scholarly journals Metabolic characterization of the bovine blastocyst, inner cell mass, trophectoderm and blastocoel fluid

Reproduction ◽  
2003 ◽  
pp. 299-308 ◽  
Author(s):  
N Gopichandran ◽  
HJ Leese
Keyword(s):  
Amino Acids ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Lactate Production ◽  
Cell Populations ◽  
Lower Glucose ◽  
Amino Acid Consumption ◽  
Production And Consumption ◽  
Acid Consumption

The formation of a viable blastocyst is dependent upon the establishment of a correct inner cell mass (ICM):trophectoderm cell ratio but little is known about the metabolism of the two cell populations or about the composition of blastocoel fluid. In this study, the metabolism of intact bovine blastocysts, isolated ICM and trophectoderm was examined in terms of glucose and pyruvate uptake, lactate production, and amino acid consumption or production. The concentration of these nutrients in blastocoel fluid was also determined. The metabolism of glucose, pyruvate and lactate differed significantly between the isolated ICM and trophectoderm. Isolated trophectoderm had a higher pyruvate (P<0.001) and lower glucose (P<0.05) consumption, and higher lactate production (P<0.05) than did ICM. The consumption or production of amino acids by ICM and trophectoderm also differed, with the trophectoderm displaying a higher turnover (the sum of production and consumption). The ICM and trophectoderm both depleted arginine, aspartate and leucine, whereas the production of alanine was consistent. Isolated ICM depleted a further six amino acids, which appeared during trophectoderm culture; the reverse trend was observed for the remaining amino acids. The concentration of lactate in blastocoel fluid was significantly higher than in synthetic oviductal fluid supplemented with amino acids and BSA (SOFaaBSA; P<0.05). However, glucose (P<0.05) and pyruvate (P<0.001) concentrations were both lower. Aspartate, glutamate, glycine, alanine and tryptophan were present at significantly higher concentrations in blastocoel fluid than in SOFaaBSA, whereas threonine and asparagine concentrations were significantly lower. The metabolism of composite blastocysts, obtained by summing the consumption and production profiles of the ICM and trophectoderm, and taking into account their respective number of cells, was higher than that of intact blastocysts, indicating that upon isolation of the two cell populations there may be disruption to paracrine interactions or the onset of culture-induced cellular stress or both.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 177
Author(s):  
J. Herrick ◽  
A. Greene ◽  
W. Schoolcraft ◽  
R. Krisher
Keyword(s):  
Amino Acids ◽  
Inner Cell Mass ◽  
Gradient Centrifugation ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Human Albumin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation

Polyamines are involved in trophectoderm attachment and outgrowth, but little is known about their role in earlier stages of development. The objective of this study was to evaluate the effects of an inhibitor of polyamine synthesis (difluoromethylornithine, DFMO) on development (blastocyst formation and hatching) and cell allocation to the trophectoderm (TE, CDX2-positive) and inner cell mass (ICM, SOX2-positive) in the bovine embryo. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and matured for 24 h in a defined maturation medium (5.0 mM glucose, 0.6 mM cysteine, 0.5 mM cysteamine, 0.1 IU mL–1 FSH, 50 ng mL–1 EGF, and 2.5 mg mL–1 recombinant human albumin). Frozen-thawed spermatozoa were processed by gradient centrifugation and co-incubated (2 × 106 mL–1) with COC [10 COC/50 µL; 7.5 µg mL–1 heparin, 2 mM caffeine, and 8.0 mg mL–1 fatty-acid free (FAF) BSA] for 20 to 22 h. After removing cumulus cells, zygotes were cultured (10 embryos/20 µL) in a medium for cleavage stage bovine embryos (0.5 mM glucose, 0.3 mM pyruvate, 6.0 mM lactate, 0.25 mM citrate, 1.0 mM alanyl-glutamine, 0.25 × MEM nonessential and essential amino acids, 5 µM EDTA, and 8.0 mg mL–1 FAF BSA). After 72 h, embryos with >4 cells were randomly allocated (5 embryos/20 µL) to a culture medium for compaction and blastocyst formation (3.0 mM fructose, 0.1 mM pyruvate, 6.0 mM lactate, 0.5 mM citrate, 1.0 mM alanyl-glutamine, 1× MEM nonessential amino acids, 0.5× MEM essential amino acids, 0.075 mM myo-inositol, and 8.0 mg mL–1 FAF BSA) containing 0 (control), 5, or 10 mM DFMO. Embryonic development was evaluated at 192 h post-insemination (96 h in the second medium containing DFMO treatments), and hatching or hatched blastocysts were fixed for analysis of cell allocation. All data were analysed by ANOVA and P < 0.05 was considered significant. Blastocyst formation and hatching (% of embryos cultured in the presence of treatments) were both inhibited (P < 0.05) when embryos (n = 157/treatment) were cultured with 5 (39.5 ± 3.9%, 14.6 ± 2.8%) or 10 (39.5 ± 3.9%, 14.0 ± 2.8%) mM DFMO compared with embryos cultured without DFMO (53.5 ± 4.0%, 26.1 ± 3.5%). The number of TE cells was also reduced (P < 0.05) in the presence of 5 (121.4 ± 7.2) and 10 (123.6 ± 6.7) mM DFMO compared with embryos cultured without DFMO (152.4 ± 9.7), but the number of ICM cells (45.2 to 54.0) and the total number of cells (TE+ICM, 168.8 to 201.1) were not affected (P > 0.05). In a second experiment (n = 163 to 165/treatment), the negative effects of DFMO on hatching (17.0 ± 2.9%; P < 0.05, v. control, 30.7 ± 3.6%) could be partially reversed when embryos were cultured with both 10 mM DFMO and an exogenous polyamine (100 µM putrescine, 23.0 ± 3.3% DFMO+Put; P > 0.05 v. control). The number of TE cells for embryos cultured with DFMO+Put (153.9 ± 8.7) was intermediate between embryos cultured with (138.0 ± 6.9) or without DFMO (control, 161.6 ± 8.7), but these differences were not significant (P > 0.05). These results provide the first evidence of a role for polyamines during blastocyst formation and hatching of bovine embryos, with specific effects on trophectoderm proliferation and hatching.

Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts

1995 ◽  
Vol 40 (4) ◽  
pp. 444-454 ◽  
Author(s):  
Annelies E. P. Van Stekelenburg-Hamers ◽  
Tanja A. E. Van Achterberg ◽  
Heggert G. Rebel ◽  
Jacques E. Fléchon ◽  
Keith H. S. Campbell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Isolation And Characterization ◽  
Permanent Cell

Characterization of goat inner cell mass derived cells in double kinase inhibition condition

2017 ◽  
Vol 483 (1) ◽  
pp. 325-331 ◽  
Author(s):  
Qiang Wei ◽  
Qihui Xi ◽  
Xiaokun Liu ◽  
Kai Meng ◽  
Xiaoe Zhao ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Kinase Inhibition

Relationship between donor animal age, follicular fluid steroid content and oocyte developmental competence in the pig

2003 ◽  
Vol 15 (2) ◽  
pp. 81 ◽  
Author(s):  
Christopher G. Grupen ◽  
Stephen M. McIlfatrick ◽  
Rodney J. Ashman ◽  
Andrew C. Boquest ◽  
David T. Armstrong ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Molar Ratios ◽  
Oocyte Developmental Competence ◽  
Trophectoderm Cells ◽  
Maturation Medium

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73%; P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL−1; P < 0.005) and androstenedione (70 v. 16 ng mL−1; P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17β-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed ‘oocyte capacitation’. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.

scholarly journals LINE-1 retrotransposition impacts the genome of human pre-implantation embryos and extraembryonic tissues

2019 ◽  
Author(s):  
Martin Muñoz-Lopez ◽  
Raquel Vilar ◽  
Claude Philippe ◽  
Raheleh Rahbari ◽  
Sandra R. Richardson ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Early Embryogenesis ◽  
Trophectoderm Cells ◽  
Human Genome Evolution ◽  
Sporadic Cases ◽  
Post Implantation ◽  
Long Interspersed Element

ABSTRACTLong Interspersed Element 1 (LINE-1/L1) is an abundant retrotransposon that has greatly impacted human genome evolution. LINE-1s are responsible for the generation of millions of insertions in the current human population. The characterization of sporadic cases of mosaic individuals carrying pathogenic L1-insertions, suggest that heritable insertions occurs during early embryogenesis. However, the timing and potential genomic impact of LINE-1 mobilization during early embryogenesis is unknown. Here, we demonstrate that inner cell mass of human pre-implantation embryos support the expression and retrotransposition of LINE −1s. Additionally, we show that LINE-1s are expressed in trophectoderm cells of embryos, and identify placenta-restricted endogenous LINE-1 insertions in newborns. Using human embryonic stem cells as a model of post-implantation epiblast cells, we demonstrate ongoing LINE-1 retrotransposition, which can impact expression of targeted genes. Our data demonstrate that LINE-1 retrotransposition starts very shortly after fertilization and may represent a previously underappreciated factor in human biology and disease.

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