Oct4 protein remains in trophectoderm until late stages of mouse blastocyst development

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

提高人胚胎干细胞建系率的优选培养体系与方法

目的:建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系,寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方法,提高人胚胎干细胞(human embryonic stem cell,hESC)建系效率,建立人胚胎干细胞库。方法:将179枚IVFDay3废弃的胚胎放入优选培养体系中培养(G2.5培养液中添加10%人血清蛋白,人白细胞抑制生长因子(hLIF),碱性成纤维细胞生长因子(bFGF))。到Day7将形成的囊胚全部用机械法分离ICM,接种于丝裂霉素C灭活处理的原代小鼠胚胎成纤维细胞(MEF)上,培养8-9天,每4-5天传代1次。结果:优选培养体系的囊胚形成率为29.1%(52/179),其中A级囊胚形成率为11.2%(20/179),50个ICM贴壁生长,20个出现克隆形态,成功建立11株hESC(FY-hES-11至FY-hES-21)。11株hESC均具有共同的多能性生物学特性。结论:优选培养体系可以明显提高囊胚形成的质量,促进ICM的增值,纯熟的机械切割法可以避免损伤ICM并提高其贴壁率,原代灭活的MEF饲养层可以明显促进细胞增殖。     

Cell allocation to the inner cell mass and the trophectoderm in rat embryos during in vivo preimplantation development

Summary The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.     

Retinoid receptor-specific agonists regulate bovine in vitro early embryonic development, differentiation and expression of genes related to cell cycle arrest and apoptosis

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 microM, 1 microM and 10 microM), ATRA 0.7 microM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 microM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 microM, while a dose-dependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r=0.93; p<0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation.     

Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo

The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.     

Effect of co-culturing of half-destroyed and intact 4-cell mouse embryos in varying ratios on subsequent in vitro development

We studied the co-culturing effect of intact and half-destroyed 4-cell mouse embryos on blastocyst formation rate and cell counts. A laser beam was used to produce a hole and destroy an adjacent blastomere in two opposite areas of the zona in the experimental group (n = 342), and to open two opposite zonal holes in the controls (n = 318). Control and half-destroyed embryos were cultured together in varying ratios of 10:0, 7:3, 5:5, 3:7, and 0:10 (group 1-5, respectively) for 48 h in 10 μl drops of cleavage medium. They were then separated and cultured in blastocyst medium for 24 h. The results showed that half-destroyed embryos had no effect on the blastulation rates of controls (97-100%, P = 0.28). Neither was there a difference in the number of ICM (27.3 ± 6.7, 29.4 ± 9.9, 27.7 ± 9.3, 26.5 ± 6.4, in group 1-4, respectively; P = 0.491), TE (47.7 ± 18.6, 52.3 ± 13.9, 48.4 ± 19.2, 57.3 ± 12.9, in group 1-4, respectively; P = 0.101), nor total cells (75.0 ± 19.5, 81.3 ± 17.1, 76.1 ± 19.6, 83.7 ± 16.2, in group 1-4, respectively; P = 0.188) in the resulting blastocysts. However, among half-destroyed embryos, cleavage arrest decreased (58.3%, 39.6%, 17.9%, and 8.3%, in group 5 to 2, respectively; P < 0.001) and blastocyst development increased (38.3%, 58.2%, 72.6%, and 88.9%, in group 5 to 2, respectively; P < 0.001) following co-culturing with intact controls. These embryos had a higher number of ICM cells (P = 0.035), but no significant changes in TE (P = 0.262) and total cell counts (P = 0.065). The findings indicate that the co-culturing of half-destroyed with intact embryos increased the blastulation rate of the first but had no effect on the latter.     

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.     

Specific PKC isoforms regulate blastocoel formation during mouse preimplantation development

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (delta, theta, iota/lambda, zeta) PKC isoforms and PKCmicro/PKD1 showed partial colocalisation with the tight junction marker ZO-1alpha+ in TE and all four PKCs (delta, theta, iota/lambda, zeta) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKCdelta and zeta activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase alpha1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase.     

Sequential expression of maternally inherited phosphoglycerate kinase-1 in the early mouse embryo

Enzyme activities of X-linked phosphoglycerate kinase (PGK-1) and autosomal glucose phosphate isomerase (GPI-1) were determined in intact mouse blastocysts and isolated inner cell masses (ICMs). Blastocysts were recovered from the uterus on day 4 of gestation and cultured overnight in vitro. ICMs were isolated by treatment with calcium ionophore A23187. On day 4, approximately 35% of the total activity of both PGK-1 and GPI-1 was located in the ICM. After overnight culture, the PGK-1 activity of the whole blastocyst nearly doubled, due to the activation of only the maternally derived gene coding for PGK-1. In the ICM, however, a pronounced decrease of PGK-1 activity was measured: only 10% of the total PGK-1 activity was measured in the ICM on day 5. In contrast to PGK-1, GPI-1 activity of the intact blastocyst remained stable from day 4 to day 5. In the ICM, the GPI-1 activity did decline, but to a lesser extent than PGK-1 activity: 20% of total GPI-1 activity was found in the ICM on day 5. These results, when compared with the data of Handyside and Hunter, suggest that the decline in GPI-1 activity in the ICM is due to a change in the ratio of trophectoderm (TE) to ICM cells. The greater reduction of PGK-1 activity in the ICM cannot, however, be explained solely by this mechanism. To explain the observed additional decrease, we postulate that Pgk-1 is not activated in the ICM prior to day 6. This implies that on day 4 maternal Pgk-1 is activated in the TE exclusively.     

Cell division and death in the mouse blastocyst before implantation

Summary The numbers of cells in the trophectoderm (TE) and inner cell mass (ICM) of mouse blastocysts were counted by differentially labelling their nuclei with two polynucleotide-specific fluorochromes. Blastocysts recovered from the uterus at intervals between their formation early on Day 4 to the initial stages of implantation on day 5 were analysed. TE cell number increase was initially rapid, indicating some synchronisation of the sixth division, but slowed down progressively and plateaued on Day 5, possibly due to the onset of primary giant cell formation. ICM cell number increase was slower than the corresponding TE cells. As a result, TE cell number more than quadrupled, whereas ICM cell number only doubled over this period. Although the mitotic index of both populations of cells fell steadily, there was no significant difference between them. The decline in the proportion of ICM cells, therefore, is likely to be due to cell death, first detected in early blastocysts and predominantly located in the ICM. In addition, however, a contribution of ICM cells to the overlying polar TE cannot be excluded.     

Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo

In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.