An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.     

Cleavage pattern and survivin expression in porcine embryos by somatic cell nuclear transfer

Mammalian embryos produced in vitro show a high rate of early developmental failure. Numerous somatic cell nuclear transfer (SCNT) embryos undergo arrest and show abnormal gene expression in the early developmental stages. The purpose of this study was to analyze porcine SCNT embryo development and investigate the cause of porcine SCNT embryo arrest. The temporal cleavage pattern of porcine SCNT embryos was analyzed first, and the blastocyst origin at early developmental stage was identified. To investigate markers of arrest in the cleavage patterns of preimplantation SCNT embryos, the expression of survivin—the smallest member of the inhibitor of apoptosis (IAP) gene family, which suppresses apoptosis and regulates cell division—was compared between embryos showing normal cleavage and arrested embryos.A total of 511 SCNT embryos were used for cleavage pattern analysis. Twenty-four hours post activation (hpa), embryos were classified into five groups based on the cleavage stage as follows; 1-cell, 2-cell, 4-cell, 8-cell and fragmentation (frag). In addition, 48 hpa embryos were more strictly classified into 15 groups based on the cleavage stage of 24 hpa; 1-1 cell (24 hpa-48 hpa), 1-2 cell, 1-4 cell, 1-8 cell, 1 cell-frag, 2-2 cell, 2-4 cell, 2-8 cell, 2 cell-frag, 4-4 cell, 4-8 cell, 4 cell-frag, 8-8 cell, 8 cell-frag, and frag-frag. These groups were cultured until 7 d post activation, and were evaluated for blastocyst formation. At 24 hpa, the proportion of 2-cell stage was significantly higher (44.5%) than those in the other cleavage stages (1-cell: 13.4%; 4-cell: 17.9%; 8-cell: 10.3%; and frag: 13.9%). At 48 hpa, the proportion of embryos in the 2-4 cell stage was significantly higher (32.4%) than those in the other cleavage stages (2-8 cell: 8.2%; 4-8 cell: 12.1%; and frag-frag: 13.9%). Some embryos arrested at 48 hpa (1-1 cell: 5.8%; 2-2 cell: 2.8%; 4-4 cell: 3.8%; 8-8 cell: 6.5%; and total arrested embryos: 18.9%). Blastocyst formation rates were higher in 2-4 cell cleavage group (20.2%) than in other groups. SCNT embryos in 2-4 cell stage showed stable developmental competence. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-cell, 4-cell stage SCNT embryos were significantly higher than those of arrested embryos. Survivin protein expression showed a similar pattern to that of survivin mRNA. Normally cleaving embryos showed higher survivin protein expression levels than arrested embryos. These observations suggested that 2-4 cell cleaving embryos at 48 hpa have high developmental competence, and that embryonic arrest, which may be influenced by survivin expression in porcine SCNT embryos.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

Co-culture of two-cell rat embryos on cell monolayers

Summary Monolayers of 2 different populations of uterine cells and of fetal fibroblasts were evaluated for the support of rat embryo development in vitro. Compared to controls, cultures performed in Earle's buffered saline solution (EBSS) alone, the cleavage rate of 2-cell embryos to the 4-cell stage was significantly increased when the embryos were cocultured for 24 h with mixed uterine stromal and myometrial cells (70.7 vs. 56.0%;P<0.01). Coculture of 2-cell embryos with either uterine epithelial-stromal or stromal-myometrial cells in medium TC 199 (M199) for 24 h significantly increased the cleavage rate to the 4-cell stage compared to controls in the same medium (respectively, 78.3 and 77.6 vs. 49.9%;P<0.01). The development was not improved when fibroblasts were used as feeder cells. After 48 h, the proportion of 4-cell embryos showing cellular fragmentation was significantly decreased in the presence of either epithelial-stromal or stroma-myometrial cells in M199 compared to controls (respectively, 18.4 and 20.0 vs. 43.8%;P<0.01). Coculture in EBSS or with fibroblasts failed to prevent embryo degeneration. In one coculture with stromal-epithelial cells in M199, 6/11 embryos proceeded beyond the 4-cell stage, two of them reaching the 8-cell stage. No embryo developed beyond that stage in our study. Although considerable efforts remain necessary to achieve further growth, these results suggest that coculture offers promise as a means of supporting the in vitro development of rat embryos.     

