Cultured Mouse Blastocysts Research Articles

Dynamic Oxygen Conditions Promote the Translocation of HIF-1α to the Nucleus in Mouse Blastocysts.

Oxygen tension is one of the most critical factors for mammalian embryo development and its survival. The HIF protein is an essential transcription factor that activated under hypoxic conditions. In this study, we evaluated the effect of dynamic oxygen conditions on the expression of embryonic genes and translocation of hypoxia-inducible factor-1α (HIF-1α) in cultured mouse blastocysts. Two-pronuclear (2PN) zygotes harvested from ICR mice were subjected to either high oxygen (HO; 20%), low oxygen (LO; 5%), or dynamic oxygen (DO; 5% to 2%) conditions. In the DO group, PN zygotes were cultured in 5% O2 from days 1 to 3 and then in 2% O2 till day 5 after hCG injection. On day 5, the percentage of blastocysts in the cultured embryos from each group was estimated, and the embryos were also subjected to immunocytochemical and gene expression analysis. We found that the percentage of blastocysts was similar among the experimental groups; however, the percentage of hatching blastocysts in the DO and LO groups was significantly higher than that in the HO group. The total cell number of blastocysts in the DO group was significantly higher than that of both the HO and LO groups. Further, gene expression analysis revealed that the expression of genes related to the embryonic development was significantly higher in the DO group than that in the HO and LO groups. Interestingly, HIF-1α mRNA expression did not significantly differ; however, HIF-1α protein translocation from the cytoplasm to the nucleus was significantly higher in the DO group than in the HO and LO groups. Our study suggests that dynamic oxygen concentrations increase the developmental capacity in mouse preimplantation embryos through activation of the potent transcription factor HIF-1α.

The effect of laser-assisted hatching on the methylation and expression pattern of imprinted gene IGF2/H19 in mouse blastocysts and offspring

This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring. The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR. The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta. These observations suggested that drilling used for hatching of in vitro cultured mouseblastocysts may improvethe production of offspring.

Leukaemia inhibitory factor increases αvβ3 integrin expression in cultured mouse blastocysts

Blocking leukaemia inhibitory factor (LIF) has a negative effect on the implantation of mouse embryos. It has been determined that LIF regulates the expression of endometrial αvβ3 integrin, but its role in trophoblast cells remains unclear. The aim of this study was to evaluate the effect of 1000 units mL−1 LIF on the expression of LIF receptor alpha (Lifr), integrin alpha V (Itgav), integrin beta 3 (Itgb3) and some apoptosis-related genes in blastocysts formed from 8-cell mouse embryos. Embryos obtained from superovulated NMRI mice were divided into four groups and cultured to the blastocyst stage. Groups 1 and 2 were simple embryo cultures in the absence and presence of LIF respectively; Groups 3 and 4 were embryo cocultures in the absence and presence of LIF respectively. The number of cells in blastocysts was counted and the expression of Itgav, Itgb3 and some apoptosis-related genes was determined using real-time polymerase chain reaction. The blastocyst rate, cell count, survival rate and expression of the Itgav and Itgb3 genes were significantly higher in the coculture groups and in the simple culture group with LIF than in the simple culture group without LIF. In conclusion, LIF improves the growth and cell count of blastocysts, increases the expression of the Itgav and Itgb3 genes in blastocysts and plays an important role in the success of implantation.

373 - Differential Effect of Nicotinamide on MMP-9 Expression Induced by High Glucose or Hydrogen Peroxide in Cultured Mouse Blastocysts

373 - Differential Effect of Nicotinamide on MMP-9 Expression Induced by High Glucose or Hydrogen Peroxide in Cultured Mouse Blastocysts

Studies on the trophoblastic invasion of the endometrium by using cultured mouse blastocysts as a model

Studies on the trophoblastic invasion of the endometrium by using cultured mouse blastocysts as a model

Ammonium Alters the Proteome of Cultured Mouse Blastocysts

Ammonium has been shown to induce several pathologies in the preimplantation mouse embryo including; reduced blastocyst cell number and ICM development, altered gene expression and impaired metabolism. It was therefore the aim of this study to investigate the effects of exposing preimplantation mouse embryos to ammonium on the protein content (proteome) of resultant blastocysts.

Ammonium alters gene expression and imprinting of H19 in cultured mouse blastocysts

Objective: Amino acids are key regulators of embryo physiology and development in culture. However, when amino acids are incubated at 37°C they spontaneously breakdown to produce ammonium. Accumulation of ammonium in culture medium perturbs embryo metabolism, reduces homeostatic regulation, inhibits blastocyst development, decreases blastocyst cell number, impairs ICM development and increases apoptosis. The effect of ammonium on gene expression and imprinting has not been determined. Therefore this study investigated the effects of ammonium on gene expression and the imprinting of H19 in mouse blastocysts.

