Mouse Morulae Research Articles

Effects of Re-Vitrification of Mouse Morula and Early Blastocyst Stages on Apoptotic Gene Expression and Developmental Potential.

Objective Re-vitrification of embryos immediately after thawing or after a culture period could be used to preserve the extra embryos after embryo transfer. This study aims to clarify the effect of re-vitrification on Bax and Bcl-2 gene expressions of compact and early blastocyst stage embryos. Materials and Methods This experimental study was performed on mouse embryos. We collected 8-cell stage embryos (n=400) from female mature mice, 60-62 hoursafter injection of human chorionic gonadotropin (hCG). The embryos were divided into 5 groups: fresh (n=80), vitrified at the 8-cell stage (n=80), vitrified at the blastocyst stage (n=80), vitrified at the 8-cell stage, and re-vitrified at the compact (n=80) and early blastocyst stages (n=80). Embryos were vitrified by cryolock. We analyzed the developmental rates of the vitrified-warmed embryos with the chi-square test. Quantitative polymerase chain reaction (qPCR) data were analyzed with SPSS version 16 using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P<0.05 were considered statistically significant. Results The expanded blastocyst formation rate showed a significant difference in re-vitrified embryos compared with fresh embryos (P<0.05). However, this result was similar between the two re-vitrified groups. Our data showed a significant difference in expression of the Bax and Bcl-2 genes between re-vitrified and fresh embryos (P<0.05). Expressions of the Bax and Bcl-2 genes showed no significant difference between the two re-vitrified groups. ConclusionBased on our study, re-vitrification could affect developmental rate and expressions of the Bax and Bcl2 genes.

Vitrification transiently alters Oct-4, Bcl2 and P53 expression in mouse morulae but does not affect embryo development in vitro

This study was conducted to determine the impact of vitrification on the expression of genes regulating pluripotency and apoptosis in mouse morulae. The morulae were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution without freezing (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In vitro development was evaluated by morphology and assessed by the blastocyst rate and the blastocyst total cell number. Gene expression in morulae and blastocysts was assessed by quantitative Real Time-PCR (qRT-PCR) and western blot. The results showed that at morulae stage, the POU class 5 homeobox1 (Oct-4) and B-cell lymphoma2 (Bcl2) mRNA levels of vitrification group were significantly lower (P < 0.05) than those of control. Strikingly, the p53 mRNA level was significantly higher in vitrification group. However, the Oct-4, Bcl2 and p53 mRNA levels in mouse blastocysts were not statistically different. Furthermore, western blot results showed that there was no significant difference in Oct-4, Bcl2 and p53 expression at protein level in mouse morulae among three groups. Additionally, the blastocyst rate (96.67%–100.00%) and the average cell number of blastocysts (89.67–92.33) were similar between all groups. The data demonstrate that vitrification transiently changes the mRNA expression of several key genes in mouse morulae regulating early embryo development but does not affect embryo developmental potential in vitro.

091 Aquaporin 9 plays a significant role in the channel-dependent movement of Me2SO and acetamide in mouse morulae

The permeability to water and cryoprotectants of the plasma membrane affects the suitable condition for cryopreservation of the cell. Previously, we showed that water, glycerol, ethylene glycol, Me 2 SO, and acetamide move through mouse morulae by facilitated diffusion. We also showed that the facilitated diffusion of water, glycerol, and ethylene glycol relies on aquaporin 3, an aquaglyceroporin (Edashige et al., 2007), and suggested that AQP9, another aquaglyceroporin, can transport Me 2 SO, acetamide, and other cryoprotectants when AQP9 was exogenously expressed in mouse oocytes (Edashige et al., in Cryo 2011). In the present study, we examined the transporting-specificity of AQP9 endogenously expressed in mouse morulae. In addition, we examined whether AQP9 expressed in morulae was involved in the tolerance to cryopreservation. As shown in previous studies, the suppression of the endogenous expression of AQP9 in morulae decreased the permeability to Me 2 SO and acetamide but not that to other cryoprotectants. And the exogenous expression of AQP9 in mouse oocytes, in which water/cryoprotectant channels are not expressed at the functional level, significantly increased not only the permeability to Me 2 SO and acetamide but also to glycerol and ethylene glycol. Cystic fibrosis transmembrane conductance regulator (CFTR), which has been reported to modulate the transporting-property of AQP9, was markedly expressed in oocytes but not in morulae. The suppression of the endogenous expression of CFTR in oocytes decreased the permeability to glycerol and ethylene glycol of oocytes enhanced by exogenous expression of AQP9 but not that to Me 2 SO and acetamide. These results indicate that the transporting-property of AQP9 is modulated by CFTR, and suggest that AQP9 endogenously expressed in morulae transports only Me 2 SO and acetamide because CFTR is not expressed in morulae. When mouse morulae were vitrified with DFS20, a Me 2 SO -based solution, by a one-step method, most of intact morulae survived and had the ability to develop into blastocysts after warming, whereas only 40% of AQP9-suppressed morulae survived and no embryos had the ability to develop, suggesting that AQP9 plays an important role in the tolerance to vitrification. These results will provide important information for understanding the movement of cryoprotectants in mouse embryos. Source of funding: The Japan Society for the Promotion of Science. Conflict of interest: None declared. keisuke@cc.kochi-u.ac.jp

