Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

The developmental potential of the inner cell mass of blastocysts that were derived from mouse ES cells using nuclear transfer technology

The present study examined the causes of the low developmental potential of enucleated oocytes that have received ES cells and consequent postnatal death of the young. The inner cell masses (ICM) of nuclear-transferred blastocysts or diploid blastocysts were injected into tetraploid blastocysts (group B) or nuclear-transferred tetraploid blastocysts (group C), respectively. The developmental potential of these groups was compared with tetraploid blastocysts injected with ICM of diploid blastocysts (group A). The potential of reconstituted blastocysts to develop into live young in group B increased slightly (5%) but was significantly lower than that in group A (45%). The rate of postnatal death of young in group B did not decrease. The implantation rate of reconstituted blastocysts in group C was very low and no live fetuses were obtained. The results of the present study indicate that the inferior potential of both ICM and trophectoderm cells of nuclear-transferred blastocysts underlies the low developmental rate of nuclear-transferred oocytes receiving ES cells and the higher rate of postnatal death of ES cell-derived young.     

Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts

Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal-stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight-cell block. © 1995 Wiley-Liss, Inc.     

Lens regeneration in axolotl: new evidence of developmental plasticity

Background

Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens.

Results

Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible.

Conclusions

Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.

     

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.     

Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice

ABSTRACT

Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (_ICRESCs). Similar to those from 129/Ola mouse blastocysts (_E14ESCs), the established _ICRESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, _ICRESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (_ICRMEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred _ICRMEFs, hybrid _B6CBAF1MEFs as feeder cells could sufficiently support in vitro maintenance of _ICRESC self-renewal. Additionally, _ICRESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in _ICRESCs cultured for 40th subpassages (164 days) on _B6CBAF1MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established _ICRESCs could be maintained on _B6CBAF1MEFs in culture.     

A simple and effective method for the isolation of inner cell mass samples from human blastocysts for gene expression analysis

The isolation of pure inner cell mass (ICM) and trophectoderm (TE) cells from a single human blastocyst is necessary to obtain accurate gene expression patterns of these cells, which will aid in the understanding of the primary steps of embryo differentiation. However, previously developed pure ICM isolation methods are either time-consuming or alter the normal gene expression patterns of these cells. Here, we demonstrate a simple and effective method of ICM samples isolation from human blastocysts. In total, 35 human blastocysts of all stages with expanded and good morphology were incubated in calcium/magnesium-free HEPES medium for 5 min before micromanipulation. With the aid of a laser, a biopsy pipette was inserted directly into the blastocoel for the suction-based removal of ICM samples. The ICM samples were obtained through simple mechanical pulling force or laser assistance, and each isolation process required 3–4 min. The isolated ICM and TE fractions were subjected to single-cell real-time quantitative RT-PCR to evaluate keratin 18 (KRT18) expression. Finally, 33 paired ICM and TE samples were verified using gene expression analysis. KRT18 was readily detectable in all TE cells but absent in 30 ICM counterparts, indicating a pure ICM isolation rate of 90.9% (30/33). The relative KRT18 expression of three TE samples compared with their three contaminated ICM counterparts was 19-fold (P?<?0.001), indicating that the contamination was very weak. These results demonstrate that our ICM isolation method is simple and effective.     

Changes in morphology and cell number of inner cell mass of porcine blastocysts during freezing

Changes in the morphology and cell number of the inner cell mass (ICM) of porcine blastocysts at the expanded and hatched stages during freezing (-6.8 degrees C, -35 degrees C and -196 degrees C) were studied by differential fluorochrome staining. The shape of each ICM cell from fresh blastocysts at the expanded and hatched stages was sharply delineated but that of ICM cells from frozen blastocysts was partially distorted. The cell-to-cell contact of the ICM from fresh blastocysts was tight, while that from frozen blastocysts was loose or scattered. The percentages (18 to 38%) of expanded and hatched blastocysts with tight-contact ICM cells from frozen groups at each step were significantly lower (P<0.05) than that (100%) from fresh blastocysts. The number of live ICM cells and their proportion from frozen expanded blastocysts (10.9, 12,4% at -36 degrees C) were significantly lower (P<0.05) than those from fresh embryos (18.4, 19.1%) and at -196 degrees C (20.6, 18.4%). At the hatched stage, the number of live ICM cells and their proportion were not significantly different between each freezing step. These results show that the ICM of porcine embryos at both the expanded- and hatched-blastocysts stages survived even after freezing at -196 degrees C and that the degree of ICM damage was lower at the hatched stage than at the expanded stage.     

Embryonic stem cells derived from morulae,inner cell mass,and blastocysts of mink: Comparisons of their pluripotencies

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley-Liss, Inc.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

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.     

