Two-cell Mouse Embryos Research Articles

Influence of Slow-rate Freezing and Vitrification on Mouse Embryos

Hredzak, R., A. Ostro, I. Maraaek, J. Kaamarik, V. Idilova, J. Vesela: Influence of Slow-rate Freezing and Vitrification on Mouse Embryos. Acta Vet. Brno 2005, 74: 23-27. The aim of the study was to compare the effect of two different methods of embryo freezing (slow-rate freezing employing programmable freezing equipment and ultra-rapid freezing by vitrification) on developmental capacity of two-cell mouse embryos on the basis of their development to blastocyst stage and implantation rate of blastocysts. Two-cell embryos were obtained from superovulated female mice and divided to three groups. The first group of embryos was frozen by the slow controlled-rate method using a programmable freezing equipment with propanediol as a cryoprotectant. Embryos from the second group were vitrified employing ethylene glycol as a cryoprotectant. The third group of embryos was cultivated in vitro without cryopreservation in a cultivation medium in an atmosphere of 95% air and 5% CO2. After thawing, the embryos from the first two groups were cultivated in vitro under conditions identical to those used for fresh embryos. The blastocysts that developed in vitro from embryos of all three groups were transferred to uteri of pseudo-gravid female mice to determine their implantation capacity. The percentage of vitrified embryos that developed into blastocysts was significantly lower than that of the fresh and slow-rate frozen embryos. The morphological appearance of embryos from all three groups was the same. The implantation rate of blastocysts that developed from vitrified embryos was significantly lower compared to the fresh and slow-rate frozen embryos. The results obtained indicate that freezing of embryos affects negatively their further development the negative effect of vitrification being more detrimental. As a “universal” vitrification protocol has not yet been defined, additional studies are needed to achieve its optimisation. Cryopreservation, cryoprotectant, implantation, slow-rate freezing, vitrification

Potassium Assay in Blastomere of Two-Cell Mouse Embryo after Equilibration and washing from Cryoprotector

The need for effective methods of cryopreservation of early mammalian embryos necessitates the study of the mechanisms of blastomere adaptation to the equilibration procedure and subsequent washing from the cryoprotector. The osmotic effects during these procedures can cause electrolyte imbalance in embryonic cells. Intracellular potassium concentrations at the stage of two-blastomere mouse embryo were studied by electron probe microanalysis.

Potassium assay in blastomere of two-cell mouse embryo after equilibration and washing from cryoprotector

Potassium assay in blastomere of two-cell mouse embryo after equilibration and washing from cryoprotector

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (m Glut12) was cloned from preimplantation embryos by 5′RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The m Glut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog m Glut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.

Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves

Aqueous extract of leaves of Indigofera suffruticosa (AELIs) were studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and AELIs at a concentration of 5 or 10 mg/ml. On Day 4 of culture, morulae and blastocysts were collected for morphological analysis of blastomeres. We found that embryos exposed to the higher concentration of AELIs (10 mg/ml) did not develop and all embryos persisted at the two-cell stage. Those embryos exposed to the lower concentration (5 mg/ml) showed development until morula, blastocyst and hatched blastocyst stages that were similar to the controls. These results suggest that use of AELIs may be hazardous to humans who make use of it in folk medicine.

Effect of Six-and Ten-Day-Old Chick Embryo Amniotic Fluid on Development of Two-Cell Mouse Embryos

The purpose of this study was to investigate the effect of six-and ten-day-old chick embryo amniotic fluid (CEAF) on the development of two-cell mouse embryos. Six- and ten-day-old CEAF (6-AF, 10-AF) were aspirated separately from the amniotic cavity and two experiments were performed. In the first experiment, two-cell mouse embryos were cultured in different supplements of heat-inactivated 6-AF, pure heat-inactivated 6-AF and pure active 6-AF. The second experiment was also carried out in the same manner using the 10-AF. The rate of the development of embryos in all groups were daily determined and statistically compared with that of control (Ham's F-10 supplemented with 5 mg/ml human serum albumin). During the cultivation period, more embryos reached to the blastocyst stage in all groups of CEAF compared to the control. The result hatched blastocysts embryos also increased from days 3 till 5 of cultivation in all groups of CEAF. The developmental potential of embryos appears to be almost identical in six- and ten-day-old CEAF. In conclusion, CEAF as a supplement or a natural medium could support the development of two-cell mouse embryos.

