Effects of light wavelength exposure during in vitro blastocyst production on preimplantation development of mouse embryos.

CONTEXT: Despite the absence of light within the body, the application of microscopy during stages of in vitro embryo production has led to the discovery of light irradiation effects on embryo preimplantation development. AIMS: To determine the optimal light irradiation wavelengths at various embryo stages for improving the preimplantation development of mouse embryos and the quality (total cell number) of blastocysts. METHOD: All in vitro procedures of zygote or 2-cell embryo manipulation, embryo monitoring, and culture medium exchange were conducted under visible (390-750nm), blue (445-500nm), green (500-575nm), yellow (575-585nm), or red (620-750nm) light irradiation wavelength. KEY RESULTS: We found that blue, green, and yellow light irradiation during in vitro blastocyst production from zygotes significantly improved blastocyst production and quality, compared to visible and red light irradiation. However, 2-cell embryos exposed to yellow light during in vitro blastocyst production produced significantly more high-quality blastocysts than did 2-cell embryos exposed to visible, blue, green, or red light. After exposure to blue and green - but not yellow - light during in vitro zygote manipulation, yellow light irradiation during embryo monitoring and culture medium exchange triggered significant retardation of preimplantation development. CONCLUSION: These results demonstrate that yellow light irradiation during in vitro blastocyst production, regardless of embryo stage, improves preimplantation development of mouse embryos. IMPLICATIONS: The present study will contribute to produce greater high-quality blastocysts and reduce experimental errors generated by light exposure during mouse embryo-related studies.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo (Bubalus bubalis) Embryos Produced Using Fibroblast Cells and Alters Their Epigenetic Status and Gene Expression.

We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 µT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

Controlled hatching at the prescribed site using femtosecond laser for zona pellucida drilling at the early blastocyst stage.

PURPOSE: To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability. METHODS: Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5-8.5 µm. Additional longitudinal incisions (5-7-µm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated. RESULTS: Femtosecond laser-assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening's small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3-94.7% vs. 83.3-85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups. CONCLUSIONS: Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

The role of Trp53 in the mouse embryonic response to DNA damage.

Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

Raman spectroscopy on live mouse early embryo while it continues to develop into blastocyst in vitro.

Laser based spectroscopic methods can be versatile tools in investigating early stage mammalian embryo structure and biochemical processes in live oocytes and embryos. The limiting factor for using the laser methods in embryological studies is the effect of laser irradiation on the ova. The aim of this work is to explore the optimal parameters of the laser exposure in Raman spectroscopic measurements applicable for studying live early embryos in vitro without impacting their developmental capability. Raman spectra from different areas of mouse oocytes and 2-cells embryos were measured and analyzed. The laser power and exposure time were varied and further embryo development was evaluated to select optimal conditions of the measurements. This work demonstrates safe laser irradiation parameters can be selected, which allow acquisition of Raman spectra suitable for further analysis without affecting the early mouse embryo development in vitro up to morphologically normal blastocyst. The estimation of living embryo state is demonstrated via analysis and comparison of the spectra from fertilized embryo with the spectra from unfertilized oocytes or embryos subjected to UV laser irradiation. These results demonstrate the possibility of investigating preimplantation mammalian embryo development and estimating its state/quality. It will have potential in developing prognosis of mammalian embryos in assisted reproductive technologies.

Effect of low temperatures on cytochrome photoresponse in mouse embryos.

Embryos cryopreservation is a widely used technology for genetic resources storage. Cryopreservation suppresses cell respiration, but very little is known about the changes that occur with mitochondria at low temperatures. We used Raman spectroscopy to investigate photoresponse and redox state of cytochromes in the respiratory electron transport chain (ETC) in early mouse embryos during cooling. Redox state of cytochromes was probed by the intensity of cytochrome resonance Raman lines. Photoinduced reactions of cytochromes were used to study the changes in the rates of redox reactions. It is found that the rate of cytochrome photoresponse detected by Raman spectra abruptly changes when embryos are cooled below -50 °C. Raman mapping revealed that the average intensity of cytochrome Raman peaks at -65 °C is higher than at -40 °C. Cytochrome b reduction was found in embryos frozen below -50 °C when irradiated with 532 nm laser radiation. These effects were observed for cells frozen in aqueous solutions of two different cryoprotectants: glycerol and propylene glycol. Raman spectroscopy of cytochromes reveals the abrupt changes in the ETC work of frozen mouse embryos at temperatures below -50 °C. We suggest that similar phenomena can be found in various cell types.

