Na+ / H+ exchanger-3 is involved in mouse blastocyst formation

The mouse blastocyst consists of the trophectoderm, the inner cell mass, and a fluid-filled cavity, the blastocoel. Formation and subsequent expansion of this cavity is important for further differentiation of the inner cell mass and successful implantation. Previous work provided evidence that vectorial transport of Na+ and CL- ions through the trophectoderm into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. As the activity of the Na+ / H+ exchanger (NHE) has been implicated as the exchanger responsible for facilitating the transtrophectodermal Na+ flux, the functional role of NHE in mouse blastocoel development was determined. Embryos were cultured in the presence of subtype-specific NHE inhibitors to examine the role of NHEs in blastocoel development. When 2-cell stage embryos were treated continuously with a specific inhibitor of NHE-1, cariporide, the embryos passed beyond the 8-cell stage and became blastocysts. However, in the presence of a specific inhibitor of NHE-3, S3226, the 2-cell stage embryos developed to the morula stage but formation of the blastocyst were inhibited in a dose-dependent manner. Cariporide did not inhibit the formation of the blastocoel cavity from the morula stage whereas S3226 did inhibit that process. S3226 also reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. An immunofluorescence study showed that NHE-3 was detected in the vicinity of the cell membrane of the trophectoderm, especially in the apical cell margins of the trophectoderm. These results suggest that NHE-3 is likely involved in blastocyst formation.     

Development of porcine embryos from the one-cell stage to blastocyst in mouse oviducts maintained in organ culture

Successful development of porcine embryos from the one-cell stage to the blastocyst stage has been accomplished using mouse oviducts in organ culture. One-cell embryos were transferred to mouse oviducts maintained in organ culture and were cultured for 6 days. Control embryos from each donor pig were cultured in a modified Krebs-Ringer bicarbonate medium. Thus control and experimental embryos obtained from the same individual pig could be directly compared. At the end of the culture period, all embryos were scored for the stage of development attained and stained to allow the cell number of each embryo to be counted. In medium alone, only 35.7% of the one-cell embryos reached the morula or blastocyst stage, whereas 78.1% of the one-cell embryos transferred to mouse oviducts reached the morula or blastocyst stage. Of those embryos reaching the morula or blastocyst stage, cell numbers were similar for the two treatments (medium alone vs. oviduct culture). The procedure described for mouse oviduct organ culture provides a simple method for culturing early-stage pig embryos to the morula or blastocyst stage prior to embryo transfer.     

ROCK inhibition prevents early mouse embryo development

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.     

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.     

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.     

The effect of quick-freezing in ethylene glycol on morphological survival and in vitro development of mouse embryos frozen at different preimplantation stages

This study was conducted to examine the effect of a quick-freezing protocol on morphological survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages. One-cell embryos were harvested from 6-to 8-wk-old CB6F1 superovulated mice, 20 to 23 h after pairing with males of the same strain and hCG injection. The embryos were cultured in human tubal fluid (HTF) containing 4 mg/ml BSA under mineral oil at 37 degrees C in 5% CO(2) plus 95% room air at maximal humidity. Twenty-four to 96 h after collection, the embryos were removed from culture and frozen at the 2 cell, 4 to 8-cell, compact morula, early blastocyst, expanding blastocyst and expanded blastocyst stages. To perform the quick-freeze procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) + 10 % fetal bovine serum (FBS) + 0.25 M sucrose + 3 M ethylene glycol (freeze medium) for 20 min at room temperature (22 to 26 degrees C) and loaded in a single column of freeze medium into 0.25-ml straws (4 to 5 embryos per straw). The straws were held in liquid nitrogen vapor for 2 min and immersed in liquid nitrogen. Embryos were thawed by gentle agitation in a 37 degrees C water bath for 20 sec and transferred to DPBS + 10 % FBS + 0.5 M sucrose (re-hydration medium) for 10 min at room temperature, rinsed 2 times in HTF plus 4 mg/ml BSA and then cultured for 24 to 96 h. Survival of embryos was based on their general morphological appearance after thawing and their ability to continue development upon subsequent culture in vitro. Survival of blastocysts after thawing also required expansion or reexpansion of the blastocoel after several hours in culture. Significant differences were found in the survival and development of mouse embryos at different developmental stages quick-frozen in ethylene glycol and sucrose: 2-cell embryos 43/84 (51%), 4 to 8-cell embryos 44/94 (47%), morulae and early blastocysts 56/70 (80%; P相似文献   

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

Asynchronous CDX2 expression and polarization of porcine trophoblast cells reflects a species‐specific trophoderm lineage determination progress model

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula‐stage embryos. In TE cells, polarization can regulate Hippo and Rho‐associated kinase (Rho‐ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula‐stage embryos but only in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species‐specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four‐cell stage porcine embryos resulted in increased CDX2‐positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, but the polarization progress is not conserved between the pig and the mouse, and results in a species‐specific trophoblast determination progress model.     

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.     

An in situ assessment of the routes and extents of colonisation of the mouse embryo by embryonic stem cells and their descendants

An embryonic stem (ES) cell line stably expressing lacZ under the control of an endogenous promoter has been isolated and used as a marker to follow the fate of ES cells injected into blastocysts and morulae, before midgestation. The results show a multisite pattern of blastocyst colonization by ES cells deposited into the blastocoel cavity and a low degree of mingling between ES cells and ICM cells. Furthermore, analysis of dispersal of ES cell descendants in postimplantation chimaeric embryos showed that colonization can be highly variable from one region of the embryo to another. In contrast, a high and reproducible degree of chimaerism was obtained when the ES cells were injected at the morula stage prior to ICM formation.     

The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro.

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.     

Rho-kinase inhibitor retards migration and in vivo dissemination of human prostate cancer cells

The Rho-kinase inhibitor, Y-27632, inhibited in vitro chemotactic migration to bone marrow fibroblast conditioned media and metastatic growth in immune-compromised mice of highly invasive human prostatic cancer (PC3) cells. Y-27632 also reduced myosin light chain phosphorylation and markedly altered the morphology of cells that developed numerous processes containing microtubules. A strikingly different, rounded phenotype was induced by an inhibitor of myosin light chain kinase, ML9. The M(110-130) subunit of the myosin phosphatase that is regulated by Rho-kinase was present in PC3 cells that contained significantly more RhoA than the less invasive, LNCaP cells. Y-27632 also inhibited angiogenesis as measured by endothelial cell tube formation on Matrigel. We conclude that invasiveness of human prostate cancer is facilitated by the Rho/Rho-kinase pathway, and exploration of selective Rho-kinase inhibitors for limiting invasive progress of prostate cancer is warranted.     

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.