Time-lapse videomicrographic analyses of contractions in mouse blastocysts.

Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts.

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 ± 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 ± 5.48 and 217.8 ± 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Differential expression of mRNAs encoding laminin chain variants during in vitro development of mouse blastocysts.

Expression of mRNAs encoding ten laminin chain variants was studied by reverse transcription-polymerase chain reaction (RT-PCR) of mRNA extracted from cultured mouse blastocysts. alpha1, beta1 and gamma1 mRNAs, of which products form laminin-1, were the only chains expressed in blastocysts before hatching. alpha4 and alpha5 mRNAs were additionally expressed after attachment of blastocysts to culture dishes. alpha2 and beta2 mRNAs started to be expressed at the stage of allantois formation. Among ten laminin chains, only the mRNA encoding components of laminin-5 (alpha3, beta3 and gamma2) was missing in embryos up to the stage of allantois formation.

Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts.

Culture of in-vivo-developed mouse blastocysts in a simple culture medium based on a balanced salt solution supplemented with carbohydrates for 3 h significantly perturbed embryo metabolism. Maximal perturbation occurred after just 6 h of culture. Similarly, culture of rat blastocysts in a simple culture medium for 3 h also resulted in perturbed metabolism. Cultured mouse and rat blastocysts both had an abnormally elevated rate of glycolysis of approximately 100% after culture (P < 0.05). Rates of pyruvate oxidation by mouse blastocysts were also significantly reduced after culture in a simple medium for 6 h (P < 0.01). Furthermore, the developmental competence of mouse blastocysts after transfer was significantly reduced by just 6 h of culture in a simple medium (P < 0.05). Addition of Eagle's amino acids or vitamins to the culture medium reduced the perturbation of both the glycolytic activity and oxidative capacity of cultured mouse blastocysts and acted in synergy to further the inhibition. Importantly, culture with amino acids and vitamins prevented any loss of viability of mouse blastocysts after culture for 6 h. It can be concluded that the mouse blastocyst is sensitive to its environment and that culture-induced stress results in the loss of normal cellular function, as manifested in this case by an abnormal pattern of glucose utilization and loss of viability.

Expression and localization of membrane type matrix metal loproteinase-1 (MT1-MMP) in trophoblast cells of cultured mouse blastocysts and ectoplacental cones

Membrane type matrix metalloproteinases (MT-MMPs) possess a C-terminal transmembrane domain and are expressed on the cell membrane. It was suspected, therefore, that MTl-MMP might play an important role in the trophoblastic invasion during implantation. The patterns of expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) were examined immunocytochemically in cultured mouse blastocysts and excised extoplacental cones (EPCs). MT1-MMP immunoreactivity was present in the giant trophoblast cells located at the periphery of the spreading trophoblast of cultured blastocysts and the outgrowths of cultured EPCs, but not in the densely packed trophoblast cells in both the blastocysts and the EPCs. It appears likely that MT 1-MMP expressed on the edge of the invading trophoblast facilitates the trophoblastic invasion by cleaving proMMP-2, a known substrate of MT1-MMP, in the decidua. Immunohistochemical examination of early conceptuses confirmed that the trophoblast cells actively invading the endometrium in vivo express MT1 .WIMP strongly. It is suggested, furthermore, that the expression of MT1-MMP might be downregulated by cell-cell contact in mouse trophoblast cells, as in the mouse mammary epithelial cell line HCII.

Enhancement of methylmercury toxicity by l-cystine in cultured mouse blastocysts

Expanded mouse blastocysts incubated with 1 to 2 μM methylmercury (MeHg) in modified Eagle's basal medium (BME + AA), which contains amino acids, collapsed and degenerated within 24 h. In contrast, blastocysts incubated with the same concentration of MeHg in egg culture medium (ECM), which does not contain amino acids, survived and remained expanded as control embryos did. By systematically omitting each BME amino acid from BME + AA and adding each BME amino acid to egg culture medium, we determined that l-cystine (0.5 mM in BME + AA) was the component of BME + AA that was responsible for the enhancement of the toxicity of MeHg. The shortest incubation time during which the cystine-enhanced MeHg toxicity became irreversible was 2 h, and the addition of any of the neutral BME amino acids (except threonine) or non-BME neutral amino acids (alanine, glycine, or serine) during the 2 h incubation eliminated or reduced the cystine-enhanced MeHg toxicity. Basic amino acids (except histidine) were less effective in protecting embryos: Glutamine and lysine reduced the toxic effect only slightly, and arginine had no effect. dl-buthionine sulfoximine (7.5 mM), a specific inhibitor of glutathione, also reduced cystine-enhanced MeHg toxicity. It therefore appears that cystine enhances MeHg toxicity indirectly, at least in part, by stimulating the synthesis of cellular glutathione, which may in turn enhance MeHg transport. In the absence of cystine, 10 μM MeHg (2 h incubation) was necessary to cause the collapse and degeneration of all blastocysts treated. The inorganic mercury compound HgCl 2 was less toxic to blastocysts: 50 μM was needed to cause the collapse and degeneration of all the blastocysts, and its toxicity was not enhanced by the addition of cystine.

