A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1353-1361
Author(s):  
J.M. Baltz ◽  
J.D. Biggers ◽  
C. Lechene
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
K Concentration ◽  
Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

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.

scholarly journals G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jan J Zylicz ◽  
Maud Borensztein ◽  
Frederick CK Wong ◽  
Yun Huang ◽  
Caroline Lee ◽  
...  
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Mouse Development ◽  
Cell Stage ◽  
Developmental Progression ◽  
Early Mouse ◽  
The Impact

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

scholarly journals ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

Development ◽  
2021 ◽  
Author(s):  
Esther Jeong Yoon Kim ◽  
Lydia Sorokin ◽  
Takashi Hiiragi
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Spatial Arrangement ◽  
Cell Types ◽  
Embryonic Cells ◽  
Primitive Endoderm ◽  
Integrin Β1 ◽  
Early Mouse Embryo ◽  
Position Sensing ◽  
Early Mouse

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their ‘inside’ position to instruct their fate is unknown. Here we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for ‘inside’ positional signalling and it is required for proper EPI/PrE patterning. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.

scholarly journals Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1550 ◽  
Author(s):  
Marwa El Sheikh ◽  
Ahmed Atef Mesalam ◽  
Muhammad Idrees ◽  
Tabinda Sidrat ◽  
Ayman Mesalam ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Akt Signaling ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Vitamin B3 ◽  
Cell Stage

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8–16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.

scholarly journals Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo.

1990 ◽  
Vol 111 (6) ◽  
pp. 2713-2723 ◽  
Author(s):  
K S O'Shea ◽  
L H Liu ◽  
L H Kinnunen ◽  
V M Dixit
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Extracellular Matrix Protein ◽  
Heparin Binding ◽  
Cell Stage ◽  
Binding Domains ◽  
Heparin Binding Domain

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.

scholarly journals ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

2021 ◽  
Author(s):  
Esther J.Y. Kim ◽  
Lydia Sorokin ◽  
Takashi Hiiragi
Keyword(s):  
Cell Polarity ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Spatial Arrangement ◽  
Cell Types ◽  
Embryonic Cells ◽  
Primitive Endoderm ◽  
Early Mouse Embryo ◽  
Position Sensing ◽  
Early Mouse

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo gives rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their inside position to instruct their fate is unknown. Here we show that cells perceive their position through extracellular matrix (ECM) and integrin-mediated adhesion. Provision of ECM to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β 1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for inside positional signalling and it is required for proper sorting of EPI/PrE cells. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.

Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation

Development ◽  
1993 ◽  
Vol 117 (2) ◽  
pp. 711-723 ◽  
Author(s):  
R.M. Albano ◽  
N. Groome ◽  
J.C. Smith
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Mouse Development ◽  
Cell Stage ◽  
Mammalian Embryo ◽  
Mesoderm Formation ◽  
Ec Cells ◽  
Early Mouse

Members of the activin family have been suggested to act as mesoderm-inducing factors during early amphibian development. Little is known, however, about mesoderm formation in the mammalian embryo, and as one approach to investigating this we have studied activin expression during early mouse development. Activins are homo- or heterodimers of the beta A or beta B subunits of inhibin, itself a heterodimer consisting of one of the beta subunits together with an alpha subunit. Our results indicate that the oocyte contains mRNA encoding all three subunits, and antibody staining demonstrates the presence of both alpha and beta protein chains. From the fertilized egg stage onwards, alpha subunit protein cannot be detected, so the presence of beta subunits reflects the presence of activin rather than inhibin. Maternal levels of activin protein decline during early cleavage stages but increase, presumably due to zygotic transcription (see below), in the compacted morula. By 3.5 days, only the inner cell mass (ICM) cells of the blastocyst express activin, but at 4.5 days the situation is reversed; activin expression is confined to the trophectoderm. Using reverse transcription-PCR, neither beta A nor beta B mRNA was detectable at the two-cell stage but transcripts encoding both subunits were detectable at the morula stage, with beta B mRNA persisting into the blastocyst. We have also analyzed activin and inhibin expression in ES and EC cells. Consistent with the observation that activins are expressed in the ICM of 3.5-day blastocysts, we find high levels of beta A and beta B mRNA in all eight ES cell lines tested. F9 EC cells express only activin beta B, together with low levels of the inhibin alpha chain. When ES and EC cells are induced to differentiate, levels of activin fall dramatically. These results are consistent with a role for activins in mesoderm formation and other steps of early mouse development.

