Evaluation of mouse preimplantation embryos cultured in media enriched with insulin-like growth factors I and II, epidermal growth factor and tumor necrosis factor alpha

Culture of preimplantation embryos is complex and requires strictly defined culture media to sustain their viability and quality. In the current study, an effort was made to evaluate comprehensively the quality of mice embryos, grown in media enriched with IGF I, IGF II, EGF and TNFalpha. For that purpose, critically chosen and thoroughly described, complex morphological methods based on contrast-phase, fluorescent and confocal microscopy were used. The study evaluated blastulation and hatching rates, total blastocyst cells, inner cell mass cell numbers (differential staining) as well as identified embryo cells with positive reactions for necrosis or apoptosis (TUNEL). The critical evaluation of the effects of the studied cytokines allowed for simultaneous, meticulous assessment of the applied study methods. Significantly more blastocysts were found in culture media enriched with IGF-I, IGF II and EGF. Significantly more hatched blastocysts were found in media with IGF-I and IGF II. Additionally, IGF I and II increased inner cell mass and total blastocyst cell numbers. Very few cells with necrosis and apoptosis were found in the culture media enriched with IGF I, IGF II and EGF. TNFalpha produced negative effects. The observed effects were dose-dependent.     

Effects of epidermal growth factor on preimplantation mouse embryos

When epidermal growth factor (EGF) was added to the medium for culture of preimplantation embryos, morphological development as determined by microscopic observation was unaffected, but 333 nM-EGF stimulated total uptake of [3H]leucine by late morulae/blastocysts which had been cultured for 24 h from morulae. Incorporation of [3H]leucine into protein by these embryos was increased by 0.33, 3.3 and 33 nM-EGF, following a quadratic relationship producing less stimulation at 333 nM, which may indicate down regulation of receptors. The estimated EC50 was approximately 0.25 nM. Manipulation of the culture period indicated that the embryos responded to EGF at the morula/blastocyst transition period and immunosurgery was used to show that the increased protein synthesis was restricted to the trophectoderm cells. No mitogenic effect was observed. The effective concentration of EGF is close to that of serum and to values which stimulate other tissues. It is suggested that EGF receptors appear at compaction and that EGF may have a role in differentiation of the trophectoderm cells.     

Effects of opioids on the development of preimplantation mouse embryos

Opioid peptide DAGO, agonist of opiate mu-receptors and naloxone antagonist of mu-, delta- and kappa-receptors in concentration 3 x 10 M inhibit embryonic development of CBA mice. Inhibition was stage-specific with maximal effect after addition of opioids to zygotes: in the presence of Naloxone no more than 6.7% of embryos reached morula and blastula stages and in the presence of DAGO--36.8%. The other embryos were arrested at two-, four- or, sometimes even, at eight-cell stages without any signs of fragmentation. Four and eight-cell embryos were less sensitive to drug action. Inhibitory effects of these opioids were reduced when they were added simultaneously to zygotes. Agonist of opiate delta-receptors, opioid peptide DADLe, failed to affect embryonic development.     

In-vivo and in-vitro effects of ethanol on mouse preimplantation embryos

In Exp. 1A, hybrid mice (N = 10) were provided with food and 25% (v/v) ethanol as the only source of liquid for 72 h, beginning at the detection of the copulatory plug (08:00 h, Day 1). Control mice received food and tap water. Food consumption (P less than 0.001) but not total caloric intake (P greater than 0.05) was less for the alcohol-treated mice than the controls. Ethanol-derived calories averaged 35% of caloric intake during the 72 h of treatment. Alcohol-treated animals showed a dramatic weight loss until Day 5 while controls gained weight (P less than 0.05). Ethanol consumption did not influence pregnancy rate, litter size or litter weight. In Exp. 1B, animals were treated as in Exp. 1A, but were killed at various times between 24:00 h, Day 1, and 08:00 h, Day 4. Trunk blood was used to determine haematocrit and serum to determine alcohol concentration. Haematocrit was greater (P less than 0.05) for all alcohol-treated mice than for controls at all time periods sampled except one. Dehydration was therefore probably responsible for the weight loss seen in Exps 1A and 1B. Average blood alcohol concentrations fluctuated with time of day and day of treatment. Average maximum concentration was 91.4 mg ethanol/100 ml serum. In Exp. 2, hybrid mouse 2-cell embryos were cultured in vitro in 0 or 0.1% ethanol (Exp. 2A) and 0 or 1.0% ethanol (Exp. 2B) for 8 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detrimental effects of prostaglandin F2alpha on preimplantation bovine embryos