Cyclin A2 is phosphorylated during the G2/M transition in mouse two-cell embryos.

In the present study, we investigated the expression of cyclin A2 in mouse two-cell embryos to elucidate the role of cyclin A2 at the G2/M transition. Two forms of cyclin A2 on SDS-PAGE (an upper and a lower band) were detected in two-cell embryos synchronized at the M phase by nocodazole. To investigate the nature of this shift, embryos synchronized at the M phase were treated with alkaline phosphatase (AP). The upper band of cyclin A2 was fainter in AP-treated embryos than in nontreated embryos. This result indicates that cyclin A2 in mouse two-cell embryos is phosphorylated and the band on SDS-PAGE shifts up during the G2/M transition. In addition, we examined the sequential expression of cyclin A2 in two-cell blocked embryos after OA treatment. The upper band of cyclin A2 was first detected at 2 hr after the treatment, corresponding to the timing of Cdc2 kinase activation. In two-cell embryos after removal from nocodazole treatment, the phosphorylated form of cyclin A2 protein decreased abruptly just before cytokinesis. These results suggest that the mechanism of cyclin A2 degradation in mouse two-cell embryos may be different from that in somatic cells.

Effects of leptin and leukemia inhibitory factor on preimplantation development and STAT3 signaling of mouse embryos in vitro.

Preimplantation mouse embryos simultaneously express receptors for leptin and leukemia inhibitory factor (LIF), both of which trigger activation of STAT3 (Signal Transducer and Activator of Transcription) protein. To examine the joint effects of leptin and LIF on embryonic development, we studied preimplantation development and activation of STAT3 signaling of mouse embryos after exposure to leptin and/or LIF in vitro. Two-cell mouse embryos (Day 2) were cultured in the presence of leptin and/or LIF. Significantly fewer leptin-exposed than control embryos hatched by Day 5 and by Day 6 of development. In addition, cells of leptin-exposed Day 5 blastocysts showed a higher rate of DNA fragmentation, which is a sign of apoptosis. Leukemia inhibitory factor alone had no effect on the rates of embryonic development or DNA fragmentation. Simultaneous exposure of embryos to leptin and LIF increased the proportion of hatching embryos and decreased the rate of apoptosis compared to embryos exposed to leptin only. Leptin treatment was associated with an increased phospho-STAT3-specific immunofluorescence in the cell membrane of blastocysts, which was not observed in LIF-exposed embryos. In conclusion, LIF modifies the effect of leptin during preimplantation embryo development in mice, presumably by interfering with activation of STAT3 signaling.

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure.

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.

Are heat shock proteins acting as modulators of pre-implantation mouse embryo development and apoptosis?

Objective: Intracellular induction of heat shock proteins (HSPs) in response to various stresses coincides with a state of increased resistance to subsequent cellular damage and apoptosis. The higher incidence of apoptosis is detrimental to blastocyst formation and leads to preimplantation embryo death. The objective of the present study was to determine the effects of HSP 60 and 70 inhibition on preimplantation mouse embryo development and apoptosis. Design: Mouse embryos cultured in the presence of anti-HSP antibody and the incidence of apoptosis in embryos was determined using confocal microscopy. Materials/Methods: Two-cell mouse embryo were cultured for 72h in human tubal fluid (HTF) media containing % serum substitute supplement (SSS) along with 100 g/mL of monoclonal antibodies to HSP60 (Group I, n = 51) or HSP70 (Group II, n = 46). Simultaneously cultured embryos in HTF-SSS were used as control group (Group I, n = 71). Since the monoclonal anti HSP antibodies belong to IgG1 subclass, HTF-SSS supplemented with purified monoclonal mouse IgG1 was used as a control immunoglobulin (Group Ia, n = 54). Total blastomere count per embryo was determined by staining with bisbenzimide (Hoechst 33258). A terminal transferase-mediated dUPT end labeling (TUNEL) assay was used to determine apoptosis in blastocysts using the confocal microscopy. To distinguish apoptosis from necrosis, embryos were stained with propidium iodide (PI) before the TUNEL assay. Results: Blastocyst development rate (BDR) was comparable between two control groups (92% and 89%, respectively). Addition of the anti-HSP60 and 70 antibodies to culture media significantly decreased the BDR (39%, p <0.0001; and 15%, p <0.0001). Compared to control embryos, total cell number per blastocyst <median (25%, 75% inter-quartile range)>was significantly lower in the presence of anti-HSP60 (p = 0.002) and anti-HSP70 (p <0.0001). Percentage of cells undergoing apoptosis was significantly higher in presence of anti-HSP60 (12.8%, p <0.0001) and anti-HSP70 (13.4%, p <0.0001) compared to control embryos. BDR for embryos cultured in presence of anti-HSP70 was significantly lower compared to embryos cultured in presence of anti-HSP60 (p = 0.04). Conclusions: The developmental alterations observed following culture of mouse embryos in presence of anti-HSP antibodies provides evidence for the role of heat shock proteins especially HSP70 on mouse embryo development and incidence of apoptosis. Supported by: None.