Morphodynamics of outgrowths of mouse trophoblast in the presence and absence of a monolayer of uterine epithelium.

Mouse blastocysts were cultured in the presence and absence of a confluent uterine, luminal, epithelial monolayer in order to assess (10 comparative developmental abilities and (2) the existence and nature of embryo-uterine intercellular contacts. Embryonic development and mural trophoblast outgrowth were examined by light, transmission and scanning electron microscopy, and by time-lapse cinematography. The results demonstrate that cultured mouse blastocysts are capable of development that is essentially equivalent to the early egg cylinder stage in the presence and absence of a uterine epithelial substratum. Penetration of the endometrium and displacement of uterine luminal epithelial cell in vivo is paralleled in vitro by the displacement of uterine cells from the advancing edge of outgrowing mural trophoblast. The ability of mural trophoblast to continue to outgrow and displace uterine cells persists for a finite period of time. The findings are discussed with respect ot (1) the nature of embryo-uterine contact in vivo and (2) mechanisms proposed to explain trophoblastic displacement of uterine luminal epithelial cells during normal implantation.

Identification and immunoprecipitation of cytoskeletal proteins from murine extra-embryonic endodermal cells.

Murine extra-embryonic endodermal cells derived from either teratocarcinomas or cultured mouse blastocysts contain two protein species of Mr = 55,000 and Mr = 50,000 endodermal cytoskeletal proteins A and B, respectively) that are insoluble in nonionic detergent and 1 M NaCl and are not found in abundance in embryonal carcinoma cells, the stem cells of teratocarcinomas. Antiserum raised against the electrophoretically purified endo B protein immunoprecipitated endo B from [35S]methionine-labeled cell lysates of three parietal endodermal cell lines, a presumptive visceral endodermal cell line, and a mouse hepatoma line. Immunoprecipitable endo B was not found in murine embryonal carcinoma cells, fibroblasts, myoblasts, keratinocytes, erythroleukemic or neuroblastoma cells. These results are consistent with the view that endo B is not tubulin, vimentin, desmin, or keratin. Amino acid composition data, partial peptide analysis of immunoprecipitated endo B, and immunoprecipitation analysis with antikeratin serum support the suggestion that endo B is not a keratin. Indirect immunofluorescent staining of parietal endodermal cells with the endo B antiserum resulted in the fluorescence of a fibrillar cytoskeletal network. The synthesis of endo B was increased dramatically when embryonal carcinoma cells were induced to differentiate by treatment with retinoic acid. Endo B appears to be a cytoskeletal protein that is synthesized when malignant embryonal carcinoma cells differentiate to benign extra-embryonic endoderm.

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth.

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B4) but no trace of LDH-5 (A4) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.

Inhibition of post-implantation development of mouse blastocysts in vitro after cyclophosphamide treatment in vivo.

IT has been widely believed that embryos, after treatment in the preimplantation period, either die before implantation or survive to term without being malformed1,2. But evaluation of the embryolethal effect of cyclophosphamide (CPA) before implantation in the rat has revealed effects of the early treatment on both embryo and mother around and after implantation3. Further investigations of the development of CPA-treated preimplantation rat embryos are impossible because rat embryos have not been cultured successfully in vitro beyond the time of implantation. On the other hand, routine culture of mouse embryos in vitro during the same period has been standardised4‐6. Such systems are sufficiently sensitive for investigations of genetically-determined abnormalities7,8 and the effects of in vitro exposure to ultraviolet and X irradiation9,10, 3H-thymidine11 and metabolic inhibitors12‐14. To elucidate further the action of CPA on the embryos after maternal treatment during the pre-implantation period, we have used mouse blastocysts to repeat our studies with CPA in the rat, and we have also cultured mouse blastocysts for 120 h from CPA-treated mothers. With concentrations of CPA that did not affect the morphology of blastocysts from treated mothers but only the cell number at the time of implantation, development of the blastocysts in vitro was inhibited in a dose-dependent manner. This system holds promise as a test for putative teratogens.

