scholarly journals Selenium Attenuates HPV-18 Associated Apoptosis in Embryo-Derived Trophoblastic Cells but Not Inner Cell Mass In Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-7
Author(s):  
Jennifer A. Tolen ◽  
Penelope Duerksen-Hughes ◽  
Kathleen Lau ◽  
Philip J. Chan
Keyword(s):  
Cell Viability ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Human Papillomaviruses ◽  
Embryonic Cells ◽  
Trophoblastic Cells ◽  
Group 2 ◽  
Apoptosis Process ◽  
Hpv 18

Objectives. Human papillomaviruses (HPV) are associated with cell cycle arrest. This study focused on antioxidant selenomethionine (SeMet) inhibition of HPV-mediated necrosis. The objectives were to determine HPV-18 effects on embryonic cells and to evaluate SeMet in blocking HPV-18 effects.Methods. Fertilized mouse embryos were cultured for 5 days to implanted trophoblasts and exposed to either control medium (group 1), HPV-18 (group 2), combined HPV-18 and 0.5 µM SeMet (group 3), or combined HPV-18 and 5.0 µM SeMet (group 4). After 48 hrs, trophoblast integrity and, apoptosis/necrosis were assessed using morphometric and dual-stain fluorescence assays, respectively.Results. HPV-18 exposed trophoblasts nuclei (253.8 ± 28.5 sq·µ) were 29% smaller than controls (355.6 ± 35.9 sq·µ). Supplementation with 0.5 and 5.0 µM SeMet prevented nuclear shrinkage after HPV-18 exposure. HPV-18 infected trophoblasts remained larger with SeMet supplementation. HPV-18 decreased cell viability by 44% but SeMet supplementation sustained cell viability. Apoptosis was lower when SeMet was present. HPV-18 decreased inner cell mass (ICM) viability by over 60%.Conclusions. HPV-18 decreased nuclear size and trophoblast viability but these effects were attenuated by the antioxidant SeMet. SeMet blocked HPV-18 associated apoptosis process in trophoblasts but not ICM cells suggesting involvement of different oxidative stress pathways.

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 127-135
Author(s):  
Mary I. Harper ◽  
Mandy Fosten ◽  
Marilyn Monk
Keyword(s):  
X Chromosome ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Phosphoglycerate Kinase ◽  
X Inactivation ◽  
Preferential Expression ◽  
Trophoblastic Cells ◽  
Extraembryonic Membranes ◽  
Early Mouse

The preferential expression of the maternal X chromosome seen in certain extraembryonic membranes of the mouse was studied by investigating the tissues from which these membranes are derived during early development. The electrophoretic variant of the X-coded enzyme PGK-1 (phosphoglycerate kinase) was used to distinguish the expression of the maternal from the paternal X chromosome in heterozygous females. Both the extraembryonic ectoderm and primary endoderm of 6½-day female egg cylinders gave almost exclusive expression of the maternal form of the enzyme whereas the epiblast gave near equal expression of the two parental alleles. No paternal PGK-1 band could be detected in samples of pooled 3½-day blastocysts, but after 3 or 4 days of culture in vitro a faint paternal band was seen in the resultant outgrowths. The activity of the maternal band in these latter samples had increased greatly from that of the blastocysts, consistent with preferential expression of the maternal Pgk-1 allele in the trophoblastic cells of the outgrowths, while both alleles are expressed in inner-cell-mass cells. The results strongly support the idea that non-random X-chromosome expression is due to preferential paternal X inactivation in trophectoderm (from which extraembryonic ectoderm is derived) and in primary endoderm, and not to cell selection.

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 39-44 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Egfp Gene ◽  
Chimeric Blastocysts ◽  
Rabbit Embryos

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Effect of culture conditions on diploid to giant-cell transformation in postimplantation mouse trophoblast

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 217-227
Author(s):  
J. Rossant ◽  
W. Tamura-Lis
Keyword(s):  
Giant Cell ◽  
Cell Transformation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Formation ◽  
Culture Conditions ◽  
Embryonic Cells ◽  
Giant Cell Formation ◽  
Ectoplacental Cone

Diploid extraembryonic ectoderm and ectoplacental cone from the 7·5-day mouse embryo were grown in vitro under a variety of culture conditions in an attempt to discover conditions which maintain trophoblast in a diploid state and prevent giant-cell formation. It was found that maintenance of tissue integrity was not enough to keep the tissues dividing and diploid, but that the presence of inner-cell-mass derivatives did have some effect. This effect was only apparent when trophoblast cells were entirely enclosed by embryonic tissues. Monolayers of embryonic or embryonal carcinoma cells did not prevent giant-cell formation. Diploid extraembryonic ectoderm and ectoplacental cone responded differently: ectoplacental cells eventually formed trophoblast giant cells even when enclosed by embryonic cells whereas extraembryonic ectoderm cells apparently could be maintained in a diploid condition. This and other differences in properties between extraembryonic ectoderm and ectoplacental cone are discussed with reference to a new model for the postimplantation trophoblast lineage in the mouse.

scholarly journals Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Gnanaratnam Giritharan ◽  
Said Talbi ◽  
Annemarie Donjacour ◽  
Francesca Di Sebastiano ◽  
Anthony T Dobson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Preimplantation Embryos ◽  
Global Pattern ◽  
Vitro Fertilization ◽  
Trophoblastic Cells

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 μm2, 113 μm2, and 86 μm2 respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

scholarly journals 50SPATIAL EXPRESSION OF OCT4 IS NORMALLY REGULATED IN PREIMPLANTATION STAGE BOVINE SOMATIC CELL CLONES

