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.     

Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: An autoradiographic study

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-³H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-³H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-³H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.     

小鼠胚胎体外发育培养基中氨基酸含量变化

通过检测哺乳动物早期胚胎体外发育过程中可以消耗或生成某些氨基酸的含量,可以了解胚胎的发育潜能。利用反相高效液相色谱法(RP-HPLC)检测KSOMaa培养基中17种氨基酸含量的变化,了解昆明小白鼠(Mus musculus)植入前胚胎体外培养过程中氨基酸含量的变化,旨在寻找一种能有效支持昆明小鼠胚胎体外发育的培养基氨基酸组成,优化小鼠胚胎体外培养体系。将180枚原核胚分为9组,体外培养至囊胚,分别于胚胎发育不同时期取样做高效液相色谱分析。这些氨基酸在胚胎发育不同时期的培养基中含量变化可分为5种类型:①在2细胞期增加但在4细胞期、8～16细胞期减少,囊胚期又增加的氨基酸(甘氨酸、亮氨酸、苏氨酸、缬氨酸、苯丙氨酸、酪氨酸);②在胚胎发育各个时期均下降(谷氨酸、甲硫氨酸、精氨酸、组氨酸);③在胚胎发育各个时期均增加(丝氨酸、赖氨酸、丙氨酸);④2细胞期含量减少而在其他时期持续增加(天冬氨酸、脯氨酸、色氨酸);⑤囊胚期减少,其他时期都有增加(异亮氨酸)。     

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

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

In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.     

Involvement of early embryonic miR‐409‐3p in the establishment of anxiety levels in female mice

Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm‐derived miRNAs appear to influence early embryos and subsequent development. For example, stress‐associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR‐409‐3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR‐409‐3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR‐409‐3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR‐409‐3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR‐409‐3p activity, possibly as a consequence of stress‐induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.     

Distribution of 5-chloromethylfluorescein diacetate staining during meiotic maturation and fertilization in vitro of mouse oocytes

The aim of this confocal microscopy study was to determine whether the pattern of CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) staining changes during meiotic maturation and fertilization in vitro of mouse oocytes. At different times during meiotic maturation and fertilization, oocytes, zygotes and two-cell embryos were stained with CMFDA to demonstrate intracellular glutathione S-transferase activity. After washing in CMFDA-free medium, most oocytes, zygotes and embryos were stained with dihydroethidium (HE) to visualize DNA structures. Meiotic maturation and fertilization in vitro of mouse oocytes were associated with changes in the pattern of intracellular CMFDA staining. In particular, accumulations of CMFDA-positive membranes were observed around the nucleus of germinal vesicle (GV) oocytes, overlaying the sperm nucleus as well as overlaying the first mitotic spindle if this approached the plasma membrane. Staining of oocytes and zygotes with the probes 3,3'-dihexyloxacarbocyanine iodine [DiOC6(3)], which stains all the intracellular membranes, and rhodamine 123, which stains active mitochondria, demonstrated that the intracellular structures evidenced by CMFDA staining did not correspond to accumulations of mitochondria. Exposure of oocytes and zygotes to the microtubule-disrupting agent nocodazole or the actin-depolymerizing drug cytochalasin D revealed an autonomous microfilament-dependent transport and relocation of CMFDA-positive membranes during meiotic maturation and fertilization. Such a transport of CMFDA-positive membranes may be envisaged as a protective shield built to prevent damage to DNA from endogenous and exogenous mutagen metabolites.     

Developmental capacity of rat embryos produced by in vivo or in vitro fertilization

This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.     