Interference with in vitro Development of Mouse Inner Cell Mass by 24-h Treatment with 5-bromodeoxyuridine

Summary Cultured mouse blastocysts were treated with 5-bromodeoxyuridine (BUdR) for 24-h periods at various stages to determine the effect on development of the inner cell mass (ICM). ICM development was most sensitive to BUdR (10 -7 –10 -6 M) when embryos were treated at the late blastocyst stage, and the sensitivity decreased at each consecutive stage. The decrease in ICM sensitivity corresponded with the time of morphological differentiation into endoderm and ectoderm. At low concentrations (10 -8 –10 -7 M), BUdR also frequently caused abnormal differentiation of ICMs, which appeared flat and had small ectodermal cores. Light microscopic examination of thick sections revealed that ectoderm cells of abnormal embryos contained numerous vacuoles, which were absent from control embryos. Endoderm cells of these embryos had fewer microvilli than control embryos and lacked apical vacuoles. Autoradiography showed that the amount of 3 H-BUdR incorporated into the DNA decreased as embryos developed, and this decrease almost completely accounted for the decrease in the BUdR effect. We conclude that BUdR treatment of mouse embryos impairs gene expression necessary for endoderm and ectoderm differentiation and that the critical period for this effect includes, but may not be limited to, the late blastocyst and attachment stages.

Morphology of mouse egg cylinder development in vitro: A light and electron microscopic study

AbstractPhase contrast, light, and electron microscopy showed that mouse egg cylinder development from the blastocyst stage in vitro closely resembled the egg cylinder stages of the fourth to seventh days of gestation in vivo. Primary endoderm and ectoderm formed on day 2 of culture, and on day 3 visceral and parietal endoderm could be distinguinshed. Mesoderm formed on days 6.5‐7 by delaminating from a thickened region of embryonic ectoderm. Extraembryonic ectoderm developed as single layer of cuboidal epithelial cells that appeared to originate from polar trophoblast cells. By day 8 of culture egg cylinders had an ectoplacental cavity, an exocoelom, a proamnionic cavity, a chorion, and an allantois and resembled egg cylinders in vivo after seven days' gestation. However, cultured blastocysts took longer to develop to these egg cylinder stages than embryos in vivo (8 days in vitro vs. 4 days in vivo) and were shorter (600 μm in vitro vs. 700 μm in vivo). The development of mural trophoblast, parietal endoderm, Reichert's membrane, and ectoplacental cone also differed from that of their counterparts in vivo. Nevertheless, because the egg cylinder itself, which produces the embryo proper, develops similarly in vitro as in vivo, we suggest that cultured mouse blastocysts provide an excellent model for studies on early postimplantation embryogenesis.

Histologic Analyses of Experimental Tumors From Mouse Blastocyst-Derived Cell Lines

Previous work has shown that established cell lines can be derived from cultured mouse blastocysts. Although these cell lines differed from one another morphologically, immunologically, and biochemically, it has not been easy to determine what embryonic cell type (if any) each cell line represents. We showed in this report that these blastocyst-derived lines could produce tumors in syngeneic mice at low yields and presented the results of histologic analyses of the tumors which we carried out to learn more about the kinds of cells present in each of the lines. Tumors derived from cell line MB2 contained two kinds of cells. Most of the cells were similar to those found in so-called "parietal yolk sac carcinomas," secreted Reichert's membrane-like material, and could be found in subsequent transplant generations. The other cell type resembled trophoblast giant cells morphologically and did not survive transplantation. These results showed that MB2 contained cells which were either parietal endoderm cells or a closely related precursor. The tumor derived from MB4 consisted of small, closely packed cells intermixed with larger cells with foamy cytoplasm and occasional giant cells. Cells in subsequent passages of the tumor were often organized into acinar clusters. Biochemical evidence has suggested that MB4 cells resembled one or both components of visceral yolk sac. Visceral yolk sac sarcomas have not previously been reported; the MB4-derived tumor may prove to be the first. Tumors derived from line MB31 contained both fibroblastic and epithelioid cell types which could not be more definitively identified. Cultures derived from each kind of tumor in each case contained cells morphologically characteristic of those in the originally inoculated population. Our results showed that tumors produced from blastocyst-derived cell lines did not resemble the teratocarcinomas which formed when blastocysts were implanted into an ectopic site in vivo: They did not contain a wide variety of cell types, nor did they possess pluripotent embryonal carcinoma "stem" cells characteristic of the teratocarcinoma. That each blastocyst-derived line gave rise to a different kind of tumor suggested that each line may have been made up of cells "trapped" at some stage characteristic of the development of a particular embryonic or extraembryonic tissue. However, each tumor did contain at least two distinct cell types; this suggested that cells in the blastocyst-derived lines, though restricted to their developmental potential, may not yet have been completely determined.