scholarly journals Disruption of Ini1 Leads to Peri-Implantation Lethality and Tumorigenesis in Mice

2001 ◽  
Vol 21 (10) ◽  
pp. 3598-3603 ◽  
Author(s):  
Cynthia J. Guidi ◽  
Arthur T. Sands ◽  
Brian P. Zambrowicz ◽  
Tod K. Turner ◽  
Delia A. Demers ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Germ Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Tumor Formation ◽  
Embryo Viability ◽  
Early Embryonic Lethality ◽  
Poorly Differentiated ◽  
Rhabdoid Tumors

ABSTRACT SNF5/INI1 is a component of the ATP-dependent chromatin remodeling enzyme family SWI/SNF. Germ line mutations ofINI1 have been identified in children with brain and renal rhabdoid tumors, indicating that INI1 is a tumor suppressor. Here we report that disruption of Ini1 expression in mice results in early embryonic lethality. Ini1-null embryos die between 3.5 and 5.5 days postcoitum, and Ini1-null blastocysts fail to hatch, form the trophectoderm, or expand the inner cell mass when cultured in vitro. Furthermore, we report that approximately 15% ofIni1-heterozygous mice present with tumors, mostly undifferentiated or poorly differentiated sarcomas. Tumor formation is associated with a loss of heterozygocity at the Ini1 locus, characterizing Ini1 as a tumor suppressor in mice. Thus, Ini1 is essential for embryo viability and for repression of oncogenesis in the adult organism.

92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?

2017 ◽  
Vol 29 (1) ◽  
pp. 153
Author(s):  
D. A. Tutt ◽  
R. E. Lyons ◽  
M. K. Holland
Keyword(s):  
Human Error ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Statistical Testing ◽  
Embryo Cryopreservation ◽  
Embryo Viability ◽  
Embryo Survival ◽  
Biopsy Procedure

The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment. Table 1. Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1

173. TRP53-DEPENDENT EXPRESSION OF GENES REGULATING EMBRYO VIABILITY

2010 ◽  
Vol 22 (9) ◽  
pp. 91
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Target Genes ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Genomic Analysis ◽  
Inbred Strains ◽  
Regulatory Control ◽  
Embryo Viability ◽  
Altered Expression ◽  
Pluripotency Genes

Embryos from inbred strains are sensitive to the stresses of culture in vitro compared to hybrid strains. This difference provides a powerful model for identifying genetic regulators of embryo viability in vitro. We showed that culture stress can cause the activation of the TRP53 stress response pathway, culminating in poor embryo viability.1 Directed genomic analysis of potential target genes was undertaken by comparing blastocysts cultured from zygotes of C57BL6.Trp53+/+, C57BL6.Trp53–/– and B6CBF2 genetic backgrounds. This comparison allowed genes with altered expression due to culture in susceptible strains to be identified and determined whether their changed expression was primarily due to the actions of TRP53. Culture had no effect on the expression of a number of house-keeping genes tested. Of known TRP53 target genes tested, only Bax showed a negative association with embryo viability. Interestingly, this was only partially reduced in C57BL6.Trp53–/–, indicating that other factors account for much of its up-regulation. Pluripotency genes showed a complex picture. Nanog was strongly negatively correlated with viability yet NANOG protein was strongly positively associated, indicating complex regulatory control; Oct4 showed no association yet OCT4 was strongly positively associated; Utf1 was the only pluripotency gene tested that showed Trp53 dependent down-regulation in cultured C57BL6.Trp53+/+. Markers of differentiation, Cdx2 (trophectodermal), Gata6 (endodermal) and Brachyury (mesodermal) did not differ between strains. The results identify Trp53-dependent alterations in gene expression in regulators of cell survival (Bax) and pluripotency (Utf1) that may account for the reduction in the capacity of susceptible blastocysts to form a proliferating inner cell mass. The study also identifies for the first time the possibility of complex post-transcriptional regulation of other pluripotency genes (Nanog and Oct4) that may shed future light on the regulation of pluripotency. (1) Li A, et al. Biol. Reprod. 2007; 76: 362–367.