Blastocysts produced by nuclear transfer between chicken blastodermal cells and rabbit oocytes

Interspecies nuclear transfer (INT) has been used as an invaluable tool for studying nucleus-cytoplasm interactions; and it may also be a method for rescuing endangered species whose oocytes are difficult to obtain. In the present study, we investigated interaction of the chicken genome with the rabbit oocyte cytoplasm. When chicken blastodermal cells were transferred into the perivitelline space of rabbit oocytes, 79.3% of the couplets were fused and 9.7% of the fused embryos developed to the blastocyst stage. Both M199 and SOF medium were used for culturing chicken-rabbit cloned embryos; embryo development was arrested at the 8-cell stage obtained in SOF medium, while the rates of morulae and blastocysts were 12.1 and 9.7%, respectively, in M199 medium. Polymerase chain reaction (PCR) amplification of nuclear DNA and karyotype analyses confirmed that genetic material of morulae and blastocysts was derived from the chicken donor cells. Analysis mitochondrial constitution of the chicken-rabbit cloned embryos found that mitochondria, from both donor cells and enucleated oocytes, co-existed. Our results suggest that: (1) chicken genome can coordinate with rabbit oocyte cytoplasm in early embryo development; (2) there may be an 8- to 16-cell stage block for the chicken-rabbit cloned embryos when cultured in vitro; (3) mitochondrial DNA from the chicken donor cells was not eliminated until the blastocyst stage in the chicken-rabbit cloned embryos; (4) factors existing in ooplasm for somatic nucleus reprogramming may be highly conservative.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

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.     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

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.     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

Effects of superovulation, embryo recovery, culture system and embryo transfer on development of rabbit embryos in vivo and in vitro

Uninterrupted development of rabbit embryos in vivo was studied in 7 superovulated and 7 normally ovulating (GnRH-treated) does, while another 7 does were superovulated and 1-cell embryos were collected from them at 19 h after LH to compare development in vivo and in vitro. Embryos from the last group were either cultured in the presence or absence of rabbit oviduct epithelial cells for 65 h in Medium 199, or were immediately transferred to recipients. At 84 h after LH or GnRH, blastomere number, embryo volume and stage of development were assessed for all embryos. Intrazonal embryo volumes were significantly reduced in embryos recovered from superovulated donors. Superovulation also had a negative effect on embryo cell numbers. However, this reduction was more severe in embryos remaining in vivo in superovulated donors until 84 h after LH than it was in embryos transferred to nonsuperovulated recipients at the 1-cell stage (19 h after LH). The embryo recovery procedure apparently caused little harm to the embryos, except that the mucin layer on flushed and immediately transferred embryos was significantly thinner than that of embryos residing continuously in vivo. Co-culture with rabbit oviduct epithelial cells resulted in improved development in vitro, but this development was still significantly retarded compared with embryos developing in vivo.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF) + sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.     

Analysis of the inhibitory effect of inorganic phosphate on development of four-cell hamster embryos in vitro

Hamster 4-cell stage embryos were cultured in a protein-free, glucose-free medium to study the nature of developmental inhibition by inorganic phosphate (Pi). In the absence of Pi, between 40 and 55% of embryos were able to develop to the blastocyst stage but addition of Pi to the medium reduced this proportion to 5-20%. The inhibition did not appear to be due to contamination of the Pi salt with heavy metals because EDTA did not relieve the effect. Inhibition by Pi showed no dose-response relationship over the range tested (1-350 microM). In contrast, another divalent anion (sulphate) produced no inhibition of 4-cell embryo development at concentrations as high as 5.6 mM. Embryos were less sensitive to inhibition by Pi after the third cleavage division had occurred, and development of mid or late 8-cell embryos was unaffected by Pi. After exposure to Pi for 1 h, embryos could recover and continue development but longer exposure was detrimental to subsequent development. These results indicate that the inhibitory effect is specific to phosphate ions, is not due to contaminants in the Pi salt, is evoked by very low concentrations of Pi, is stage-specific, and is reversible following brief exposure of embryos to Pi. These effects may be artifacts of the culture milieu, or they may reflect some unknown characteristic of the early cleavage stage hamster embryo.     

Expression of a glucose-regulated cell surface protein in early mouse embryos

A 100,000-Da glucose-regulated surface protein (100K-GRP) has previously been isolated from the cell surface and culture medium of human fibroblasts. A rabbit antiserum directed against this protein reacts with the cell surface of both human and murine cultured cells and with a broad spectrum of mammalian tissues. It is shown, via indirect immunofluorescence, that this protein is also present on cells of the developing mouse embryo and can be detected as early as the 4-cell stage. The 8-cell embryo and morula show positive surface labeling; the inner cell masses of both the pre- and postimplantation blastocysts are also positive but the trophectoderm is not. At the 6-day egg cylinder stage, the embryonic and extra-embryonic ectoderm label intensely with the antiserum and visceral endoderm shows faint labeling. No labeling can be detected on parietal endoderm or on the trophoblastic giant cells invading the uterine decidua. However, the internal cells of the ectoplacental cone exhibit bright fluorescence. The same pattern is observed on 7- to 8.5-day embryos, except that at this stage no label is associated with the visceral endoderm. In addition, mesodermal cells emerging from the primitive streak are also labeled.