The effect of light exposure on the cleavage rate and implantation capacity of preimplantation murine embryos.

During assisted reproduction the embryos are subjected to light. We investigated the relationship between light exposure and the developmental- and implantation capacity of mouse embryos. In vitro cultured embryos were exposed to white or red filtered light, then transferred to the uteri of pseudo-pregnant females. The mice were sacrificed on day 8.5 and implantation sites were counted. The number of nucleic acid containing (PI+) extracellular vesicles (EVs) in culture media of light-exposed and control embryos, as well as, the effect of the EVs on IL-10 production of CD8+ spleen cells was determined by flow cytometry. DNA fragmentation in control and light exposed embryos was detected in a TUNEL assay. The effect of light on the expression of apoptosis-related molecules was assessed in an apoptosis array. Light exposure significantly reduced the implantation capacity of the embryos. The harmful effect was related to the wavelength, rather than to the brightness of the light. Culture media of light exposed groups contained significantly higher number of PI + EVs than those of the control embryos, and failed to induce IL-10 production of spleen cells. The number of nuclei with fragmented DNA, was significantly higher in embryos treated with white light, than in the other two groups. In conclusion exposure to white light impairs the implantation potential of in vitro cultured mouse embryos. These effects are partly corrected by using a red filter. Since there is no information on the light sensitivity of human embryos, embryo manipulation during IVF and ICSI should be performed with caution.

Influence of wideband visible light with an padding red component on the functional state of mice embryos and embryonic stem cells.

It is known that visible light, including sunlight and laboratory lighting, adversely affect the development of embryos in vitro. In with article we present a technology for the synthesis of composite screens, capable to photoconvert UV and a part of the blue spectrum into red light with the maximum ~630 nm. It is established that the application of such transformed light with an evident red component raises the chances of embryos to survive and protects embryonic stem cells. To create photoconversion screens, the CdZn/Se quantum dots were obtained, the average size being about 7 nm. When the quantum dots are excited by electromagnetic waves of the UV and blue spectral range, photoluminescence is observed. The average photon energy for photoluminescence is of the order of 2 eV. On the basis of CdZn/Se quantum dots and methylphenylsiloxane polymer, light-transforming composite screens were made. In case of the light-transforming composite screen, the UV component disappeared from the energy spectrum, and the intensity of the blue region of the spectrum was reduced. On the contrary, in the red region (λmax = 630 nm) one can see a little more than two-fold increase of intensity. It is shown that when exposed to 2-cell embryos by transformed light, the proportion of normally developing embryos increases by 20%, the number of dead embryos decreases twice, and number of dead and apoptotic cells was lower in blastocysts, what's decreased by 70%, as compared to the control group. When blastocysts are transferred to the feeder substrate, colonies of embryonic stem cells are formed. Cells obtained from blastocysts irradiated with transformed visible light are in a normal state in 90% of cases and did not change expression levels, biochemistry and morphology for at least 20 passages. It is assumed that the data obtained can be used for the design of systems of efficient cultivation of embryonic cells for tissue engineering and cell therapy.

Radiation-induced bystander effect in large Japanese field mouse (Apodemus speciosus) embryonic cells.