Slow freezing and vitrification of mouse morula and early blastocysts

To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing. Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined. Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively). Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.

Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers

BackgroundIt is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers.MethodsIn experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls.ResultsNo difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls.ConclusionsFull-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.

2P135 マウス胚盤胞と桑実胚間での異なるメカニカルストレス応答(09.発生・分化,ポスター,日本生物物理学会年会第51回(2013年度))

2P135 マウス胚盤胞と桑実胚間での異なるメカニカルストレス応答(09.発生・分化,ポスター,日本生物物理学会年会第51回(2013年度))

The Impact of Ovarian Stimulation on the Expression of Candidate Reprogramming Genes in Mouse Preimplantation Embryos

Ovarian stimulation with gonadotrophins is an integral part of assisted reproductive technologies in human subfertility/infertility treatment. Recent findings have associated ovarian stimulation with the increased incidence of imprinting disorders in humans as well as defects in genome-wide methylation reprogramming and, in particular, imprinting in mice. Here, we present the first study that determined the impact of ovarian stimulation on the expression of developmentally important reprogramming genes (Apex1, Lig1, Lig3, Mbd2, Mbd3, Mbd4, and Polb) in single early mouse morula embryos (16-cell stage). Using absolute quantification of mRNA by quantitative real-time PCR, we observed an association of ovarian stimulation with a downregulation of mRNAs encoding the base excision repair proteins APEX1 and POLB as well as the 5-methyl-CpG-binding domain protein MBD3 in individual morula embryos. Whole mount immunofluorescence staining of early and late morula embryos with an antibody against APEX1 revealed individual embryos with lower protein expression levels after ovarian stimulation and a correlation of mRNA expression with protein abundance. Our data argue for a negative impact of ovarian stimulation during female gametogenesis and/or early embryo development affecting the expression of candidate reprogramming factors.

The Role of Aquaporin 9 in the Movement of DMSO and Acetamide in Mouse Morulae.

The permeability to water and cryoprotectants of the plasma membrane affects the suitable condition for cryopreservation of the cell. Previously, we showed that water, glycerol, ethylene glycol, DMSO, and acetamide move through mouse morulae by facilitated diffusion. We also showed that aquaporin 3, an aquaglyceroporin, plays an important role in the facilitated diffusion of water, glycerol and ethylene glycol, but that other channels are involved in the facilitated diffusion of DMSO and acetamide. In this study, we try to clarify whether aquaporin 9, another aquaglyceroporin, is involved in the movement of DMSO and acetamide first by suppressing the expression of aquaporin 9 in morulae by injecting double stranded RNA of mouse aquaporin 9 at the 1-cell zygote stage, and secondly by artificial expression of aquaporin 9 in oocytes. The suppression of the expression of aquaporin 9 in morulae significantly decreased the permeability to DMSO and acetamide, but not the permeability to water, glycerol and ethylene glycol, suggesting that aquaporin 9 plays an important role in the facilitated diffusion of DMSO and acetamide in morulae. The exogenous expression of aquaporin 9 in oocytes significantly increased the permeability to DMSO and acetamide. However, the exogenous expression also significantly increased the permeability to glycerol and ethylene glycol. It has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) is a regulator of aquaporin 9 and can modulate the permeability-property of aquaporin 9. Therefore, we examined the expression of CFTR in oocytes and morulae. CFTR was markedly expressed in oocytes but not in morulae. Compared to the permeability of oocytes injected with aquaporin 9 cRNA alone, the additional injection of CFTR si-RNA significantly decreased the permeability to glycerol and ethylene glycol but not that to DMSO and acetamide. These results suggest that aquaporin 9 plays an important role in the movement of DMSO and acetamide in mouse morulae, and that aquaporin 9 also transports ethylene glycol and glycerol but only when CFTR is present. These results will provide important information for understanding the movement of water and cryoprotectants in mouse embryos. This research was supported by Grants-in-Aid for Scientific Research from JSPS (KAKENHI).

