A comparison of the number of inner cell mass and trophectoderm cells of preimplantation Meishan and Yorkshire pig embryos at similar developmental stages

Whole 8-cell morulae can be aggregated with isolated inner cell masses from blastocysts. On examining semithin light microscope sections of such aggregates we found that cells of the morula changed shape and spread over the surface of the ICM, thus translocating it to the inside of the aggregate. Using single cells from 8-cell embryos in combination with single cells from other stage embryos or isolated ICMs we show that 1/8 blastomeres spread over other cells providing a suitably adhesive surface. The incidence of spreading is high with inner cells from 16-cell embryos (56 %) and 32-cell embryos (62%) and isolated inner cell masses (64%). In contrast, the incidence of spreading of 1/8 blastomeres is low over outer cells from 16-cell embryos (26%) and 32-cell embryos (13%). Blastomeres from 8-cell embryos do not spread over unfertilized 1-cell eggs, 1/2 or 1/4 cells or trophectoderm cells contaminating isolated ICMs. When 1/8 cells are aggregated in pairs they flatten on one another (equal spreading) as occurs at compaction in whole 8-cell embryos. However, if 1/8 is allowed to divide to 2/16 in culture one of the cells engulfs the other (51-62/ pairs). Based on the ideas of Holtfreter (1943) and Steinberg (1964,1978) these results are interpreted to indicate an increase in adhesiveness at the 8-cell stage as well as cytoskeletal mobilization. Following the 8-cell stage there is an increase in adhesiveness of inside cells while the outside cells decrease in adhesiveness. The difference in adhesiveness between inside and outside cells in late morulae is probably central to the divergent differentiation of (inner) ICM and (outer) trophectoderm cell populations.

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Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst

Development ◽

10.1242/dev.84.1.63 ◽

1984 ◽

Vol 84 (1) ◽

pp. 63-90

Author(s):

Tom P. Fleming ◽

Paul D. Warren ◽

Julia C. Chisholm ◽

Martin H. Johnson

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Central Area ◽

Ultrastructural Changes ◽

Trophectoderm Cells ◽

Junctional Complexes ◽

Secondary Lysosomes ◽

Cytoplasmic Processes ◽

Cellular Cover

Mouse blastocysts, aged 0, 2, 6 and 12 h from the onset of cavitation, were examined by transmission (TEM) and scanning (SEM) electron microscopy. In TEM sections, trophectoderm cells (TE) differed morphologically from those of the inner cell mass (ICM) by their flattened shape, paler cytosol staining and polarized disposition of both junctional complexes (apicolateral) and intracellular secondary lysosomes (SL; basal). Throughout this period of development, cytoplasmic processes, characterized by abundant SLs, cover approximately 80 % of the juxtacoelic face of the ICM. These processes are shown to be derived from the basal surface of TE cells intermediately placed between polar and mural regions. In SEM preparations of the juxtacoelic ICM surface, revealed by ‘cracking open’ blastocysts, the processes appear as tongue-shaped, centripetally oriented structures which terminate collectively at a central area on the ICM surface. The potential of cultured ICMs to generate TE was demonstrated following their immunosurgical isolation from blastocysts aged up to 12 h post cavitation and by examining the sequence of ultrastructural changes associated with TE generation by ICMs from 2 h blastocysts. In contrast, the juxtacoelic cells of similarly aged ICMs observed in situ in ultrasections of intact embryos showed little or no evidence of totipotency expression as judged by the absence of TE characteristics. Since TE expression within presumptive ICM cells is thought to be generated by an asymmetry of cell contacts (Johnson & Ziomek, 1983), we propose that the juxtacoelic TE processes, by providing a cellular cover to the ICM, function in suppressing the expression in situ of ICM totipotency.

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Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development

Development ◽

10.1242/dev.115.1.305 ◽

1992 ◽

Vol 115 (1) ◽

pp. 305-312

Author(s):

M. Aghayan ◽

L.V. Rao ◽

R.M. Smith ◽

L. Jarett ◽

M.J. Charron ◽

...

Keyword(s):

Western Blot ◽

Cellular Localization ◽

Glucose Transporter ◽

Developmental Stages ◽

Molecular Level ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Preimplantation Development ◽

Developmental Expression

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.

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Defects in the allocation of cells to the inner cell mass and trophectoderm of parthenogenetic mouse blastocysts

Reproduction Fertility and Development ◽

10.1071/rd9961193 ◽

1996 ◽

Vol 8 (8) ◽

pp. 1193 ◽

Cited By ~ 9

Author(s):

B Mognetti ◽

D Sakkas

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Substantial Reduction ◽

Cell Lineage ◽

Blastocyst Stage ◽

Trophectoderm Cells ◽

Parthenogenetic Development ◽

Preimplantation Stage ◽

Parthenogenetic Mouse Embryos ◽

Parthenogenetic Embryos

Diploid parthenogenetic mouse embryos (which possess two maternally-derived genomes) can develop only as far as the 25-somite stage when transferred in utero and exhibit a substantial reduction in trophoblast tissue. The loss of cultured parthenogenetic embryos during postimplantation indicates that a defect in cell lineage may be evident as early as the blastocyst stage. The possibility that a defect may already be reflected at the preimplantation stage was investigated by examining the allocation of cells to the trophectoderm (trophoblast progenitor cells) and the inner cell mass of haploid and diploid parthenogenetic mouse blastocysts. Utilizing a differential labelling technique for counting cells, diploid parthenogenetic blastocysts were found to have fewer inner cell mass cells and trophectoderm cells than their haploid counterparts and normal blastocysts. In addition, both haploid and diploid parthenogenetic blastocysts had a lower inner cell mass: trophectoderm ratio than normal blastocysts. Thus, the relatively poor development of the trophectoderm lineage at the postimplantation stage is not reflected by a reduction in its allotment of cells at its first appearance. Nevertheless, the findings indicate that parthenogenetic development is already compromised at the blastocyst stage, and provide evidence that the expression of imprinted genes has significance for the development of the embryo at the preimplantation stage.