Although evidence suggests that ionizing radiation can induce the bystander effect (radiation-induced bystander effect: RIBE) in cultured cells or mouse models, it is unclear whether the effect occurs in cells of wild animals. We investigated medium-mediated bystander micronucleus (MN) formation and DNA damage in un-irradiated cells from a large Japanese field mouse (Apodemus speciosus). We isolated four clones of A. speciosus embryonic fibroblasts (A603-1, A603-2, A603-3, and A603-4) derived from the same mother, and examined their radiation sensitivity using the colony-forming assay. A603-3 and A603-4 were similar, and A603-1 and A603-2 were highly sensitive compared with A603-3 and A603-4. We examined RIBE in the four clones in autologous medium from cell cultures exposed to 2 Gy X-ray radiation (irradiated cell conditioned medium: ICCM). We only observed increased MN prevalence and induction of DNA damage foci in A603-1 and A603-3 cells after ICCM transfer. The ICCM of A603-3 (RIBE-induced) was able to induce MN in A603-4 (not RIBE-induced). To assess the possible contribution of reactive oxygen species (ROS) or nitric oxide (NO) in medium-mediated RIBE, dimethyl sulfoxide (DMSO; a ROS scavenger) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; an NO scavenger) were added to the medium. A suppressive effect was observed after adding DMSO, but there was no effect after treatment with c-PTIO. These results suggest that an enhanced radiosensitivity may not be directly related to the induction of medium-mediated RIBE. Moreover, ROS are involved in the transduction of the RIBE signal in A. speciosus cells, but NO is not. In conclusion, our results suggest that RIBE may be conserved in wild animals. The results contribute to better knowledge of radiation effects on wild, non-human species.

A detailed characterization of congenital defects and mortality following moderate X-ray doses during neurulation.

BACKGROUND: Both epidemiological and animal studies have previously indicated a link between in utero radiation exposure and birth defects such as microphthalmos, anophthalmos, and exencephaly. However, detailed knowledge on embryonic radiosensitivity during different stages of neurulation is limited, especially in terms of neural tube defect and eye defect development. METHODS: To assess the most radiosensitive stage during neurulation, pregnant C57BL6/J mice were X-irradiated (0.5 Gy or 1.0 Gy) at embryonic days (E)7, E7.5, E8, E8.5, or E9. Next, the fetuses were scored macroscopically for various defects and prenatal resorptions/deaths were counted. In addition, cranial skeletal development was ascertained using the alcian-alizarin method. Furthermore, postnatal/young adult survival was followed until 5 weeks (W5) of age, after X-irradiation at E7.5 (0.1 Gy, 0.5 Gy, or 1.0 Gy). In addition, body and brain weights were registered at adult age (W10) following X-ray exposure at E7.5 (0.1 Gy, 0.5 Gy). RESULTS: Several malformations, including microphthalmos and exencephaly, were most evident after irradiation at E7.5, with significance starting respectively at 0.5 Gy and 1.0 Gy. Prenatal mortality and weight were significantly affected in all irradiated groups. Long-term follow-up of E7.5 irradiated animals revealed a reduction in survival at 5 weeks of age after high dose exposure (1.0 Gy), while lower doses (0.5 Gy, 0.1 Gy) did not affect brain and body weight at postnatal week 10. CONCLUSIONS: With this study, we gained more insight in radiosensitivity throughout neurulation, and offered a better defined model to further study radiation-induced malformations and the underlying mechanisms.

Cerebral impact of prenatal irradiation by 131I: an experimental model of clinical neuroradioembryological effects. / VPLYV PRENATAL'NOGO OPROMINENNIa 131I NA GOLOVNYY̆ MOZOK: EKSPERYMENTAL'NA MODEL' KLINIChNYKh NEY̆RORADIOEMBRIOLOGIChNYKh EFEKTIV.