Involvement of oocyte-coded message in cell differentiation control of early human embryos

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 317-322 ◽  
Author(s):  
J. Tesarik
Keyword(s):  
Plasma Membrane ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Gene Activity ◽  
Cell Junctions ◽  
Preimplantation Embryos ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Continuous Increase ◽  
Embryonic Genome

Considerable evidence indicates that the first phenotypical diversification of embryonic cells during mammalian preimplantation development is achieved in two successive steps: (i) generation of cell asymmetry and (ii) unequal cell division. This paper shows that ultrastructural signs of blastomere surface regionalization in human preimplantation embryos are evident as early as the 2-cell stage when modifications of the plasma membrane (loss of microvilli and endocytotic activity, formation of cell junctions) are induced in places of blastomere contact. The capacity of the plasma membrane to undergo these cell-contact-dependent changes precedes any detectable activity of the embryonic genome. The area of the modified plasma membrane shows a continuous increase during the first three cleavage stages. The progression of these membrane modifications is the same in embryos that have properly enhanced their transcriptional activity at the 8-cell stage and in those that have not. In spite of the failure of this early-cleavage-progressed-cleavage transition of gene activity, the formation of zonula adherens and gap junctions goes on apparently normally in the respective embryos and morphologically distinct inner cell mass and trophectoderm cell lineages are subsequently segregated in 16-cell morulae. However, tight junctions do not develop under these conditions. The occurrence of the progressed-cleavage pattern of gene activity in the majority of embryonic cells is a necessary prerequisite for the appearance of the blastocyst cavity. Thus, oocyte-coded message is apparently involved in the control of relatively late stages of human preimplantation development including the differentiation of the first two embryonic tissues, but the embryonic genome is required for the full achievement of this early differentiative event.

170 EXPRESSION PATTERN OF THE Sox2 GENE IN BOVINE OOCYTES AND IN VITRO-DERIVED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 165 ◽  
Author(s):  
T. A. L. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
G. Pennarossa ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Sox2 Expression ◽  
Bovine Oocytes

Sox2 is a member of the Sox (SRY-related HMGbox) family. It acts to maintain developmental potential and marks the pluripotent lineage of the early mouse embryo; in particular, as in the case of Oct-4 and Nanog, Sox2 is expressed specifically in the inner cell mass (ICM) and in the epiblast of this species. Moreover, it plays an important role in the transcription network that maintains stem cell pluripotency, interacting with other factors such as Oct-4 and Nanog. Little information is available on this gene in bovine; therefore aims of the present study were: a) to identify and characterize the Sox2 expression profile in bovine oocytes and preimplantation embryos; and b) to investigate its expression pattern in ICM and trophectoderm (TE). Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. mRNA was isolated from 3 pools, each consisting of 5 MII oocytes, 2-, 4-, 8-, and 16-cell embryos, morulae, blastocysts, ICMs, and TEs. Semi-quantitative analysis of Sox2 expression was performed in the exponential phase of PCR amplification using rabbit globin as exogenous control. Data were analyzed with one-way ANOVA, followed by multiple pairwise comparisons with Tukey test (SigmaStat 2.03, SPSS, Inc., Chicago, IL, USA). Values are presented as mean � SEM and differences of P ≤ 0.05 are considered significant. In order to rule out false negative results, PCR amplifications of isolated ICMs and TEs were extended to the plateau phase. Fragment identity was confirmed by sequencing. Comparison of bovine Sox2 cDNA sequence (EMBL AM774325) with databases revealed a 98%, 93%, and 87% homology with sheep, human, and mouse, respectively. Sox2 mRNA was detectable in oocytes as well as in embryos at the different developmental stages analyzed. Semi-quantitative expression studies revealed that Sox2 was present as both maternal and embryonic transcript; in particular, a statistically significant increase from the 8-cell stage, concomitant with embryo genome activation, was observed. Differently from the mouse, Sox2 was expressed in both bovine ICM and TE, resembling the profile previously shown for Oct-4 (van Eijk et al. 1999 Biol. Reprod. 60, 1093–1103), and suggesting that Sox2 expression might be regulated by Oct-4 also in bovine, as described in mouse and human. These findings also suggest that its expression may become restricted to the ICM only at the expanded hatched stage, as previously described for Oct-4 in pig embryos (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). This work was supported by PRIN 2006, FIRST 2005, TECLA-MIUR, and EUROSTELLS-ESF.

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