Two studies were performed to determine effects of prostaglandin F2alpha (PGF2alpha) on continued development of pre-compacted (in vitro-produced) and compacted (in vivo-derived) bovine embryos. In Experiment 1, pre-compacted embryos were placed in KSOM media supplemented with polyvinyl alcohol (0.3%) and assigned to the following treatments: (1) control; (2) PGF-1 (1 ng/mL PGF2alpha); (3) PGF-10 (10 ng/mL PGF2alpha); (4) PGF-100 (l00 ng/mL PGF2alpha); or (5) PGE-5 (5 ng/mL PGE2). Following 4 days of incubation in assigned treatments, continued development of pre-compacted embryos to blastocysts was reduced by addition of PGF2alpha in culture medium (P = 0.002). Development did not differ between control and PGE2 treatments (P > 0.10). In Experiment 2, compacted morula' s were placed in KSOM-PVA supplemented media and assigned to one of four treatments: (1) control; (2) PGF-0.1 (0.1 ng/mL PGF2alpha); (3) PGF-1 (1 ng/mL PGF2alpha); and (4) PGF-10 (10 ng/mL PGF2alpha). After 24h in culture, embryos were washed and placed in KSOM-BSA (0.5%) without PGF2alpha for an additional 48 h until assessment for development. Continued development of compacted morula to blastocyst was not affected by addition of PGF2alpha to the culture medium (P > 0.10). However, hatching rates of embryos cultured with PGF2alpha were lower (P = 0.05). In conclusion, it is suggested that PGF2alpha has a direct negative effect on continued embryonic development of pre-compacted and compacted bovine embryos.     

Expression of tumor necrosis factor at a specific developmental stage of mouse embryos

We investigated the expression of the tumor necrosis factor (TNF) gene during development of mouse embryos, and observed its transient expression on Days 9 and 10 of gestation. We also detected a 25-kDa protein showing immunological cross-reactivity with mouse TNF antibody in an extract of 10-day embryos. These results suggest that TNF plays a role in mammalian ontogenesis.     

Effect of superoxide dismutase on the development of preimplantation mouse embryos

The role of superoxide dismutase (SOD) was tested on preimplantation development of mouse embryos in vitro. The presence of SOD in ovarian antral follicles and in oviductal and uterine secretions was also investigated. Zygotes from superovulated ICR female mice were cultured in modified Whittingham's T6 medium supplemented with SOD (0 to 370 U) or EDTA (100 muM) at 37 degrees C under 5% CO(2) in air. Supplementation of SOD (370 U) significantly promoted the development of zygotes to the blastocyst stage (45%) as compared to that of the controls (1.4%). This favorable effect of SOD was comparable to that of EDTA and completely suppressed by anti-SOD antibody. Blastocysts cultured with SOD consisted of 78.2+/-10.4 blastomeres and possessed as many blastomeres as those (81.6+/-9.3) developing in vivo; blastocysts cultured with EDTA had significantly fewer blastomeres (42.6+/-13.7). These findings suggest that SOD protects embryos against oxidative insults and that it can be an effective substitute for EDTA for supporting mouse embryo development in vitro. The SOD activity was detected in 3 different lumina from mouse reproductive organs, and SOD was identified as a cytosolic Cu,Zn-SOD on photochemically stained polyacrylamide gels. Our results suggest that oxidative injury may be responsible for developmental retardation of preimplantation-stage mouse embryos in vitro and that Cu,Zn-SOD may play a crucial role in protecting embryos against oxygen toxicity in vivo as well as in vitro.     

The effects of carbon-13 incorporation into preimplantation mouse embryos on development before and after implantation

Preimplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled ¹³C-glucose. The ¹³C content of the blastocysts was 20 atom % according to incorporation studies with ¹⁴C-glucose. No embryotoxic effects of carbon-13 incorporation could be detected on the basis of these criteria of normal development: the percentage of embryos reaching the blastocyst stage during the culture period; the number of cells in these blastocysts; and the development after transplantation to pseudopregnant foster mothers.     

Patterns of organelle distribution in mouse embryos during preimplantation development

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.     

Effects of metabolic factors in the diabetic state on the in vitro development of preimplantation mouse embryos

The effects of insulin, glucagon, beta-hydroxybutyrate, and acetoacetate on the in vitro development of preimplantation mouse embryos were studied. In controls, 24% of blastocysts failed to develop successfully when grown for 72 h in Eagle's medium supplemented with 10% fetal calf serum. Insulin at concentrations of 1.0 and 2.0 IU/ml of culture medium interfered with development in 62-63% of the blastocysts. Preimplantation embryos showed a threshold pattern in their reaction to glucagon: its addition in concentrations of 0.0015 mM (5 micrograms/ml) did not significantly inhibit blastocyst development, while concentrations of 0.003 mM (10 micrograms/ml) inhibited 70% of blastocysts. The embryotoxic effects of ketone bodies were manifested only in relatively high doses. beta-hydroxybutyrate was embryotoxic at concentrations greater than 5 mg/ml, and its effects were dose dependent: 48 mM (6 mg/ml) inhibited 45% of blastocysts, while 80 mM (10 mg/ml) arrested 87% of embryos from further development. Acetoacetate at concentrations of 0.1 mM (10 micrograms/ml) inhibited the development of 50% of the blastocysts, and its effects were not dose dependent: concentrations of 1 mM (100 micrograms/ml) inhibited development in 63% of the embryos. The combination of the diabetic metabolic factors in relatively low concentrations was highly embryotoxic, especially when accompanied by hyperglycemia.