Human brain in prenatal period is a most vulnerable to ionizing radiation body structure. Unlike atomic bombings or radiological interventions in healthcare leading at most to external irradiation the intensive internal exposure may occur upon nuclear reactor accidents followed by substantial release and fallout of radioactive 131I. The latter can lead to specific neuroradioembryological effects. OBJECTIVE: To create an experimental model of prenatal cerebral radiation effects of 131I in human and to determine the experimental and clinical neuroradioembryological effects.Study object. The neuroradioembryological effects in Vistar rats exposed to 131I in prenatal period. Nervous system status and mental status in 104 persons exposed to ionizing radiation in utero due to the ChNPP accident and the same in 78 not exposed subjects. METHODS: Experimental i.e. behavioral techniques, including the spontaneous locomotive, exploratory activity and learning ability assessment, clinical i.e. neuropsychiatric, neuro and psychometric, neuropsychological, neurophys iological methods, both with dosimetric and statistical methods were applied. RESULTS: Intrauterine irradiation of Wistar rats by 131I was simulated on a model of one time oral 27.5 kBq radionu clide administration in the mid gestation period (0.72±0.14 Gy fetal thyroid dose), which provides extrapolation of neuroradioembryological effects in rats to that in humans exposed to intrauterine radiation as a result of the Chornobyl catastrophe. Abnormalities in behavioral reactions and decreased output of conditioned reflex reactions identified in the 10 month old rats suggest a deterioration of cerebral cognition in exposed animals. Specific cog nitive deficit featuring a disharmonic intellectual development through the relatively decreased verbal intelligence versus relative increase of nonverbal one is remained in prenatally exposed persons. This can indicate to dysfunc tion of cortical limbic system with especial involvement of a dominant hemisphere hippocampus. Decreased theta band spectral power (4-7 Hz range) of cerebral bioelectrical activity in the left frontotemporal area is suggestive of hippocampal dysfunction mainly in dominant hemisphere of prenatally irradiated persons. Disorders of hippocam pal neurogenesis due to prenatal exposure by radioactive iodine can be a biologic basis here. Innovative approach es in social adaptation, psychoprophylaxis and psychorehabilitation involve the maximum effective application and development of just the most developed psychological and cognitive abilities in survivors.

Transgeneration effects of N-stearoylethanolamine in irradiated rats. / TRANSGENERATsIY̆NI EFEKTY N-STEAROÏLETANOLAMINU V OPROMINENYKh ShchURIV.

OBJECTIVE: to explore possible transgeneration effects in the rats offspring of the first generation of parents subject ed to the combined effects of N stearoylethanolamine (NSE) and external exposure.Materials and metods. In the first generation rats of both sexes born to parents who have experienced the combined influence of ionizing radiation at a dose of 2.0 Gy and NSE a daily dose of 50.0 mg/kg, administered before or after exposure indicators of pro and antioxidant systems (the concentration of TBA reactive products, catalase and glu tathione peroxidase activity in plasma) were defined, concentrations of sex hormones testosterone and estradiol and nitrite anion were studied. RESULTS: Irradiation of parents caused a three fold reduction of testosterone in the blood plasma of males progeny, increased activity of catalase in plasma of female offsprings, as well as significantly increased the concentration of protein in the offsprings' blood plasma of both sexes. Introduction of NSE to parents before exposure caused the acti vation of lipid peroxidation in plasma of both sexes offsprings' against the background of a trustworthy decrease in activity of antioxidant enzymes (catalase and glutathione peroxidase), however, prevented a sharp reduction of testosterone content in the blood plasma of males offsprings, conditioned by the influence of radiation on the body of their parents. NSE introduction to parents after exposure caused no significant violations of pro/ antioxidant bal ance in the body of both sexes progeny, but did not eliminate the negative impact of parental exposure to testos terone levels in male offsprings. CONCLUSION: The transgeneration impact of NSE is manifested by radio sensitizing properties in the first generation offsprings in case of application to parents before irradiation.

Direct evidence for cell adhesion-mediated radioresistance (CAM-RR) on the level of individual integrin ß1 clusters.