Apoptosis during mouse blastocyst formation: evidence for a role of high levels of reactive oxygen species

Objective: Apoptosis is a common mode of programmed cell death occurring during embryonic development as well as in many pathological conditions. In the mouse blastocyst, apoptosis eliminates redundant cells from the inner cell mass (ICM) to maintain appropriate ratio of ICM/trophectoderm (TE) and plays an important role in normal development. However, cell death is increased under sub-optimal culture and stressful conditions. In vitro culture of embryos results in production of higher amounts of reactive oxygen species (ROS) which can influence the in vitro development of early mammalian embryos. The objective of this study was to determine the role of ROS in induction of apoptosis in early mouse embryos. Design: A prospective study in a reproductive research laboratory. Materials/Methods: Two-cell mouse embryos (n = 72) were cultured for 72h in HTF medium. Blastocyst development rate (%BDR) and total cell number in blastocyst were recorded. Total blastomere count per embryo was determined by staining with bisbenzimide (Hoechst 33258). The number of nuclei in ICM and TE in blastocyst were determined using confocal microscopy. Control group (n = 71) consisted of the embryos cultured in HTF alone and HTF medium supplemented with 10% serum supplement as a ROS scavenger. The levels of ROS produced by embryos in culture medium were measured at 24h, 48h, and 72h by chemiluminescence assay using luminol as the probe and the results were expressed as × 106 counted photons per minute (cpm). The incidence of apoptosis was detected by terminal transferase-mediated DNA end labeling (TUNEL) using confocal microscopy. Results: The median (25th, 75th percentile) value of total cell number (TCN) of blastocysts in HTF + serum group <53.64 (45.20, 60.75)>was similar to blastocysts cultured in HTF-alone <55.38 (53.71, 57.6)>(p = 0.31). Levels of ROS in HTF and HTF + serum media after 24h was 0.28 (0.13, 0.43) vs. 0.0 (0.0, 0.01) (p = 0.02), at 48h was 0.24 (0.21, 0.26) vs. 0.02 (0.01, 0.02) (p = 0.02), and was 0.26 (0.09. 0.32) vs. 0.02 (0.02, 0.03) (p = 0.01) at 72h, respectively. There was a significant positive correlation between the percentage of apoptosis and the levels of ROS after 24h (r = 0.76, p = 0.01), 48h (r = 0.87, p = 0.001), and 72h (r = 0.53, p = 0.05). Results of BDR and apoptosis in both groups are provided in the table. TableGroupBDR (%)Apoptotic blastocyst (%)Apoptosis per blastocystApoptosis in ICM (%)Apoptosis in TE (%)HTF-alone92985.84 (5.19, 6.60)∗Values are median and interquartile range.25.13.5HTF + serum92942.27 (1.89, 3.30)15.21.2P-value†P < 0.05 was considered significant using repeated measures ANOVA.0.80.3<0.001<0.001<0.001∗ Values are median and interquartile range.† P < 0.05 was considered significant using repeated measures ANOVA. Open table in a new tab Conclusions: Our results indicate that higher amounts of reactive oxygen species induce apoptosis in cultured mouse pre-implantation embryos. Addition of serum may help scavenge the excessive amounts of ROS in culture media and decrease the incidence of apoptosis. Supported by: None.

