Coculture of human oviductal cells maintains mitochondrial function and decreases caspase activity of cleavage-stage mouse embryos

We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells (∼40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+ concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 μM), an inhibitor of the mitochondrial F0F1-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion (∼2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.

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Vitrification of cleavage stage mouse embryos by the cryoloop procedure

Acta Veterinaria Hungarica ◽

10.1556/avet.57.2009.3.6 ◽

2009 ◽

Vol 57 (3) ◽

pp. 399-410 ◽

Cited By ~ 3

Author(s):

Philip Klambauer ◽

Zsuzsa Keresztes ◽

Katalin Kanyó ◽

Erika Varga ◽

Rita Kriston ◽

...

Keyword(s):

Side Effects ◽

Ethylene Glycol ◽

Propylene Glycol ◽

Mouse Embryos ◽

Cleavage Stage ◽

Control Groups ◽

Step Procedure ◽

Step Loading ◽

Cryoprotective Solution

By decreasing the volume of the cryoprotective solution it is possible to increase dramatically the freezing speed and — at the same time — reduce the toxicity and osmotic side effects of cryoprotectants (CPA). The objective of our study was to vitrify Day-3 cleavage stage mouse embryos (n = 229) with the cryoloop technology using a new composition of vitrification media. Embryos were exposed to a 2-step loading of CPA, ethylene glycol (EG) and propylene glycol (PG), before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop, then they were rapidly submerged into liquid nitrogen. After warming, the CPA was diluted out from the embryos by a 3-step procedure. Survival of embryos was based on morphological appearance after thawing and continued development to expanded blastocysts upon subsequent 48-hour culture. Embryos of the two control groups were either treated likewise except that they were not vitrified, or cultured in vitro without any treatment. Our data show that a high percentage of embryos survived (92.7%) vitrification in the mixture of EG and PG combined with cryoloop carrier and developed normally (89.1%) in vitro after thawing. To our knowledge this is the first report of the successful vitrification of cleavage stage mouse embryos using VitroLoop vitrification procedure.

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Molecular control of mitochondrial function in preimplantation mouse embryos

Molecular Reproduction and Development ◽

10.1002/mrd.20260 ◽

2005 ◽

Vol 71 (4) ◽

pp. 405-413 ◽

Cited By ~ 133

Author(s):

Jacob Thundathil ◽

France Filion ◽

Lawrence C. Smith

Keyword(s):

Mitochondrial Function ◽

Mouse Embryos ◽

Molecular Control ◽

Preimplantation Mouse Embryos ◽

Preimplantation Mouse

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Developmental potential of teratocarcinoma stem cells in utero following aggregation with cleavage-stage mouse embryos

Development ◽

10.1242/dev.74.1.79 ◽

1983 ◽

Vol 74 (1) ◽

pp. 79-96

Author(s):

Joanne T. Fujii ◽

Gail R. Martin

Keyword(s):

Embryonal Carcinoma ◽

Normal Development ◽

Mouse Embryos ◽

Carcinoma Cells ◽

Abnormal Development ◽

Clear Correlation ◽

Embryonal Carcinoma Cells ◽

Embryonic Cells ◽

Cleavage Stage ◽

Developmental Potential

Embryonal carcinoma cells were aggregated with cleavage stage mouse embryos, cultured briefly, and transferred as morulae to the uteri of pseudopregnant females. When midgestation foetuses were examined, many were morphologically abnormal. The severity of this abnormal development was correlated with the extent of contribution by embryonal carcinoma cells to the foetuses as indicated by GPI (glucose phosphate isomerase) analysis. This was true for all three of the cell lines studied, NG-2, PSA-1, and LT1-2D. The clear correlation between increasingly abnormal development and more extensive participation by the embryonal carcinoma cells was not observed in control experiments in which embryos of different stages of development were aggregated together. The data therefore suggest that the embryonal carcinoma cells studied here are unable to support normal development in the absence of a substantial number of host embryonic cells. It remains unclear whether this is a consequence of the karyotypic abnormalities of the cells tested, or whether it reflects a characteristic limitation in the ability of embryonal carcinoma cells to independently direct normal development. When aggregates were allowed to develop to term and the extent of chimaerism was examined in the live-born animals, it was found to be sporadic and limited. This is consistent with the results indicating that large contributions by embryonal carcinoma cells are not compatible with normal development at midgestation. The chimaerism observed in the live-born animals was comparable in both frequency and in tissue distribution to that generally obtained in other studies using either the aggregation or blastocyst injection techniques.

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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

Frontiers in Physiology ◽

10.3389/fphys.2020.613840 ◽

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.

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