The cellular interaction with the extracellular matrix (ECM) modulates many key processes such as proliferation, migration, differentiation and survival. In addition, cells cultured under 3D conditions in presence of an ECM display a marked radioresistance towards ionizing radiation (IR) in comparison to conventionally 2D cultured cells. This process, also known as "cell-adhesion-mediated-radio-resistance" (CAM-RR), has been linked to the chromatin structure that differs between cells cultured on stiff surfaces versus cell grown on soft planar supports or in 3D environments. As integrins are the key mediators of cell adhesion and mechanosensing, they originate the molecular signalling towards chromatin remodelling in response to a cell's microenvironment. We aimed to investigate this molecular origin that leads to CAM-RR by investigating the distribution of integrins at the single molecule level and show that cells cultured in 2D keep a lower fraction of integrin ß1 in clusters and maintain a less defined cluster status than 3D cultured cells. Upon X-irradiation this nanoscale distribution of integrin ß1 is disturbed at much lower dosages in 2D versus 3D cultured cells. Radioresistance is thus linked to the ability to maintain a well defined organization of integrins in clusters, making integrin distribution a potential drug target for radiosensitization.

Inhibiting DNA-PKcs in a non-homologous end-joining pathway in response to DNA double-strand breaks.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase. Inhibition of DNA-PKcs prolonged IR-induced G2/M phase arrest because of sequential activation of cell cycle checkpoints. DSBs were introduced, and cell cycle checkpoints were recruited after exposure to IR in nasopharyngeal carcinoma SUNE-1 cells. NU7441 radiosensitized MEF cells and SUNE-1 cells by interfering with DSB repair. Together, these results reveal a mechanism in which coupling of DSB repair with the cell cycle radiosensitizes NHEJ repair-deficient cells, justifying further development of DNA-PK inhibitors in cancer therapy.

Effect of low-dose X-ray irradiation on micronucleus formation in human embryo, newborn and child cells.

PURPOSE: It is well known that a high-dose of ionizing radiation is sufficient to break DNA strands, which leads to elevated genotoxic risks; however, the risks associated with low doses of ionizing radiation remain unclear. In addition, there is little data about the effect of low-dose ionizing radiation on human-derived embryo, newborn and child cells. We investigated the frequency of micronucleus (MN) formation in these cells to understand the genotoxic effects of ionizing radiation. MATERIALS AND METHODS: We irradiated the cells with X-rays from 0.02-2 Gy at a rate of 0.0635 Gy/min. After irradiation, we investigated the effect of low-dose X-ray irradiation on cellular viability and frequency of MN formation. RESULTS: Increases in MN formation were largely dose-dependent; however, there were no differences between controls and doses lower than 0.2 Gy, except in KMST-6 human transformed embryo cells. CONCLUSION: We could not detect an obvious effect of low-dose X-ray irradiation at doses lower than 0.1 Gy. The embryonic cells were more sensitive to X-ray irradiation than newborn and child cells. The threshold for X-ray-induced MN formation appears to be in the range of 0.05-0.1 Gy in cultured human embryo, newborn and child cells.

Differential DNA damage signalling and apoptotic threshold correlate with mouse epiblast-specific hypersensitivity to radiation.

Between implantation and gastrulation, mouse pluripotent epiblast cells expand enormously in number and exhibit a remarkable hypersensitivity to DNA damage. Upon low-dose irradiation, they undergo mitotic arrest followed by p53-dependent apoptosis, whereas the other cell types simply arrest. This protective mechanism, active exclusively after E5.5 and lost during gastrulation, ensures the elimination of every mutated cell before its clonal expansion and is therefore expected to greatly increase fitness. We show that the insurgence of apoptosis relies on the epiblast-specific convergence of both increased DNA damage signalling and stronger pro-apoptotic balance. Although upstream Atm/Atr global activity and specific γH2AX phosphorylation are similar in all cell types of the embryo, 53BP1 recruitment at DNA breaks is immediately amplified only in epiblast cells after ionizing radiation. This correlates with rapid epiblast-specific activation of p53 and its transcriptional properties. Moreover, between E5.5 and E6.5 epiblast cells lower their apoptotic threshold by enhancing the expression of pro-apoptotic Bak and Bim and repressing the anti-apoptotic Bcl-xL. Thus, even after low-dose irradiation, the cytoplasmic priming of epiblast cells allows p53 to rapidly induce apoptosis via a partially transcription-independent mechanism.

