scholarly journals A preliminary note upon the question of the nutrition of the early embryo, with special reference to the guinea-pig and man

1905 ◽  
Vol 76 (508) ◽  
pp. 164-165 ◽  
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Early Embryo ◽  
Uterine Horn ◽  
Dense Layer ◽  
Preliminary Note ◽  
Trophoblastic Cells ◽  
Decidual Cells ◽  
Uterine Secretion

During the progress of a research into the earliest implantation of the embryo of the guinea-pig, I have been particularly struck with the way in which the nutrition of the embryo is anticipated and provided for during the time it remains free in the uterine horn. The so-called yolk-granules of the ovum are obviously insufficient to provide for the growth of the embryo to the stage prior to differentiation of the inner cell-mass, to which it attains during the five or six days which elapse before it comes into contact with the maternal tissues. It is clear that it must derive nourishment from the medium in which it lies―the product of the secretion of the uterine or other glands, which, during the period of pro-œstrum, exhibit such marked activity. I suggest that this secretion, which consists of mucus and probably albumin, is assimilated by the embryo after having undergone a process of digestion, the result of a secretory activity on the part of the outermost cells of the embryo―the cells of the Trophoblast. This suggestion I base on my observations in the guinea-pig, where I am able to demonstrate a breaking-down of maternal cells before the Trophoblastic cells are in actual contact; likewise in human placentation where a more or less dense layer of fibrin and broken-down leucocytes and decidual cells, the result of Trophoblastic activity, affords a barrier interposed between the invading Trophoblastic cells and the Decidua. This layer I purpose naming the “Protective Layer.” Looked at from a comparative point of view, there is in all probability a close analogy between the uterine secretion of mammals, and the secretion of the oviducts of the lower vertebrata. In the case of birds the analogy is very striking, on account of the direct and important share in the nutrition of the embryo afforded by this secretion, commonly known as the white of the egg. In the case of the frog the ovum receives in its passage down the oviduct, corresponding to the uterine horn of the guinea-pig, a coating of mucus and probably albumin, comparable to the uterine secretion referred to above; when it reaches the water and becomes fertilised, this swells up by absorption, forming a gelatinous covering. The embryo for nutriment depends upon the yolk contained in the ovum before fertilisation, upon the covering of mucus and probably albumin, and lastly upon the water in which it lies. In certain mammals, as, for example, the rabbit and the mole, a distinct gelatinous envelope is described as surrounding the embryo before implantation occurs; this envelope is, I suggest, possibly due to some digestive action of the cells of the Trophoblast upon the adjacent medium, producing a form of coagulation.

The control of trophoblastic growth in the guinea pig

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 405-418
Author(s):  
E. B. Ilgren
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
The Other ◽  
In Vivo Analysis

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 743-753 ◽  
Author(s):  
J.E. Collins ◽  
J.E. Lorimer ◽  
D.R. Garrod ◽  
S.C. Pidsley ◽  
R.S. Buxton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Splice Variants ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 175-198
Author(s):  
R. L. Gardner
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Early Embryo ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Developmental Potential ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Endodermal Cells

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

186 LASER CAPTURE MICRODISSECTION FOR GENE EXPRESSION ANALYSIS OF INNER CELL MASS AND TROPHOBLAST FROM BOVINE BLASTOCYSTS

2011 ◽  
Vol 23 (1) ◽  
pp. 194
Author(s):  
M. Filliers ◽  
W. de Spiegelaere ◽  
L. J. Peelman ◽  
K. Goossens ◽  
C. Burvenich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Serial Sections ◽  
Trophoblastic Cells ◽  
Keratin 18 ◽  
Laser Capture

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
C. Sauvegarde ◽  
D. Paul ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Immature Oocyte ◽  
Cell Stage ◽  
Morula Stage ◽  
Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

97 Loss of aggregation capacity of bovine invitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

2020 ◽  
Vol 32 (2) ◽  
pp. 174
Author(s):  
V. Alberio ◽  
M. Yauri Felipe ◽  
D. Salamone
Keyword(s):  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Culture Conditions ◽  
Sodium Pyruvate ◽  
Trophoblastic Cells ◽  
Stereoscopic Observation ◽  
Fetal Bovine ◽  
Humidified Air ◽  
Oviductal Fluid

Embryo aggregation consists of placing more than one zona-free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared with nonaggregated embryos. One way to obtain these benefits in IVF embryos would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the inner cell mass (ICM). Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10µgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. We performed IVF with 16×106 spermatozoa per mL for 5h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O2, 5% CO2, and 90% N2. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n=22) or with ZF embryos of Days 4 (n=23), 5(n=25), or 6 (n=22) and blastocysts (n=25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n=18), 4 (n=18), 5 (n=47), 6 (n=48), and 7 (n=45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the invitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.

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

2019 ◽  
Vol 31 (12) ◽  
pp. 1758 ◽  
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.

scholarly journals A further note on the nutrition of the early embryo: with special reference to the chick

1908 ◽  
Vol 80 (540) ◽  
pp. 332-338 ◽  
Keyword(s):  
Special Reference ◽  
Royal Society ◽  
Early Embryo ◽  
Accurate Analysis ◽  
Preliminary Note ◽  
Series Of Experiments ◽  
Uterine Secretion ◽  
Do So

In furtherance of the views put forward regarding the nutrition of the early embryo by the author in a preliminary note read before the Royal Society in February, 1905, the following series of experiments was carried out. The changes that take place between the growing embryo and the maternal secretion are in the mammalia, not easy to study, on account of the difficulties to be encountered, such as the minute size of the embryo, and the small amount of uterine secretion available. In birds, on the order hand, the uterine secretion, viz., the white of the egg, is abundant. The growing embryo can be examined easily at any stage which may be desired, and since all the changes which take place do so within the limits of the shell, the products of these changes are capable of accurate analysis.

scholarly journals Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

2003 ◽  
Vol 358 (1436) ◽  
pp. 1403-1409 ◽  
Author(s):  
Wolf Reik ◽  
Fatima Santos ◽  
Kohzoh Mitsuya ◽  
Hugh Morgan ◽  
Wendy Dean
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Lineage Commitment ◽  
Imprinted Genes ◽  
Mammalian Development ◽  
Paternal Genome ◽  
Developmental Defects ◽  
Set Up

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X–chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.

scholarly journals Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos

Reproduction ◽  
1995 ◽  
Vol 104 (2) ◽  
pp. 251-258 ◽  
Author(s):  
T. Tao ◽  
B. Reichelt ◽  
H. Niemann
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblastic Cells
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