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155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab155 ◽

2013 ◽

Vol 25 (1) ◽

pp. 226 ◽

Cited By ~ 1

Author(s):

E. P. López-Damián ◽

T. Fiordelisio ◽

M. A. Lammoglia ◽

M. Alarcón ◽

M. Asprón ◽

...

Keyword(s):

Lipid Droplets ◽

Embryo Quality ◽

Developmental Stages ◽

Bos Taurus ◽

Inner Cell Mass ◽

Cell Mass ◽

Bos Indicus ◽

Nile Red ◽

Blastocyst Stage ◽

Transfer Program

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P < 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P < 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P < 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P < 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P < 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P < 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P < 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs. Research funding provided by UNAM-DGAPA-PAPIIT IN200810.

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Morphology, developmental stages and quality parameters of in vitro-produced equine embryos

Reproduction Fertility and Development ◽

10.1071/rd19257 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1758 ◽

Cited By ~ 1

Author(s):

Elaine M. Carnevale ◽

Elizabeth S. Metcalf

Keyword(s):

Embryo Development ◽

Developmental Stages ◽

Inner Cell Mass ◽

Early Stage ◽

Cell Mass ◽

Quality Parameters ◽

Early Embryo ◽

Developmental Competence ◽

Limited Information

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

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Nuclear Transplantation of Mouse Inner Cell Mass and Trophectoderm Cells into Enucleated Two-Cell Embryos.

Journal of Reproduction and Development ◽

10.1262/jrd.44.1 ◽

1998 ◽

Vol 44 (1) ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Yusuke SOTOMARU ◽

Yoko KATO ◽

Yukio TSUNODA

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Nuclear Transplantation ◽

Trophectoderm Cells

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205 RNA-SEq PROFILING OF BOVINE PRE-IMPLANTATION EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab205 ◽

2013 ◽

Vol 25 (1) ◽

pp. 251

Author(s):

S. Krebs ◽

A. Graf ◽

Z. Valeri ◽

H. Blum ◽

E. Wolf

Keyword(s):

Differentially Expressed Genes ◽

Developmental Stages ◽

Inner Cell Mass ◽

Cell Mass ◽

Differentially Expressed ◽

Rna Seq ◽

Cell Stage ◽

Total Rna ◽

Genome Activation ◽

Zygotic Genome

In order to provide a comprehensive view of the transcriptome changes during the earliest stages of bovine development, we sequenced the total RNA content of bovine oocytes, 4-cell, 8-cell, and 16-cell embryos and the inner cell mass and trophoblast envelope of expanded blastocysts on the Illumina Genome Analyzer IIx. For each experiment pools of in vitro matured oocytes from the German Simmental cows were fertilized by sperm of a single bull, and 10 oocytes or embryos per developmental stage were collected to generate total RNA pools used for sequencing. Synthesis of cDNA was initiated directly in the cell lysate in order to avoid any losses during RNA preparation and was random primed in order to capture all RNA species. Amplified cDNA and unstranded sequencing libraries were prepared using kits from Nugen (Ovation RNA-Seq, Nugen, San Carlos, CA, USA). Biological replicates were generated by inseminating the oocytes with sperm from the distant breeds Jersey (n = 3) and Brahman (n = 3). This cross-breeding design allowed tracking of single sequencing reads back to the maternal or paternal genome, where breed-specific SNP are present in the expressed transcripts. The analysis of this dataset resulted in monitoring of zygotic genome activation and parent-specific expression for single transcripts, a catalogue of splicing isoforms, novel transcripts, and non-coding RNAs and differentially expressed genes between the single developmental stages. Using the program DESEqn, 2784 genes showed differential expression between any of the stages at a false discovery rate of 1%. Specifically, we found 200 genes differentially expressed between immature and matured oocytes, 209 genes between matured oocytes and 4-cell embryos, 580 genes between the 4-cell and 8-cell stage, 567 genes between the 8-cell and 16-cell stage, 987 genes between the 16-cell stage and the inner cell mass, and 1569 genes between the 16-cell and the trophoblast. Functional analysis revealed stage-specific functions of the differentially expressed genes. In summary, by fully exploiting the single-nucleotide resolution of the RNA-Seq method, this dataset provides an invaluable resource for the study of zygotic genome activation, imprinting, transcript annotation, and gene expression in the earliest developmental stages of bovine embryos.

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Ultrastructural observations of lethal yellow (Ay/Ay) mouse embryos

Development ◽

10.1242/dev.35.1.73 ◽

1976 ◽

Vol 35 (1) ◽

pp. 73-80

Author(s):

Patricia G. Calarco ◽

Roger A. Pedersen

Keyword(s):

Developmental Stages ◽

Inner Cell Mass ◽

Structural Characteristics ◽

Cell Mass ◽

Blastocyst Stage ◽

Cell Organelles ◽

Cell Stage ◽

Wide Range ◽

Lethal Yellow ◽

And Control

Ay/Ay embryos were identified by the presence of large excluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of Ay/Ay embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage or slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Nonexcluded cells of Ay/Ay embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +Ay gene was not determined, but evidence from this study and others indicates that the effects of +Ay gene action occur over a wide range of time in early cleavage and implantation.

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