The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts.

The methylation of cytosine at CpG dinucleotides (5 meC) is an important epigenetic mechanism that governs genome stability and gene expression. Important ontological and pathological transitions are associated with marked global changes in detectable levels of methylation. We have previously found two pools of immune-detectable 5 meC exist within cells, a pool that can be detected after acid treatment of fixed cells to denature chromatin and another large but variable pool that requires a further tryptic digestion step for complete epitope retrieval. The trypsin-sensitive pool has been shown to be largely associated with the heterochromatic fraction (by a heterochromatin marker, HP1-ß) of the genome, and the size of this pool varies with the growth disposition of cells. Since DNA damage imposes large changes on chromatin structure the present study analyzed how such changes influences the faithful immunological detection of 5 meC within mouse embryonic fibroblasts. DNA damage was induced by either UV-irradiation or doxorubicin treatment, each of which resulted in increased levels of immune-detectable 5 meC at 24-48 h after treatment. There was a marked trypsin-sensitive pool of 5 meC in these cells which was significantly increased after DNA damage. The increased levels of 5 meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-ß staining. The relative amount of masked 5 meC after DNA damage was positively associated with increased levels of HP1-ß. The methyl binding protein, MBD1, was a less reliable measure of changes in 5 meC, with a significant fraction of 5 meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5 meC within the nuclei of fibroblasts in response to DNA damage.

No adverse effects were identified on the perinatal outcomes after laser-assisted hatching treatment.

The aim of this study was to evaluate the safety of laser-assisted hatching (LAH) by comparing obstetric and neonatal outcomes between assisted hatching and control groups in cryopreserved embryo transfer cycles. A retrospective cohort analysis was carried out. A total of 699 women with 392 infants delivered were included. Laser- assisted hatching was carried out on D-3 thawed and warmed embryos before transfer in 480 cryopreserved embryos transfer cycles. Obstetric outcomes, neonatal outcomes, and congenital birth defects were recorded. A total of 815 cryopreserved embryo transfer cycles (480 in LAH group and 335 in control group) in 699 patients were analysed. Statistically significantly higher implantation (31.85% versus 16.95%), clinical pregnancy (53.96% versus 33.43%) and live delivery (44.58% versus 23.88%) rates were observed in the LAH group (all P < 0.001). For either singleton or multiple gestations, no statistically significant differences were found in mean gestational age, mean birth weight and mean Apgar score. Four major malformations occurred in the assisted hatching group and three malformations (one major and two minor) in the control group. This study did not identify any harmful effect of LAH on neonates, which suggested that LAH may be a safe treatment in cryopreserved embryo transfer cycles.

Effect of red light on the development and quality of mammalian embryos.

PURPOSE: To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on embryonic development and quality in pig. METHOD: Spectral scalar irradiance (380-1050 nm) was measured by a fiber-optic microsensor in the focal plane of a dissection microscope, an inverted microscope and a time-lapse incubation system. Furthermore, the effect of three different red light levels was tested in the time-lapse system on mouse zygotes for 5 days, and on porcine zona-intact and zona-free parthenogenetically activated (PA) embryos for 6 days. RESULTS: The time-lapse system used red light centered at 625 nm and with a lower irradiance level as compared to the white light irradiance levels on the dissection and inverted microscopes, which included more energetic radiation <550 nm. Even after 1000 times higher total energy dose of red light exposure in the time-lapse system, no significant difference was found neither in blastocyst development of mouse zygotes nor in blastocyst rates and total cell number of blastocysts of porcine PA embryos. CONCLUSIONS: Our results indicate that red light (625 nm, 0.34 W/m(2)) used in the time-lapse incubation system does not decrease the development and quality of blastocysts in both mouse zygotes and porcine PA embryos (both zona-intact and zona-free).