Culture of mouse embryos during neurulation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 109-116
Author(s):  
T. W. Sadler ◽  
D. A. T. New
Keyword(s):  
Growth And Development ◽  
Calf Serum ◽  
Mouse Embryos ◽  
Rat Serum ◽  
Developmental Abnormalities ◽  
Time Period ◽  
Lower Protein ◽  
Made In

A comparison between static versus rotator culture systems and a variety of media (rat serum, new born calf serum, DMEM and Waymouth's) was made in an attempt to promote in vitro growth of mouse embryos from the beginning of neurulation (headfold stage) to the closure of the neural tube and formation of the limb buds (48 h). The results demonstrate that good development can be achieved for 48 h using a rotator system and that 80% of embryos cultured on rotators show growth and differentiation similar to that obtained for the same time period in vivo. Static cultures are less successful and embryos grown in this system show lower protein content and somite numbers than those maintained on rotators. Undiluted rat serum is superior to all other media tested and supports better growth and development as monitored by total protein and developmental abnormalities.

Comparison of in vivo vs. in vitro Bonding of Composite Resin to the Dentin of Canine Teeth

1988 ◽  
Vol 67 (2) ◽  
pp. 467-470 ◽  
Author(s):  
E.L. Pashley ◽  
L. Tao ◽  
J.R. Mackert ◽  
D.H. Pashlety
Keyword(s):  
Composite Resin ◽  
Bonding Agents ◽  
Dentin Bonding ◽  
Dentin Bonding Agents ◽  
Time Period ◽  
Bond Strengths ◽  
Made In

Dogs were utilized in a study to compare the bond strengths of dentin bonding agents made to dentin in vivo and then again in vitro in the same teeth 30 min, one day, one week, and one month post-extraction. No statistically significant differences were observed between bonds made in vivo and those made in vitro at any time period. Contamination of the dentin surfaces with blood or saliva lowered the bond strengths, but these could be restored to control values by re-surfacing of the dentin with a bur.

Response of preimplantation rat blastocysts in vitro to extracellular uterine luminal components, serum and hormones

1977 ◽  
Vol 25 (1) ◽  
pp. 265-277
Author(s):  
M.A. Surani
Keyword(s):  
Calf Serum ◽  
Giant Cells ◽  
Biologically Active ◽  
Foetal Calf Serum ◽  
Active Components ◽  
Rat Serum ◽  
Pregnant Females ◽  
Trophoblast Giant Cells

The influence of extracellular environmental factors on preimplantation rat blastocysts was tested by determining the number of embryos which escaped from their zonae pellucidae, followed by attachment and outgrowth of trophoblast giant cells, after 72 h in culture Uterine luminal ocmponents from individual females, or hormones, were included in Dulbecco's medium which contained 4 mg/ml bovine serum albumin. In about 20% of cases, uterine fluids were embryotonic. However, uterine fluids from day-5 pregnant females, the day of implantation in the rat, were more potent in these tests than uterine fluids obtained from ovariectomized females treated with progesterone alone. The potency of a mixture of the 2 fluids was also high. Uterine fluids obtained at 14 h after an injection of oestradiol and progesterone to the ovariectomized females, were also effective in these tests. Rat serum and foetal calf serum were effective too, but steroids or insulin alone in the medium had no detectable influence on embryos. Serum or uterine luminal proteins appear to be essential for maintaining the viability of the blastocysts and for inducing the responses observed here. In the uterine fluids, some proteins released into the lumen after treatment of females with oestradiol and progesterone appear to be the biologically active components. Differences in the responses of blastocysts in vitro are compared with those in vivo.

scholarly journals Perspective: One-Cell and Cleavage-Stage Mouse Embryos Thrive in Hyperosmotic Oviductal Fluid Through Expression of a Glycine Neurotransmitter Transporter and a Glycine-Gated Chloride Channel: Clinical and Transgenerational Implications

2020 ◽  
Vol 11 ◽  
Author(s):  
Lon J. Van Winkle
Keyword(s):  
Chloride Channel ◽  
Well Being ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Cleavage Stage ◽  
Developmental Abnormalities ◽  
Neurotransmitter Transporter ◽  
Oviductal Fluid

The osmolality of mouse oviductal fluid ranges from about 300 mOsmol/kg in the ampulla 0–3 h post coitus (h p.c.) to more than 350 mOsmol/kg in the isthmus 34–36 h p.c. Thus, it has been surprising to find that development of one-cell and cleavage-stage mouse embryos arrests in vitro in media exceeding 300 mOsmol/kg, and they develop best in unphysiological, hypotonic media. The glycine concentration in oviductal fluid can, however, rescue development in hypertonic media, so physiological conditions in vivo and in vitro likely work together to foster embryo well-being. Glycine acts on one-cell and cleavage-stage mouse embryos through the glycine-gated chloride channel, GLRA4, and uptake via the glycine neurotransmitter transporter, GLYT1. Since these processes lead to further signaling in neurons, the presence and function of such signaling in preimplantation embryos also should be investigated. The more we know about the interactions of physiological processes and conditions in vivo, the better we would be able to reproduce them in vitro. Such improvements in assisted reproductive technology (ART) could improve patient outcomes for IVF and potentially help prevent unwanted developmental abnormalities in early embryos, which might include undesirable epigenetic DNA and histone modifications. These epigenetic modifications may lead to transgenerational adult disorders such as metabolic syndrome and related conditions.