Embryo implantation and embryonic stem cell development in primates

2001 ◽  
Vol 13 (8) ◽  
pp. 517 ◽  
Author(s):  
John P. Hearn
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Embryo Implantation ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Passive Immunization ◽  
Embryo Viability ◽  
Successful Establishment

The endocrine dialogue that results in implantation and the successful establishment of pregnancy in primates relies on embryonic secretion of chorionic gonadotrophin (CG). This hormone is a signal of embryo viability and capacity to support the corpus luteum. The expression of CG is apparently restricted to primates. Active or passive immunization of marmoset monkeys against the beta subunit of CG prevented implantation and early pregnancy, without disrupting the ovarian cycle. Studies of individual embryos cultured in vitro showed that CG is secreted at low levels by the blastocyst from before attachment, with secretion increasing exponentially after attachment. Gonadotrophin releasing hormone (GnRH) was also secreted, from mid-blastocyst stages, before the detection of CG. The secretion of GnRH by the embryo continued through the attachment and outgrowth stages of embryonic differentiation in vitro. The hypothetical role of GnRH in regulating CG release during implantation was tested in recently completed experiments. Individual embryos cultured with GnRH, or with agonist or antagonist to GnRH, showed significant variations in their secretion of CG and in their survival in culture, suggesting a causal relationship between these hormones. Embryos cultured with natural GnRH showed enhanced growth and development. Embryonic stem cells, from the inner cell mass of marmoset and rhesus monkeys, were the first primate embryonic stem cells to be isolated and characterized, enabling the subsequent isolation of human embryonic stem cells.

131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 213
Author(s):  
V. Ghaffarilaleh ◽  
F. Ghafari ◽  
M. Teresa-Paramio ◽  
A. Fouladi-Nashta
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Apoptotic Cell ◽  
Cell Mass ◽  
Differential Staining ◽  
Embryo Viability ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Large Size

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.

scholarly journals Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle

2016 ◽  
Vol 37 (5) ◽  
pp. 3189
Author(s):  
Paula Alvares Lunardelli ◽  
Luciana Simões Rafagnin Marinho ◽  
Camila Oliveira Rosa ◽  
Amauri Alcindo Alfieri ◽  
Marcelo Marcondes Seneda
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Embryo Viability ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Trophectoderm Cells ◽  
Preimplantation Stage ◽  
First Time

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P < 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P < 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 h after fertilization and from those produced from follicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattle embryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at preimplantation stage.

scholarly journals Establishment of a primed pluripotent epiblast stem cell in FGF4-based conditions

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Jin Young Joo ◽  
Hyun Woo Choi ◽  
Min Jung Kim ◽  
Holm Zaehres ◽  
Natalia Tapia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Germ Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Germ Layers ◽  
Homogeneous Population

Abstract Several mouse pluripotent stem cell types have been established either from mouse blastocysts and epiblasts. Among these, embryonic stem cells (ESCs) are considered to represent a “naïve”, epiblast stem cells (EpiSCs) a “primed” pluripotent state. Although EpiSCs form derivatives of all three germ layers during in vitro differentiation, they rarely incorporate into the inner cell mass of blastocysts and rarely contribute to chimera formation following blastocyst injection. Here we successfully established homogeneous population of EpiSC lines with efficient chimera-forming capability using a medium containing fibroblast growth factor (FGF)-4. The expression levels of Rex1 and Nanog was very low although Oct4 level is comparable to ESCs. EpiSCs also expressed higher levels of epiblast markers, such as Cer1, Eomes, Fgf5, Sox17 and T, and further showed complete DNA methylation of Stella and Dppa5 promoters. However, the EpiSCs were clustered separately from E3 and T9 EpiSC lines and showed a completely different global gene expression pattern to ESCs. Furthermore, the EpiSCs were able to differentiate into all three germ layers in vitro and efficiently formed teratomas and chimeric embryos (21.4%) without germ-line contribution.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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