New Insights into the Biophysics of Cellular Polarization during Embryogenesis

New Insights into the Biophysics of Cellular Polarization during Embryogenesis

Ouabain Stimulates a Na+/K+-ATPase-Mediated SFK-Activated Signalling Pathway That Regulates Tight Junction Function in the Mouse Blastocyst

The Na+/K+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

74. The role of aquaporin 9 in the movement of Me 2SO and acetamide in mouse morulae

74. The role of aquaporin 9 in the movement of Me 2SO and acetamide in mouse morulae

Governing cell lineage formation in cloned mouse embryos

Blastomeres of the pre-implantation mouse embryo form trophectoderm and inner cell mass via a process that requires the transcription factors Tead4, Cdx2, Oct4 and Nanog. In mouse morulae cloned by somatic cell nuclear transfer, we observed that the trophectoderm transcription factor Cdx2 is expressed very differently at the protein level compared to time- and stage-matched fertilized counterparts. Protein levels of Cdx2 in cloned embryos appear ‘erratic,’ i.e. are widely distributed, when plotted as histograms. In contrast to Cdx2, protein levels of the upstream factor Tead4 and of inner cell mass transcription factors Oct4 and Nanog are similar in cloned and fertilized embryos. These observations suggest that trophectoderm formation is initiated but not maintained correctly in cloned mouse morulae, which is consistent with cloned blastocysts' limited implantation and post-implantation success. Because a cell's ability to differentiate is greatly enhanced if it is surrounded by more cells differentiating the same way, a concept designated community effect by Gurdon, we reasoned that the insufficient cell numbers often observed in cloned embryos might lead to premature Cdx2 expression and differentiation of blastomeres into trophectoderm. Therefore, we created larger cloned embryos by aggregating them at the 4-cell stage. Homologous aggregation stimulates expression of multiple signaling pathways' components and results in cloned embryos with levels of Cdx2 similar to fertilized embryos. Most of the resultant morulae and blastocysts consist of cells of all three founders, indicating that aggregation increases stability of all of the individual components. We conclude that the induction of pluripotency in cloned embryos is more efficient than previously assumed, and we propose that a minimum cell number is necessary to stabilize pluripotency and inhibit premature expression of Cdx2 in cloned mouse embryos.

Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival

In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10–EFS50) contained 10–50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at −80 °C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24 h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30–120 s at 25 °C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79–93%) and EFS40 (96–99%). After slow warming, survival decreased with both EFS30 (48–62%) and EFS40 (44–64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at −80 (25–75%), −60 (7–49%), −40 (0–41%), or −20 °C (26–60%). When samples were kept at −80 °C for 24 h, the survival decreased to 0–14%. These results suggest that ICI forms at a wide range of temperatures including −80 and −20 °C, more likely between −60 and −40 °C, and the ice forms not only quickly but also slowly.

Effects of sucrose treatment on the development of mouse nuclear transfer embryos with morula blastomeres as donors

In this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180+/-10 s, 130+/-10 s and 120+/-10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p>0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.

33. The role of urea transporters in the movement of cryoprotectants across the plasma membrane in mouse morulae

33. The role of urea transporters in the movement of cryoprotectants across the plasma membrane in mouse morulae

The Role of Aquaporin 3 in the Movement of Water and Cryoprotectants in Mouse Morulae1

The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.

Effect of In-Straw Thawing on In Vitro- and In Vivo-Development of Vitrified Mouse Morulae

For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25°C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.

135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (&amp;lt;−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P &amp;gt; 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P &amp;lt; 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P &amp;lt; 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C

35. The role of aquaporin-3 in the movement of water and cryoprotectants across the plasma membrane in mouse morulae

35. The role of aquaporin-3 in the movement of water and cryoprotectants across the plasma membrane in mouse morulae

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos.

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.