Effects of paternal heat stress on the in vivo development of preimplantation embryos in the mouse

The objective of this study was to examine the effect of paternal heat stress on the in vivo development of preimplantation embryos in the mouse. Synchronised B6CBF1 female mice were mated either to a control male mouse or to one that had been exposed at 7, 21 or 35 days previously, for 24 h to an ambient temperature of 36+/-0.3 degrees C and 66+/-5.6% relative humidity. Embryos were collected from the oviducts of mice at 14-16 h, 34-39 h or 61-65 h after mating or from the uterus at 85-90 h after mating and their developmental status was evaluated morphologically. The number of cells within blastocysts was also determined using bisbenzimide-propidium iodide staining. Paternal heat stress 7 days before mating reduced the proportion of embryos developing from 4-cell (4-C) to morulae (M), hatched blastocysts, total blastocysts and the number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocyst. Paternal heat stress 21 days prior to mating reduced the proportion of 2-C and 4-C to M embryos with no embryos developing to blastocysts. There were also increases in the number of 1-C and abnormal embryos recorded at this time. Paternal heat stress 35 days before mating decreased the proportion of 2-C embryos, expanded blastocysts and ICM and TE cells in the blastocyst. These results support previous work demonstrating that both the sperm in the epididymis and germ cells in the testis are susceptible to damage by environmental heat stress, with spermatocytes being the most vulnerable. This study also demonstrates that subtle effects on the male such as a short exposure to elevated environmental temperatures can translate to quite profound paternal impacts on early embryo development.     

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.     

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 methylmercury and mercuric chloride on preimplantation mouse embryos in vivo

This report compares the effects of methylmercuric chloride (MMC) and mercuric chloride (MC) on the development of mouse preimplantation embryos in vivo. Female mice were injected with a single intravenous dose of 0.5-20.0 mg Hg/kg MMC or 0.5-2.5 mg Hg/kg MC on day 0 of gestation. The embryos were recovered by flushing excised oviduct and uterus on day 3.5 of pregnancy, and were examined for abnormalities. In the groups treated with doses of 0.5 and 1.0 mg Hg/kg of both compounds, the rates of abnormal embryos were not significantly different from that in the control group. The 50% effective dose of MMC was twice as great as that of MC. With increasing dose, the difference became more obvious; the 80% effective doses differed by a factor of ten. The body weight of dams decreased in terms of the dose of mercury in MC-treated groups, but did not vary in MMC-treated groups. The sensitive developmental stage for mercury toxicities could not be determined clearly, although the high sensitivity was reported in the blastocyst stage in vitro. The embryos treated in vivo were less sensitive than those reported in vitro.     

Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Mouse interferon produced in vivo does not inhibit the development of preimplantation mouse embryos

Mouse embryos were cultured in vitro in medium with serum containing interferon which had been induced in vivo by intravenous administration of polyinosine-polycytidylic acid. Two-cell and blastocyst-stage embryos were incubated for 72 and 24 h respectively before embryo transfer, or fixation to determine cell number. Further, blastocysts were outgrown on coverslips in embryo culture medium with fetal calf serum and interferon. Expression of an intermediate filament protein (Mr 55 000) in blastocyst outgrowths was examined with a monoclonal antibody. Embryos appeared morphologically normal and after treatment the mean cell number did not differ from that of controls. Implantation was unaffected by any of the treatments, but culture before transfer in medium containing mouse serum reduced the number of normal fetuses recovered on Day 14 of gestation compared to those cultured in medium without serum. Exposure to interferon did not modify the expression of filaments in the outgrown blastocyst. It is therefore unlikely that interferon induced by viral infection during pregnancy is responsible for preimplantation embryonic loss.     

Effects of human diabetic serum on the in vitro development of mouse preimplantation embryos

The effects of sera from different types of human diabetes (type I with and without ketoacidosis; type II treated with insulin or Daonil or untreated) on the in vitro development of early preimplantation mouse embryos were studied. In controls, 20% of blastocysts failed to develop successfully when grown for 72 h in RPMI medium supplemented with 10% fetal bovine serum and 50% nondiabetic human serum. In experiments using 50% diabetic serum, the highest embryotoxic effect was found in type-I diabetes with and without ketoacidosis: The percents of undeveloped embryos were 66 and 58, respectively. In type-II diabetes, embryotoxic effects were found among all studied types: The percent of undeveloped blastocysts varied from 36% in insulin-treated type-II diabetes to 44% in untreated type-II diabetes. A high correlation was found between the number of undeveloped embryos and the blood concentrations of metabolic diabetic factors: glucose (r = .53-.64 in type-I diabetes), B-HOB (r = .7-.77 in type-II diabetes untreated or treated with Daonil), acetoacetate (r = .66 in insulin-treated type-II diabetes), and HbA1c (r = .89 in insulin-treated type-II diabetes or .99 in Daonil-treated type-II diabetes). A concentration of 80% serum was embryo-toxic when obtained from nondiabetic or from diabetic human. The possible role of diabetic metabolic factors in causing increased risk of spontaneous abortions and infertility among diabetic women is discussed.     

