Development of 1-cell embryos from different strains of mice in CZB medium

One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB medium. CZB medium can be used to culture CF1 x B6SJLF1/J 1-cell embryos to the blastocyst stage provided glucose is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range of 5.5 to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65-85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CF1 and DBA/2J (both x B6SJLF1/J) mice, which exhibit a "2-cell block" to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2F1/J and CD1 female mice (both x B6SJLF1/J males), which do not exhibit a "2-cell block" to in vitro development, optimum development to morula and blastocyst stages (95% and 50%) was in CZB medium containing both glutamine and glucose from the start of culture.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (～42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67–73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley-Liss, Inc.     

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

克服昆明小鼠体外受精卵发育阻滞方法的研究

本研究应用CZB和WM培养液进行昆明小鼠体外受精胚胎的发育培养,建立了一个可行的胚胎体外培养的新方法,并通过改变培养液的成分及其含量,对胚胎发育的阻滞机理和突破方法进行了初步的探索。培养于WM中的受精卵发生阻滞,有48％停留于2细胞阶段;而CZB中的胚胎有81％发育为桑椹胚和囊胚。在WM中添加EDTA和谷氨酰胺得到了66％的囊胚;加大WM中乳酸钠和丙酮酸钠的比值未能克服发育的阻滞现象。实验结果表明,EDTA和谷氨酰胺在克服阻滞时具有协同作用。     

Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro

Pig embryos at the 1- or 2-cell stage (before the 'block' to development in vitro) were cultured in 8 different media derived from Krebs'-Ringer-bicarbonate medium. A 2 x 2 x 2 factorial arrangement was used for the treatments, with glucose, glutamine and phosphate being the major effects tested. Embryos were obtained from sows approximately 44-48 h after the observation of oestrus, with the majority being at the 1-cell stage. Embryos from each female were randomly assigned to each treatment. After in-vitro culture, all embryos were scored for the stage of development attained and stained to determine final cell number. Significant effects were evident due to female, glucose, glutamine, a phosphate x glucose interaction and a glutamine x glucose interaction. None of the media components tested was inhibitory to embryo development. The greatest development (45-60% morula or blastocyst) was achieved with glucose and glutamine (both alone and in combination) in the media, demonstrating that an amino acid can serve as the sole energy source for complete preimplantation embryonic development in vitro.     

Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose

The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied. Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate. In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days. Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration. In a second experiment, phosphate levels were varied in the absence of glucose. Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate. After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher. In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate. However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate. These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate. Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro. The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production. The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.     

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.     

Implication of glucose 6 phosphate isomerase (EC 5.3.1.9) activity in blocking segmentation of the mouse ovum at the 2 cell stage in vitro

The mouse embryo, when grown in media containing glucose, synthesizes and accumulates glycogen. In certain strains, glucose could be responsible for the 2 cell block; it can be replaced for a short time by glutamine, but with an increase in embryo degeneration. We have tested the hypothesis according to which the problems of glycogen accumulation and the developmental arrest could be due to a metabolic lock involving glucose 6 phosphate isomerase (EC 5.3.1.9). Fructose is located immediately downstream from this metabolic lock. We have exactly replaced the glucose in Whittingham M16 culture medium by fructose: Medium F.M. With this culture medium, Swiss one cell embryos, which normally block in M16 medium (greater than 90%), do not block any longer at the 2 cell stage (11.6%). They can be cultured for 4 days with high rates of blastocyst formation (53.6%). Embryo degeneration (23.6%) is lower than that observed in CZB (31%), and not different from our control degeneration in vivo. Moreover fructose seems to be adequate all through the culture period as there is no need for further addition of metabolites, to obtain blastocysts, as observed for CZB. This demonstrates that a lock at the Glucose phosphate isomerase level is responsible for glycogen accumulation and the 2 cell block in the mouse embryo in vitro.     