scholarly journals The in vitro culture of primitive-streak-stage mouse embryos

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 131-143
Author(s):  
P. P. L. Tam ◽  
M. H. L. Snow
Keyword(s):  
Culture Method ◽  
Primitive Streak ◽  
Defined Medium ◽  
Mouse Embryos ◽  
Mouse Serum ◽  
Rat Serum ◽  
Total Growth ◽  
Size And Morphology

The in vitro growth and morphogenesis of mouse embryos from early primitive-streak stage to early-somite stage is described. The embryo culture method employs a static culture system, a conventional chemically defined medium (Dulbecco's modified Eagle's medium), supplemented with additional glucose, glutamine and suitably prepared serum. The method of serum preparation is important for successful culture. Both mouse serum and rat serum support good development of primitive-streak-stage mouse embryos. Over 60% of earlystreak stage and about 90 % of late-streak stage grow and develop for 48 h in vitro. During the first 24 h in culture, total growth of the embryos, as reflected by protein content, size and morphology is the same as in vivo.

49 PRACTICAL APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD FOR CRYOPRESERVATION OF MAMMALIAN EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Uchikura ◽  
T. Wakayama ◽  
S. Wakayama ◽  
H. Matsunari ◽  
M. Maehara ◽  
...  
Keyword(s):  
High Performance ◽  
Sucrose Solution ◽  
Calf Serum ◽  
Hollow Fibre ◽  
Mouse Embryos ◽  
Embryo Cryopreservation ◽  
Cell Stage ◽  
Common Marmosets

We recently developed the hollow fibro vitrification (HFV) method, which is a novel, high-performance embryo cryopreservation method (Matsunari et al., 2012). In this study, we aimed to verify the applicability of the HFV method for cryopreserving various types of embryos; BDF1 mouse embryos at the 2-cell stage, porcine parthenogenetic morulae derived from in vitro-matured oocytes, bovine morulae produced by in vitro maturation/fertilization (LIAJ Animal Biotechnology Center, Tokyo, Japan), and in vivo-derived blastocysts of common marmosets were vitrified, and their survival was assessed by culture or transfer. The embryos were vitrified using 20 mM HEPES-buffered TCM-199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibres (25 mm) containing 1 to 20 embryos were placed in an equilibration solution containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 5 to 7 min, followed by incubation for 1 min in vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose. The embryos were then vitrified by immersion in LN. The embryos were devitrified by immersing the hollow fibre in a 1 M sucrose solution at 38.5°C, which was followed by stepwise dilution of the cryoprotectants and washing. For a subset of the vitrified mouse embryos, rewarming in a non-ultra-rapid manner by melting a hollow fibre in air at room temperature for 5 s was tested. Embryo transfer was performed to assess the viability of the vitrified mouse embryos. For porcine embryos, vitrification in LN vapor (–150°C) was tested. Development of the vitrified mouse embryos to blastocysts was equal to that of the non-vitrified embryos [105/110 (95.5%) v. 109/110 (99.1%)]. Post-transfer development to fetuses was also equal between the vitrified and non-vitrified embryos [pregnancy rates: 4/4 v. 2/2; developmental rates: 55/80 (68.8%) v. 35/40 (87.5%)]. Non-ultra-rapid rewarming did not decrease the survival of the vitrified mouse embryos [blastocysts: 94/100 (94.0%); pregnancy: 4/4; fetuses: 55/80 (68.8%)]. Blastocyst formation was equivalent for vitrification of porcine embryos in LN vapor [27/34 (79.4%)], direct immersion into LN [28/35 (80.0%)], and the non-vitrified control [31/32 (96.9%)]. Vitrification of 191 bovine morulae resulted in 153 (80.1%) blastocysts. In preliminary experiments, survival of marmoset blastocysts was 100% (n = 6). These data demonstrate that the HFV method is (1) effective for embryos of various species and production methods; (2) effective even for porcine in vitro-derived morulae, which are highly cryosensitive; and (3) amenable to modifications such as non-ultra-rapid warming and cooling in LN vapor, increasing the potential applicability of the HFV method. For instance, vitrification in LN vapor may allow embryo cryopreservation with high hygienic standards. This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.

Culture of early somite mouse embryos during organogenesis

Development ◽  
1979 ◽  
Vol 49 (1) ◽  
pp. 17-25
Author(s):  
T. W. Sadler
Keyword(s):  
Total Protein ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Normal Growth ◽  
Protein Analysis ◽  
Mouse Embryos ◽  
Culture Period ◽  
Similar Time ◽  
Rat Serum

Early somite (2—4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum + Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells

1989 ◽  
Vol 1 (2) ◽  
pp. 127 ◽  
Author(s):  
D Sakkas ◽  
AO Trounson ◽  
I Kola
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Mouse Embryos ◽  
Early Cleavage ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Mouse Embryo Development ◽  
Embryo Culture Medium

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

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