Leukemia inhibitory factor antisense oligonucleotide inhibits the development of murine embryos at preimplantation stages

Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. In this study, changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. Although no significant differences were found in the percentages between the untreated group and the 0.25-fmol-treated group, the 0.5- or 1.0-fmol-treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol-treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol-treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol-treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the inner cell mass (ICM) or trophectoderm (TE) and ICM:TE ratio. The 1.0- or 2.0-fmol-treated groups had significantly lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significantly higher percentage than those in the LIF gene-impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.     

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.     

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.     

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.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Intercellular communication in in vivo- and in vitro-produced bovine embryos.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.     

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.     

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.     

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.     

DNA methylation pattern in pig in vivo produced embryos

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine “in vivo” produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200–300 cells and is not caused by the difference in the cell volume of ICM and TE cells.An erratum to this article can be found at     

Impact of adding 5.5 mM glucose to SOF medium on the development,metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.     

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.     

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.