Amelioration of Mitochondrial Dysfunction and Apoptosis of Two-Cell Mouse Embryos after Freezing and Thawing by the High Frequency Liquid Nitrogen Infusion

Amelioration of Mitochondrial Dysfunction and Apoptosis of Two-Cell Mouse Embryos after Freezing and Thawing by the High Frequency Liquid Nitrogen Infusion

Relationship between oxidative stress and embryotoxicity of hydrosalpingeal fluid.

Oxidative stress mechanisms are involved in the pathophysiology of many reproductive disorders. The objective of this study was to characterize oxidative stress parameters in hydrosalpingeal fluid (HSF) and examine their possible role in early embryo development. HSF was aspirated at laparoscopic salpingectomy in 11 infertile women. Reactive oxygen species (ROS), total (non-enzymatic) antioxidant capacity (TAC) and lipid peroxidation (LPO) were assayed. Two-cell mouse embryos were incubated with 25, 50 or 75% HSF and the blastocyst development rate was observed. ROS was detected in five of 11 (45%) HSF samples with a mean of 4.2 x 10(4) c.p.m. LPO was detected in all samples at a mean (+/- SD) value of 5575.4 +/- 6091.9 micromol/l malonaldehyde. The mean blastocyst development rate at 25, 5+/- 0 and 75% HSF and in the control group was 88.9 +/- 9.4, 65.7 +/- 19.1, 45.7 +/- 5.7 and 96.7% respectively (P < 0.0001). The blastocyst development rate was positively correlated to ROS concentrations (P < 0.02) but was not significantly related to LPO. The blastocyst development rate decreased with increasing concentrations of HSF. For the first time, the presence of ROS, LPO and TAC activity in human HSF was characterized. A possible role of oxidative stress in the embryotoxicity of HSF is suggested.

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos.

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.

Improvement of development of vitrified two-cell mouse embryos by vero cell coculture.

The purpose of this study was to determine the developmental potential of two-cell mouse embryos resulting from vitrification could increased using monolayer of Vero cells. Two-cell mouse embryos were divided into vitrified and nonvitrified groups. Embryos in the vitrified group were frozen with a combination of 10% ethylene glycol, 30% ficoll, and 0.5% M sucrose (EFS10) as cryoprotectants, and thawed rapidly with 0.5 M sucrose. The survived embryos were cultured either with Vero cells monolayer or in T6 medium. Accordingly the embryos of the nonvitrified group were also cultured. The rates of the development in all the groups were daily determined and statistically compared. At the end of the cultivation period, several expanded blastocysts from each group were stained with ethidium bromide and the mean number of the blastomers were counted and statistically compared. After 4 days of culture, the developmental potential of vitrified-thawed embryos were significantly reduced in Vero cell-free medium, and the mean cell number of embryos reaching the expanded blastocyst stage were also lower than that of nonvitrified embryos. With exception of last day of culture, Vero cell coculture, resulted in a significant increase in the rate of development of vitrified-thawed embryos as well as improved the mean cell number of expanded blastocysts. On the other hand, the mean cell number of expanded blastocysts of nonvitrified group was significantly improved in coculture group. However, the rate of embryo development except for the first day of culture was similar to that of medium alone. The developmental potential of vitrified-thawed embryos appears to be retarded in conventional medium and Vero cell monolayer is capable to eliminate the postthaw deleterious effect of vitrification during the first 3 days of cultivation, but not for a longer period.

Effect of protein phosphatase inhibitors on the development of mouse embryos: protein phosphorylation is involved in the E-cadherin distribution in mouse two-cell embryos.

