Carboxylated ε-poly-L-lysine,a cryoprotective agent,is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.     

Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M).     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis

A study was conducted to identify the embryonic stage when the zygotic genome begins to direct development and to characterize protein synthesis in pig oocytes and embryos. Reproductive tracts of gilts were flushed to obtain unfertilized oocytes (UFO), zygotes (Z), 2-, 4-, and 8-cell embryos, compact morulae (M), initial blastocysts (IB), blastocysts (B), and hatched blastocysts (HB). Pig eggs and embryos were cultured in medium containing 1 microM L-[35S]methionine and evaluated for amino acid uptake, incorporation of the radiolabel into protein, and qualitative changes in protein profiles specific to each cleavage stage. Unfertilized oocytes sequestered 65.7 fmol methionine/4 h/embryo. Uptake of methionine decreased (p less than 0.05) from the Z (49.4), 2-cell (41.8), and 4-cell (37.6) embryonic stages to the M (8.97 fmol/4 h/embryo) stage. This downward trend was reversed at the IB, B, and HB stages when uptake increased to 37.3, 50.3, and 84.2 fmol/4 h/embryo, respectively. Incorporation of methionine into protein followed a similar pattern, being relatively higher in the UFO (21.0), Z (20.5), and 2-cell stages (16.0); decreased (p less than 0.05) at the 4-cell (6.67), 8-cell (6.84), and M (6.16) stages; and increased (p less than 0.05) at the IB (28.0), B (41.5), and HB (69.6 fmol/4 h/embryo) stages. Differences in protein profiles were observed for UFO, Z, 4-cell, and M stages using lysates of single embryos, one-dimensional SDS-PAGE, and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acquisition of essential somatic cell cycle regulatory protein expression and implied activity occurs at the second to third cell division in mouse preimplantation embryos

It is clear that G1-S phase control is exerted after the mouse embryo implants into the uterus 4.5 days after fertilization (E4.5); null mutants of genes that control cell cycle commitment such as max, rb (retinoblastoma), and dp1 are embryonic lethal after implantation with proliferation phenotypes. But, a number of studies of genes mediating proliferation control in the embryo after fertilization-implantation have yielded confusing results. In order to understand when embryos might first exert G1-S phase regulatory control, we assayed preimplantation mouse embryos for the acquisition of expression of mRNA, protein, and phospho-protein for max, Rb, and DP-1, and for the proliferation-promoting phospho-protein forms of mycC (thr58/ser62) and Rb (ser795). The key findings are that: (1) DP-1 protein was present in the nucleus as early as the four-cell stage onwards, (2) max protein was in the nucleus, suggesting function from the four-cell stage onwards, (3) both mycC and Rb all form protein was present at increasing quantities in the cytoplasm from the 2 cell and 4/8 cell stage, respectively, (4) the phosphorylated form of mycC phospho was present in the nucleus at high levels from the two-cell stage through blastocyst-stage, and (5) the phosphorylated form of Rb was detected at low levels in the two-cell stage embryo and was highly expressed at the 4/8-cell stage through the blastocyst stage. Taken together, these data suggest that activation of mycC phospho/max dimer pairs, (E2F)/DP-1 dimer pairs, and repression of Rb inhibition of cell cycle progression via phosphorylation at ser795 occurs at the earliest stages of embryonic development. In addition, the presence of max, mycC phospho, DP-1, and Rb phospho in the nuclei of embryonic and placental lineage cells in the blastocyst and in trophoblast stem cells suggests that a similar type of cell cycle regulation is present throughout preimplantation development and in both embryonic and extra-embryonic cell lineages.     

Phosphorylation of histone H3 serine 10 in early mouse embryos: Active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

DNA damage-sensing kinases mediate the mouse 2-cell embryo's response to genotoxic stress

A critical function of cells is the maintenance of their genomic integrity. A family of phosphoinositide-3-kinase-related protein kinases, which includes ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) kinases, play key roles in sensing DNA damage. ATM and ATR were demonstrated in the cleavage stages of mouse embryo development. Genotoxic stress was imposed by exposure to ultraviolet (UV) radiation (causes DNA strand breaks) or cisplatin (causes strand cross-links). UV irradiation or cisplatin treatment of 2-cell embryos in the G(2) phase of the cell cycle caused DNA damage as defined by increased phosphorylation of the H2A histone family, member X (H2AFX; previously H2AX) variant. UV irradiation caused a stable G(2)-M arrest, and cisplatin treatment allowed progression through mitosis followed by activation of a G(1)-S checkpoint. Both checkpoints were transformation-related protein 53-independent. Caffeine (inhibits both ATM and ATR), but not KU55933 (ATM-selective inhibitor), reversed the G(2)-M block induced by UV, inferring a primary role for ATR in sensing this form of DNA damage. Caffeine and KU55933 were equally effective in reversing the cisplatin-induced G(1)-S block, implicating ATM as the primary sensing enzyme. Breaching of either checkpoint by treatment with caffeine or KU55933 allowed embryos to progress through several further cell cycles, yet none developed to blastocysts. The results show, to our knowledge for the first time, that the G(2)-M and G(1)-S cell-cycle checkpoints in the early embryo are differentially regulated by ATM and ATR in response to genotoxic stress and that they act as an initial point for containment of genomic damage. Under conditions of extensive or persistent DNA damage, the demise of the embryo is the ultimate method of protecting genomic integrity.     

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.