Hypoxia during mammalian preimplantation development: Extreme circumstance vs. typical environment

The given paper summarizes the data on the early mammalian embryo development in culture media containing low oxygen concentration. Experimental results on in vitro modeling the hypoxia for preimplantation development are reviewed. Hypoxic conditions were shown to be available in the female reproductive tract of different mammalian species. The estimation of the embryo developing in vitro exhibits that lower oxygen level in culture media improves embryonic quality.     

Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.     

Relationship between stage of development and sex of bovine IVM-IVF embryos cultured in vitro versus in the sheep oviduct

We have confirmed more rapid development of male compared with female in vitro-cultured bovine embryos during the first 7 d after in vitro fertilization. The male-to-female sex ratio of expanded blastocysts after 10 d of in vitro culture was 1.37:1.00, which was significantly different from the expected 1:1 ratio, but no deviation from a 1:1 ratio was observed for male and female expanded blastocysts derived from culture of bovine embryos in the sheep oviduct (1.11:1.00). When embryos that developed only to the morula stage were analyzed for sex, a greater number of female than male bovine embryos was observed from in vitro culture but not after culture in the sheep oviduct. Possible causes of these sex-related differences in development of cultured bovine embryos are discussed.     

Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo

We describe a fluorimetric method for measuring the level of H2O2 in individual mouse oocytes and early embryos. Levels of H2O2 are low but detectable in unfertilized oocytes recovered freshly from the female reproductive tract. The levels in early cleaving embryos (1-cell to 8-cell stages) immediately after recovery from the female tract seem to be slightly higher the later the stage examined. However, when embryos are cultured in vitro from the 1-cell or early 2-cell stage, H2O2 levels rise when the embryos reach the mid-2-cell stage and remain elevated until they enter the early 4-cell stage. No equivalent elevation of H2O2 is seen during the transition from 1-cell to 2-cell or from 4-cell to 8-cell stages. Embryos that are able to develop successfully in vitro, as well as those that show a developmental block at the 2-cell stage on culture in vitro, both show this rise in H2O2 levels after in vitro culture. The relationship between the rise in H2O2 and the '2-cell block' to development is discussed.     

Development of porcine embryos in vivo and in vitro; evidence for embryo 'cross talk' in vitro

The in vitro development of zygotes of domestic species to the blastocyst stage is facilitated by culture in groups, suggesting a role for autocrine/paracrine factors. A novel method was used to investigate the potential role of such factors using in-vitro-produced and in-vivo-derived porcine embryos. The development of individual zygotes to the blastocyst stage was optimal when they were cultured 81-160 mum apart. As the distance between the embryos was increased, blastocyst rates declined significantly, reaching zero beyond 640 mum. Blastocyst volume and cell number (both inner cell mass and trophectoderm) were also increased when the distance apart was between 81 and 160 mum. Culturing embryos in groups at different stages of development suggested that group culture confers a greater advantage to development after the activation of the genome. Group culture of in-vivo-derived embryos showed a weak distance effect. The results suggest a role for as yet unknown diffusible paracrine/autocrine factors released by early porcine embryos in promoting the growth of neighbouring embryos in vitro. This advantage is observed to a lesser extent by in-vivo-derived zygotes which are likely to have been better conditioned for development in vitro by being conceived in the female reproductive tract.     

Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells

Human "spare" embryos, judged unsuitable for freezing because of their poor quality, were cocultured for 5 days on a "Vero" cell layer. These epithelial cells were selected because kidney and genital tract have a common embryologic origin and "Vero" cells are a safe and highly controlled cellular support used for vaccine production. In the control group, the embryos were cultured in culture medium alone (B2 + 15% serum). At the end of the culture, the number of blastocysts was significantly higher in the coculture group: 61% vs. 3%. Moreover, at least half of the blastocysts were expanding and hatching (13/25), with a chronologically normal development. These observations suggest that (1) the coculture system improves human embryonic development; (2) it can rescue early degenerating embryos; (3) beneficial effects of coculture are not strictly genital-tract specific, but rather epithelium dependent. This coculture system could be used for in vitro fertilization to prolong in vitro culture and thus make it possible to transfer embryos at a more appropriate time, to eliminate early-blocked eggs, and to freeze embryos at the blastocyst stage, when freezing procedures are most successful.     

In vitro production of embryos in swine.

In recent years, progress has been achieved in the production of pig embryos through IVM and IVF techniques. Cytoplasmic maturation of oocytes has been improved by modifications to IVM procedures. However, the historical problem of polyspermic penetration still remains a major issue to be solved. Recent studies indicate that the type of IVF medium and certain modifications to that medium can reduce polyspermy. Efforts should be directed to increase the developmental competence and quality of embryos. At present, many embryo culture (EC) media are available that can overcome the historical 4-cell block and support development of early in vivo derived embryos to the blastocyst stage. In contrast, blastocyst development of in vitro produced embryos in these culture media varies significantly. Furthermore, morphology and cell numbers in in vitro produced blastocysts are inferior to their in vivo counterparts. However, several modifications to EC techniques have improved embryo quality and developmental competence. Testing embryo viability through surgical transfer to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although reliable in vitro systems are available for the generation of pig embryos, the problem of polyspermy and poor embryo development hamper their large-scale implementation. Further research efforts should be directed to improve oocyte/embryo quality and the methods to minimize polyspermy through development of novel IVM, IVF, and EC techniques.     

