1/6NCr × C3H/HeNCr). Results were compared with in vivo data for each strain, and between media. There was a significant difference in the rate of preimplantation development in vivo of the three strains chosen, which was mirrored in vitro, regardless of the medium. The two random-bred strains suffered from a glucose-related two-cell block which was primarily mediated by inorganic phosphate. Inorganic phosphate was detrimental to embryo development regardless of strain or the presence of glucose. Although glucose, in the absence of inorganic phosphate, resulted in some blocking in development in the inbred strains initially, its presence in media was associated with increased rates of development at later stages in embryos that did not block. Glucose, but not inorganic phosphate, was beneficial but not essential to the development of the F2 embryos. The results of this study demonstrated that mouse embryos from different strains have differential rates of development in vivo and in vitro, and different sensitivities to glucose and inorganic phosphate. The two-cell block was primarily induced in the combined presence of glucose and inorganic phosphate. Glucose was beneficial in the absence of inorganic phosphate, and inorganic phosphate was detrimental to the rate of in vitro development. © 1996 Wiley-Liss Inc.     

Energy substrate requirements for in vitro development of early cleavage-stage bovine embryos

Energy substrate preferences of bovine cleavage-stage embryos produced by in vitro maturation and in vitro fertilization were examined in a chemically-defined (protein-free) culture medium modified hamster embryo culture medium-3, (mHECM3). Few inseminated ova cleaved without energy substrates. Glucose and/or glutamine could not support embryo development, but lactate alone was effective (37% 5–8-cells), equivalent to complex medium TCM-199 (44%). Addition of 11 selected amino acids to lactate increased embryo cleavages, although this treatment was not significantly different from pyruvate alone. Addition of glucose to lactate or to pyruvate depressed development. Lactate + amino acids was significantly better than TCM-199 (54% and 26% ≤8-cells, respectively). Blastocyst development was evaluated after transferring ≤8-cell embryos into a complex medium (TCM-199) containing serum. Cleavage-stage embryos produced with pyruvate alone or with lactate + amino acids yielded the highest proportions of blastocysts (36% and 41%, respectively, of inseminated ova). Between 33–63% of blastocysts derived from embryos that were initially developed in mHECM-3 supplemented with various substrates escaped from their zonae (hatched) depending on the treatment, but none of the embryos from the pyruvate + glucose combination hatched. This study shows that optimal energy substrates for bovine cleavage-stage embryo development can be determined using a chemically-defined culture medium, that a simple medium with selected substrates can support early development as well as or better than a complex medium, that a two-step culture system can be used to evaluate blastocyst development from these cleavage-stage embryos, and that timing and hatching of embryos may provide additional information about discriminating between the suitabilities of different substrates for early embryo development. © 1996 Wiley-Liss, Inc.     

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.