Incidence of embryos with chromosomal anomalies in the inner cell mass among bovine blastocysts fertilized in vitro

Two experiments were performed to study chromosomal anomalies. In Experiment 1, chromosome complements of the inner cell mass (ICM) were investigated that had been separated immunosurgically from 169 and 83 bovine blastocysts cultured either in vitro or in vivo in rabbit oviducts from the four-cell stage following in vitro fertilization of in vitro-matured follicular oocytes. The incidence of embryos with chromosomal anomalies in the ICM cells was 18.2% (4/22) for in vitro cultured embryos and 22.2% (4/18) for in vivo cultured embryos and did not differ significantly from those of entire embryos. One haploid (4.5%), two triploid (9.1%) and one 2N/3N (4.5%) in vitro and three 2N/3N (16.7%) and one 2N/4N mosaic in vivo, respectively, were observed in the two culture systems. In Experiment 2, the origin of chromosomal anomalies observed in ICM cells was investigated using early bovine embryos derived from the same bull semen used in Experiment 1. Both the 2N/3N and 2N/4N anomalies were also observed in two-cell embryos. These results indicate that chromosomal anomalies were not restricted to ICM cells and that the 2N/3N anomaly in ICM cells may have been fertilization-derived chimera.     

Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo

The morphology and proportion of inner cell mass (ICM) of bovine blastocysts cultured in vitro or in vivo in rabbit oviducts after in-vitro fertilization of in-vitro matured follicular oocytes were compared with those of blastocysts fertilized in vivo by a differential fluorochrome staining technique. The delineation of each ICM cell was improved by the transfer of embryos derived from in-vitro fertilization to a rabbit oviduct although the cell-cell contacts of ICM cells were not as tight as those from in-vivo fertilization. The proportions (15.8 and 14.9%) of ICM in blastocysts cultured in vitro at early and expanded stages were significantly lower than those cultured in rabbit oviducts after in-vitro fertilization and fertilized in vivo. These results show that the transfer of bovine embryos derived from in-vitro fertilization to the rabbit oviduct increased the proliferation of ICM cells to the level of embryos fertilized in vivo although the cell-cell contact of ICM cell is not improved by the process.     

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.     

Polystoma gallieni: Experimental evidence for chemical cues for developmental plasticity

Among monogeneans that display direct life cycles, plastic developmental strategies may have been selected to counter the lack of transmission opportunities. Within amphibian polystomatids, some species of the genus Polystoma develop into two different phenotypes depending on the host physiological stage to which free swimming larvae attach. When oncomiracidia infest old tadpoles, they develop slowly and migrate during host metamorphosis towards the bladder where they reach maturity. On the other hand when larvae infest young tadpoles, they develop rapidly into neotenic phenotypes that reproduce in the branchial chamber. These alternative developments are explored through experimental infestations with Polystoma gallieni, the specific polystome of the stripeless tree frog Hyla meridionalis. When oncomiracidia were left for 6 h in water in which young tadpoles had been previously maintained for one day, they mainly developed into the neotenic phenotype regardless of the tadpole stage they encountered later. This indicates that P. gallieni could collect released host-derived signals before physical contact with its host.     

In vivo oocyte IGF-1 priming increases inner cell mass proliferation of in vitro-formed bovine blastocysts

Studies addressing the effects of supraphysiological levels of IGF-1 on oocyte developmental competence are relevant for unravelling conditions resulting in high bioavailability of IGF-1, such as the polycystic ovary syndrome (PCOS). This study investigated the effects of supraphysiological levels of IGF-1 during in vivo folliculogenesis on the morula-blastocyst transition in bovine embryos. Compacted morulae were non-surgically collected and frozen for subsequent mRNA expression analysis (IGF1R, IGBP3, TP53, AKT1, SLC2A1, SLC2A3, and SLC2A8), or underwent confocal microscopy analysis for protein localization (IGF1R and TP53), or were cultured in vitro for 24 h. In vitro-formed blastocysts were subjected to differential cell staining. The mRNA expression of SLC2A8 was higher in morulae collected from cows treated with IGF-1. Both IGF1R and TP53 protein were present in the plasma membrane and cytoplasm. IGF-1 treatment did not affect protein localization of both IGF1R and TP53. In vitro-formed blastocysts derived from morulae recovered from IGF-1-treated cows displayed a higher number of cells in the inner cell mass (ICM). Total cell number (TCN) of in vitro-formed blastocysts was not affected. A higher mean ICM/TCN proportion was observed in in vitro-formed blastocysts derived from morulae collected from cows treated with IGF-1. The percentage of in vitro-formed blastocysts displaying a low ICM/TCN proportion was decreased by IGF-1 treatment. In vitro-formed blastocysts with a high ICM/TCN proportion were only detected in IGF-1 treated cows. Results show that even a short in vivo exposure of oocytes to a supraphysiological IGF-1 microenvironment can increase ICM cell proliferation in vitro during the morula to blastocyst transition.     

Cellular 'neoteny': a possible developmental basis for chromaffin cell plasticity

Adrenal medullary chromaffin cells, SIF cells and sympathetic neurons are derived from the sympatho-adrenal sublineage of the neural crest, and represent a range of cellular phenotypes extending from endocrine to neuronal. It is suggested here that these cell types may represent different stages of developmental 'arrest' along a linear pathway whose endpoint is a cholinergic sympathetic neuron. This model explains the 'transdifferentiation' of mature cells seen in this system as simply a delayed realization of transitions that normally occur between these stages during development. Such a 'linear model' of phenotypic diversification may be applicable to other developing systems that generate closely related but distinct cell types.