Protein phosphorylation plays many important roles in cell functions and cell differentiation. To clarify the roles of protein phosphorylation in early embryonic development in mice, 2-cell embryos were cultured in the presence of various protein phosphatase inhibitors such as calyculin A, okadaic acid, cyclosporin A, tacrolimus (FK506) and benzyl-phosphonic acid. Calyculin A potently inhibited the 2-cell cleavage to the 4-cell stage. The concentration for 50% inhibition was 0.26 nM. At the same time, we found that calyculin A-treated 2-cell embryos showed a morula-like shape at a concentration of 2 nM in 24 h. It is well known that E-cadherin plays a key role in the compaction of late 8-cell stage embryos. In this report, we observed the distribution of E-cadherin protein using anti-E-cadherin antibody with a fluorescence microscope, and also evaluated the relative E-cadherin mRNA content at various stages of embryos by RT-PCR and ABI PRISM 7700 System (a real time PCR apparatus). The fluorescence intensity of E-cadherin increased along with the embryonic development. During the embryonic development from the 2-cell stage to the blastocyst stage, the relative E-cadherin mRNA content greatly increased in a time-dependent manner, while the mRNA did not increase with the addition of calyculin A at the 2-cell stage. Therefore, we observed the localization of the E-cadherin protein in calyculin A-treated embryos with a laser microscope. The distribution pattern of E-cadherin was altered by the addition of calyculin A from a scattered pattern throughout the embryos to a localized pattern at the cell-cell boundary region. These results strongly suggest that the distribution of E-cadherin protein is regulated by protein phosphorylation and/or dephosphorylation.

Nuclear accumulation of cyclin B1 in mouse two-cell embryos is controlled by the activation of Cdc2.

In the present study, the sequential expression and cellular localization of cyclin B1 was examined in two-cell mouse embryos to elucidate the mechanism of the two-cell block. One-cell embryos derived from in vitro fertilization were cultured with oviductal tissue (nonblocking condition) or without oviductal tissue (blocking condition) to establish the experimental conditions in which the embryos either overcome the two-cell block or do not. The amount of cyclin B1 gradually increased through the second cell cycle (through S to G2 phase). However, the difference was not observed between culture conditions. This showed that even embryos exhibiting the two-cell block normally synthesize cyclin B1 through the cell cycle. Cyclin B1 in embryos cultured under nonblocking condition accumulates in the nucleus during the transition from the G2 to the M phase, whereas that in embryos cultured in blocking condition localizes in the cytoplasm throughout the cell cycle. These data indicate that two-cell embryos cultured in blocking condition are able to normally synthesize cyclin B1 but have defects in nuclear accumulation of the protein. However, when two-cell blocked embryos were treated with okadaic acid, an activator of Cdc2 kinase, part of cyclin B1 in the embryos translocated into the nucleus. Moreover, treatment with butyrolactone I, a specific inhibitor of Cdc2 kinase, inhibits nuclear translocation of cyclin B1 in those embryos. These results suggest that Cdc2 kinase regulates the nuclear accumulation of cyclin B1 in mouse two-cell embryos.

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development.

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.

RINGO efficiently triggers meiosis resumption in mouse oocytes and induces cell cycle arrest in embryos

RINGO was identified as a Cdc2-binding and activating protein which is necessary and sufficient to trigger G2/M progression in Xenopus oocytes. We have investigated whether the function of RINGO is conserved in mouse oocytes. We show that RINGO induces Germinal Vesicle BreakDown (GBVD) in mouse oocytes. Mos is known to induce GVBD in mouse oocytes, and is also involved in the metaphase II arrest, which is due to the CSF (CytoStatic Factor) activity. We found that RINGO also has CSF activity and induces cleavage arrest after injection into one blastomere of a late two-cell mouse embryo, like Mos. However, RINGO also inhibits polar body extrusion of wild type mouse oocytes. The same effect of RINGO on first and second polar body extrusion was observed in Mos - /- mouse oocytes. The injection of RINGO mimics Mos effects: GVBD induction and efficient cleavage arrest. However, our results in mouse oocytes suggest that RINGO may have additional functions in meiosis regulation.

Betadine (povidone-iodine) is toxic to murine embryogenesis.

Objective: Exposure of gametes or embryos to potential toxins at any point during the IVF cycle—from vaginal egg retrieval to embryo transfer—may have a detrimental effect on embryogenesis and consequent pregnancy rates. Toxicity testing of a wide range of such factors, among which are: vaginal-disinfecting solutions; media; disposable equipment and ambient air, is the backbone of any stringent quality control program in IVF. Developmental rates of a single-cell and two-cell mouse embryo system have evolved into the “gold-standard” of quality control for IVF. This system was utilized in the current study, designed to assess the potential embryo toxic effect of the common practice of painting the external introitus with betadine solution prior to commencing ultrasound-guided oocyte retrieval.