A comparison of mouse and rabbit embryos for the production of transgenic animals by pronuclear microinjection

Procedures for the production of transgenic animals have low overall efficiency. To evaluate factors responsible for low efficiency, zygotes of two species, varying intensities of microscope light, different qualities of injection pipettes, and six different genes were tested for their influence on the efficiency of pronuclear gene injection for the production of transgenic rabbits and mice. Rabbit zygotes were less sensitive to mechanical manipulation during injection than mouse zygotes. Exposing zygotes to a microscope light intensity of 5550 lx significantly reduced their cleavage rate, while a lower intensity (2280 lx) did not. Using pipettes with a filament for pronuclear gene injection of mouse zygotes resulted in a higher cleavage rate of zygotes after injection than when pipettes were used without filament (30.3 vs 20.6%). Implantation rates varied between 2.9% (HB72CAT) and 23.1% (ts 58-2) depending on the gene used. No transgenic animals were obtained after injection of uteroglobin-CAT-hybrid genes (B2B3UGCAT, HB72CAT), while all other genes used (UG 11.8, UGTAg, RSV lacZ, ts 58-2) resulted in transgenic embryos, fetuses, and newborn animals.     

Factors influencing chromosome condensation and development of cloned rat embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.     

Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro

Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.     

Estimates of mouse oviductal fluid tonicity based on osmotic responses of embryos

Zygotes and early cleavage-stage embryos are very sensitive to increased osmolality in vitro, although the tonicity of their in vivo environment, oviductal fluid, is unknown. A preference for low osmolality in vitro might imply similar conditions in vivo or be specific to culture. Previous electron probe x-ray microanalysis measurements of total ion content predicted oviductal fluid osmolalities of 310-360 mOs/kg, higher than osmolalities tolerated by mouse zygotes in vitro. However, such indirect estimates may not reflect the tonicity experienced by embryos. We have now used embryos themselves as osmosensors to determine the tonicity of mouse oviductal fluid. In one method, we measured the mean volume of zygotes in undiluted oviductal fluid and compared this to the mean volumes measured for zygotes in media spanning a range of osmolalities. The osmolality corresponding to the measured mean volume in oviductal fluid was taken to be isotonic. In another, independent method, the sizes of zygotes and two-cell embryos were measured as a function of time beginning immediately after removal from oviducts. The osmolality in which the embryos neither swelled nor shrank was taken to be isotonic. Both methods yielded approximately the same range for the tonicity of oviductal fluid: around 290-300 mOs/kg.     

Production and breeding of transgenic cattle using in vitro embryo production technology

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.     

Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote.     

Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos

The nuclei from four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos. It was found that such embryos not only developed to the blastocyst stage in vitro (72% and 35%), but also developed to full term (22% and 8%) after transfer to recipient mice. However, development of embryos which contained nuclei from the inner cell mass was not observed. Since the development of enucleated zygotes which contain advanced nuclei is limited (the present study; McGrath and Solter: Science, 226:1317-1319, '84; Robl, Gilligan, Critser, and First: Biol. Reprod., 34:733-739, '86), it appears that cytoplasmic factors are important for the development of nuclei from advanced cells.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

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.     

C-type virus-lymphocyte interactions in developing mouse thymus.

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.     

Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption

Pyruvate has been considered the sole substrate that can support development of the mouse zygote to the two-cell stage, with lactate able to support development from the two-cell stage. This study has determined for the first time that mitochondrial reducing equivalent shuttles regulate metabolism in the early embryo. Activity of the malate-aspartate shuttle was found to be essential for the metabolism of lactate in the two-cell embryo. Furthermore, the inability of the mouse zygote to use lactate as an energy source was a result of a lack of malate-aspartate shuttle activity. The mRNA for the four enzymes for shuttle activity were detected at all stages of development. It was determined that aspartate was a rate-limiting factor in the activity of the malate-aspartate shuttle in mouse zygotes probably due to the high K(m) of the cytoplasmic aspartate aminotransferase. Addition of high concentrations of exogenous aspartate to the culture medium enabled mouse zygotes to utilize lactate in the absence of pyruvate and develop normally to the blastocyst stage as well as produce normal viable offspring. This study determined that the malate-aspartate shuttle is a key regulator of embryo metabolism and therefore viability and is the first report that mouse zygotes can develop normally to term in the absence of pyruvate.