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.     

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.     

Bioactivity of selected plant essential oils against listeria monocytogenes

Ninety-three different commercial essential oils were screened for activity against 20 Listeria monocytogenes strains in vitro and the results correlated against the actual chemical composition of each oil. There was a substantial difference in the activity between different essential oils as expected, but there was also a difference in activity between different samples of the same essential oil. Strong anti- Listeria activity was often correlated with essential oils containing a high percentage of monoterpenes, eugenol, cinnamaldehyde, thymol, and sometimes with citronellol, limonene and geraniol. However, as there was often no correlation between the anti- Listeria activity and the main chemical components, it is possible that either there is a more complex relationship with the chemical composition (which includes the minor components) or that substantial adulteration had occurred in some essential oil samples. The possible use of a number of essential oils in a dual role as flavours and antimicrobials is discussed.     

Effects of thioredoxin on the preimplantation development of bovine embryos

Thioredoxin (TRX) is an ubiquitous protein disulfide reductase, which is known to be involved in the implantation development of mouse embryos. In the present study, recombinant human TRX was used to evaluate its effect on the promotion of preimplantation development of bovine embryos derived from in vitro maturation and fertilization. Supplementation of the medium 24h post insemination with TRX significantly (P<0.05) enhanced the frequency of development to the blastocyst stage in 5% O(2) concentration. The optimal concentration was 0.5 microg/ml (P<0.05, compared with 0, 0.1 and 1.0 microg/ml). This effect of TRX was evident only when added around the time of the first cleavage stage (24 h post insemination); no promotion was found with treatment at 6h (one-cell) or 44 h (six- to eight-cell) after insemination. Moreover, it is of interest that even with the best combination of the dose and timing of TRX treatment (0.5 microg/ml, at 24 h post insemination), no promotion of development was observed when embryos were cultured under 20% O(2). However, a preincubation of TRX in the culture medium under 20% oxygen for 24h did not diminish the promoting effect in the subsequent TRX treatment under optimal conditions, thus suggesting that the possible oxidation of TRX alone may not be the reason for the disappearance of the effect under a high oxygen concentration. These results indicate that TRX does improve the development of bovine embryos in vitro, though unlike the general reducing reagents such as beta-mercaptoethanol or cysteamine, TRX may have to exert its effect at specific times and in more physiologic oxygen environments.     

RBM19 is essential for preimplantation development in the mouse

Background  

RNA-binding motif protein 19 (RBM19, NCBI Accession # NP_083038) is a conserved nucleolar protein containing 6 conserved RNA recognition motifs. Its biochemical function is to process rRNA for ribosome biogenesis, and it has been shown to play a role in digestive organ development in zebrafish. Here we analyzed the role of RBM19 during mouse embryonic development by generating mice containing a mutation in the Rbm19 locus via gene-trap insertion.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

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.     

Metabolism in preimplantation mouse embryos

The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.     

Antibiofilm activity of selected plant essential oils and their major components

The aim of the study was to examine the antibiofilm activity of selected essential oils (EO): Lavandula angustifblia (LEO), Melaleuca alternifolia (TTO), Melissa officinalis (MEO) and some of their major constituents: linalool, linalyl acetate, alpha-terpineol, terpinen-4-ol. Biofilms were formed by Staphylococcus aureus ATCC 29213 and Escherichia coli NCTC 8196 on the surface of medical biomaterials (urinary catheter, infusion tube and surgical mesh). TTC reduction assay was used for the evaluation of mature biofilm eradication from these surfaces. Moreover, time-dependent eradication ofbiofilms preformed in polystyrene 96-well culture microplates was examined and expressed as minimal biofilm eradication concentration (evaluated by MTT reduction assay). TTO, alpha-terpineol and terpinen-4-ol as well as MEO, showed stronger anti-biofilm activity than LEO and linalool or linalyl acetate. Among the biomaterials tested, surgical mesh was the surface most prone to persistent colonization since biofilms formed on it, both by S. aureus and E. coli, were difficult to destroy. The killing rate studies of S. aureus biofilm treated with TTO, LEO, MEO and some of their constituents revealed that partial (50%) destruction of 24-h-old biofilms (MBEC50) was achieved by the concentration 4-8 x MIC after 1 h, whereas 2-4 x MIC was enough to obtain 90% reduction in biomass metabolic activity (MBEC90) after just 4 h of treatment. A similar dose-dependent effect was observed for E. coli biofilm which, however, was more susceptible to the action of phytochemicals than the biofilms of S. aureus. It is noteworthy that an evident decrease in biofilm cells metabolic activity does not always lead to their total destruction and eradication.