昆明白小鼠1细胞胚胎体外培养系统的研究

研究发现在有或者没有磷酸盐的条件下,葡萄糖均抑制昆明白小鼠ｌ－４细胞期胚胎的体外发 育。在不含葡萄糖和磷酸盐的ＨＥＣＭ－ｌ中,桑椹率为４０．０５％（７４／１６８）,而对照Ｇ－ＨＥＣＭ－１仅为 ８．１４％（７／８６）;不含葡萄糖含有磷酸盐的ＣＺＢ中,桑椹率为６７．１１％（９３／１５２）,而对照ＴＡＬＰ仅 ６· ６７％（６／９０）。用不含葡萄糖而含有１． ０ｍｍｏ１／Ｌ谷氨酸肢和０． １１ｍｍｏｌ／Ｌ ＥＤＴＡ的ＣＺＢ液,与兔输 卵管上皮单层培养细胞（ＲＯＥＣ）协同培养小鼠１细胞胚,７３．３３％（１１０／１５０）胚胎发育至桑椹胚, 但没有观察到囊胚形成、用上述ＣＺＨＲＯＥＣ系统培养小鼠１细胞胚４８小时（３－４细胞）,再移入 ＴＣＭ１９９＋１０％ＦＣＳ＋ＲＯＥＣ系统,有７６．７４％（６７／８６）胚胎发育至桑椹胚,９６／小时后,４０．７０％ （３５／８６）发育至囊胚。     

Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos.     

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.     

A culture system for the development of the preimplantation rat embryo

We describe a system for the culture of 1-cell rat embryos through to the blastocyst stage, using co-culture on specific feeder cell layers and particular defined media. We show that the use of rat uterine epithelial cells as a feeder layer, together with either M16M, CZB or HECM-1 media at 38.5 degrees C can improve the in vitro development of cultured rat embryos. There was considerable variation in the culture conditions, which were optimal for each strain of rat tested. We show that the 4 to 8-cell embryos are viable after reimplantation and that the second 4 to 8-cell block in the rat can be overcome using HECM-1 in a co-culture system, thus enabling the in vitro culture of rat embryos up to the blastocyst stage.     

In vitro development of embryos from Sinclair miniature pigs: a preliminary report

The goal of this project was to identify conditions that result in development from the zygote or the 2-cell stage Sinclair miniature pig embryos to the blastocyst stage. Four media were selected, 2 that have been shown to result in in vitro development in domestic pigs (Hepes buffered Tyrode's medium and Whitten's medium), 1 that is compatible with similar development in the cow (CR-1), and 1 that is compatible with development in the mouse (CZB). One- and two-cell stage embryos from Sinclair miniature pigs were flushed from oviducts in Hepes buffered Tyrode's medium, allocated to 1 of the 4 media and cultured for 120 h. At the end of the culture period, embryos were morphologically scored and nuclei were counted. Morphology scores were lowest for Hepes buffered Tyrode's medium but were not different for Whitten's medium, CZB or CR-1. The highest (P < 0.07) number of nuclei was present in the oocytes cultured in Whitten's medium (21.3), with CR-1 (15.7) and CZB (16.5) not differing significantly. Similar to the morphology scores, Hepes buffered Tyrode's medium resulted in the lowest number nuclei (5.5). In a parallel experiment, domestic pig embryos were cultured in Hepes buffered Tyrode's medium versus Whitten's medium. The domestic pig embryos, while also developing better in Whitten's Medium, developed better in the Hepes buffered Tyrode's medium than did the embryos from Sinclair pigs. Thus, the Sinclair pig embryo develops best if placed in Whitten's Medium.     

Effect of culture media on in vitro development of cloned mouse embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.     

Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro.

Analysis over the first 48 h of development in vitro from the one-cell stage to the early four-cell stage indicated that (i) ethylenediaminetetraacetic acid (EDTA) exerts the major beneficial effect on culture to the blastocyst stage of F1 and MF1 embryos, (ii) glutamine assists development of MF1, but not F1, embryos to the blastocyst stage and probably functions as part of a metabolic response to oxidative damage to mitochondria and (iii) exposure to glucose at some time during early cleavage is essential for full development to blastocysts. None of the culture conditions examined affected significantly the increase in concentration of reactive oxygen species in late two-cell embryos in vitro, although F1 embryos in vitro often had lower peroxide concentrations than MF1 embryos. A decline in oxygen tension from 20 to 50% had no consistent effect on culture to the blastocyst stage or production of reactive oxygen species. Aminooxyacetate, an inhibitor of transaminase activity, prevented non-blocking embryos from developing beyond G2 of the second cell cycle. It is concluded that the chelation of transitional metals provides the most effective method of overcoming the block to development in vitro.     

Oviductal fluid and growth factors failed to enhance development of porcine embryos

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).     

The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro.

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.