Two-phase chemically defined culture system for preimplantation rat embryos

A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos. Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains. Here we report the use of commonly available, chemically defined culture media to overcome these limitations. In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG). This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage. Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers.     

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.     

Culture of ovine embryos in the absence of bovine serum albumin

Bovine serum albumin (BSA), a relatively impure protein, is routinely used as a component of embryo culture media. Since media containing BSA are chemically undefined, it would be desirable to replace BSA with substitutes of similar activity which are either chemically better defined and/or better standardized than BSA. Two commercial products, Ultroser((R)) G (USG) and Solcoseryl((R)) (SOL), were evaluated as replacements for BSA in culture with respect to the development of ovine embryos in vitro. A total of 126 late 8-cell and early 16-cell embryos were distributed among modified Brinster's medium for ovum culture (BMOC-2) containing either 1.5% BSA, 2.0% USG or 2.0% SOL. All three culture media supported development of ovine embryos. Results indicate that 8- and 16-cell embryos will develop into blastocysts in a BSA-free medium containing either USG or SOL. A higher number of embryos developed into blastocysts in media containing BSA than in media containing USG or SOL, and more blastocysts hatched in media containing BSA. Although the overall degree of embryonic development was more advanced in BSA-supplemented media, the concentrations of USG and SOL that were used in this study may not have been optimal for ovine embryo culture.     

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.     

Lower total cell numbers in mouse preimplantation embryos cultured in human assisted reproductive technique (ART) media are not induced by apoptosis

A common feature of assisted reproductive techniques such as IVF or intracytoplasmic sperm injection is the IVC of oocytes or preimplantation embryos in artificial culture media. The IVC conditions are selected to mimic the environment of the female genital tract. We have shown that murine preimplantation embryos respond to different culture media with changes in developmental rates, cellular lineage composition, and gene expression patterns. In this study, we explored whether apoptosis is responsible for cell loss in mouse preimplantation embryos after exposure to different human culture media. We examined total embryonic cell count as well as the pattern of protein expression for caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and the executioner caspase-3 via immunohistochemical staining. Total cell counts decline in embryos cultured either in innovative sequential medium 1 and Blast Assist (Origio) or human tubal fluid and MultiBlast (Irvine Scientific) when compared to KSOM(aa). Few cells were caspase-9 and -3 positive in all experimental groups. Staining for caspase-8 was almost undetectable. We conclude that embryonic cell loss is not associated with higher rates of intrinsic apoptotic cell loss. Our results suggest that the culture medium–dependent decline in total cell count and the developmental restriction in embryos cultured in innovative sequential medium 1/Blast Assist and human tubal fluid/MultiBlast are related to processes affecting cell proliferation.     

Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.     

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.     

Energy and protein sources for development of pig embryos cultured beyond hatching in vitro

The effects of supplementation of synthetic culture media with different energy and protein sources on in vitro development of pig embryos beyond the 4–8-cell stage have been explored.Minimal Essential Medium (MEM) supplemented with glucose (1 mg/ml) proved superior to Krebs-Ringer Bicarbonate (KRB) supplemented with glucose (1 mg/ml) in its capacity to support embryonic development to the expanded blastocyst stage (P < 0.05). Inclusion of pyruvate (0.25 mM) or lactate (25 mM) in either MEM or KRB based media inhibited embryonic development. As pyruvate and lactate are important and readily utilizable energy sources for development of most other mammalian embryos in vitro, it is suggested that the observed inhibitory effects of these substrates reflect comparatively lower critical ranges of concentrations of pyruvate and lactate for optimum development of pig embryos in vitro.As a supplementary protein source to MEM, heat inactivated (HI) human serum (10% υ/υ) was superior (P < 0.05) to HI-pig serum (10% υ/υ), HI-foetal calf serum (10% υ/υ) or bovine serum albumin (5 mg/ml). The proportion of 4–8-cell pig embryos which developed beyond hatching in MEM supplemented with HI-human serum (> 56%) was higher than any other reported for in vitro culture of pig embryos through the same developmental period and this medium is recommended for future studies on in vitro development of pig embryos from the four-cell through the hatched/expanded blastocyst stages.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been considered as laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.     

Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts

In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.     

The extended survival of tw5/tw5 mouse embryo cells in vitro

The tw5 haplotype is a recessive mutation which is lethal when homozygous in mouse embryos following implantation. This series of studies was undertaken to determine the effect of the tw5/tw5 genotype on embryos developing in vitro. Blastocyst embryos from +/tw5 inter se matings were compared with control blastocysts obtained from matings between T/+ and +/+ females and +/tw5 males for their abilities to continue development in vitro in two culture media. The data show that there are no significant differences between the percentages of experimental and control blastocyst embryos which attach and outgrow or which contain inner cell masses on any day of culture up to equivalent gestation day 21 in either media. These findings show that the life span of cells from tw5/tw5 embryos can be extended significantly by in vitro culture.     

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.