2004 ◽  
Vol 16 (2) ◽  
pp. 147
Author(s):  
S. Kurosaka ◽  
S. Eckardt ◽  
M.K. Friez ◽  
N.A. Leu ◽  
R. Reinbold ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Inner Cell Mass ◽  
Marker Gene ◽  
Cell Mass ◽  
Calf Serum ◽  
Ectopic Expression ◽  
Cell Lineage ◽  
Embryonic Cells ◽  
Oct4 Protein

Oct4 is a member of the POU family of transcription factors and is expressed in blastomeres, pluripotent embryonic cells and the germ cell lineage. In the mouse blastocyst, expression of Oct4 becomes restricted to the inner cell mass (ICM). In bovine, Oct4 has not been considered to be a marker for pluripotency because Oct4 protein is expressed in both the ICM and the trophectoderm (TE) of bovine blastocysts (van Eijk MJT et al., 1999 Biol. Reprod. 60, 1093–1103; Kirchhof N et al., 2000 Biol. Reprod. 63, 1698–1705). Oct4 has been used as a marker gene for nuclear reprogramming in cloned embryos. Aberrant spatial distribution and levels of Oct4 have been observed in the majority of mouse clone blastocysts and blastocyst outgrowths (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), indicating reprogramming failure of mouse clones. Lack of or abnormal Oct4 protein expression was also observed in cloned monkey embryos (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159). The spatial distribution of Oct4 mRNA and protein in bovine clones has not been reported. Bovine oocytes were obtained from a commercial supplier (BOMED, Inc., Madison, WI, USA), and were matured in vitro. Enucleated oocytes were fused with fibroblasts from ear skin and then treated with 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D for 6h. Embryos were cultured in SOF supplemented with 1% fetal calf serum (FCS) at 39°C under 5% CO2, 5% O2 and 90% N2 for 2 days. At Day 2, embryos were transferred to SOF supplemented with 5% FCS and cultured under the same conditions until Day 7. Blastocysts were analyzed at Day 7. Oct4 mRNA expression was visualized by whole-mount in situ hybridization using a bovine Oct4-specific antisense riboprobe. Oct4 protein was detected by immunocytochemistry. Control embryos were produced by IVF and were cultured under the same conditions to the blastocyst stage (Day 7). We found that Oct4 mRNA signal was restricted to the ICM in bovine blastocysts. Bovine clones were not different from control embryos in that distribution of Oct4 mRNA signal was typically restricted to the ICM (14 of 16). In contrast to our previous report on mouse clones (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), ectopic expression of Oct4 mRNA in the TE was rarely detected in bovine clones (2 of 16). Distribution of Oct4 protein was also similar between clones and controls with distribution in both the ICM and TE (clones: 9 of 9; controls: 9 of 9). It is unclear why defects in Oct4 distribution should differ between bovine and other species tested including monkey (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159–160); however, the higher rate of normal Oct4 distribution is consistent with the generally higher rates of postimplantation development of bovine clones (Shi W et al., 2003 Differentiation 71, 91–113).

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

195 DEVELOPMENT OF INNER CELL MASS AND FORMATION OF EMBRYOID BODIES ON A GELATIN-COATED DISH AND ON THE FEEDER LAYER IN BUFFALO (BUBALUS BUBALIS)

2006 ◽  
Vol 18 (2) ◽  
pp. 205 ◽  
Author(s):  
M. S. Chauhan ◽  
V. Verma ◽  
R. S. Manik ◽  
P. Palta ◽  
S. K. Singla ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Embryoid Bodies ◽  
Feeder Layer ◽  
Fetal Fibroblast ◽  
Group 2 ◽  
Group 1

Isolation and culture of embryo-derived cell lines have been reported in many mammals, however, there is not even a single report toward initiation of such work in buffalo (Bubalus bubalis). Therefore the present study was carried out to isolate the inner cell mass from in vitro-produced buffalo blastocysts and to grow this inner cell mass for formation of embryoid bodies in a gelatin-coated dish and on an homologous fetal fibroblast feeder layer. Immature buffalo oocytes were isolated from the slaughterhouse ovaries. In vitro production of blastocysts was carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). A total of 26 buffalo blastocysts were produced in vitro. These blastocysts were transferred into a 100-mL drop of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) for further culturing. Thirteen blastocysts hatched on the next day of culture. The hatched mass was separated, suspended in Dulbecco's phosphate buffer saline (DPBS, without Ca++ and Mg++) containing 5% FBS + 0.25% trypsin and examined under the zoom stereomicroscope until disappearance of the trophectoderm cells. The remaining cells of seven blastocysts were cultured in DMEM supplemented with 20% FBS on 0.2% gelatin coated culture dish (group 1), and cells of six blastocysts in DMEM medium supplemented with 20% FBS were cultured on a mitomycin-c-treated (10 �L/mL) feeder layer (group 2), for 14 days. The isolated cells attached to the bottom of the dish in both the groups, spreading was noticed on Day 5 of the culture in group 1 and on Day 3 of culture in group 2. The attached cells were trypsinized using DPBS with 0.25% trypsin, isolated, and subcultured further. Attachment and spreading was noticed only in group 2 subcultured cells. The cellular integrity was homogeneous and the plasma membrane was clearly visible in group 2, but not in group 1. Less than 10% of the attached cells formed embryoid bodies in group 1, where as more than 30% attached cells in group 2 formed embryoid bodies; the latter expressed alkaline phosphatase activity and were blue after staining. These results indicate that the culturing of the inner cell mass on an homologous fetal fibroblast feeder layer is a better choice for production of embryonic stem cells in buffaloes.

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