ABSTRACT
BACKGROUND: The timing of the origins of fetal alcohol syndrome has been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development. METHODS: Rhesus monkey ESC lines (ORMES-6 and ORMES-7) were treated with 0, 0.01, 0.1, or 1.0% ethanol, 1.0% ethanol with estradiol, or 0.00025% acetaldehyde with or without estradiol for 4 weeks. RESULTS: Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers (TRA-1-81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6. CONCLUSIONS: Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and ORMES-7 cell line. The morphological changes and labeling for pluripotency, proliferation, and apoptosis demonstrated that how ethanol affects these early cells that develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.
Subject(s)
Acetaldehyde/toxicity , Cell Differentiation/drug effects , Central Nervous System Depressants/toxicity , Embryonic Stem Cells/drug effects , Estradiol/toxicity , Estrogens/toxicity , Ethanol/toxicity , Acetaldehyde/metabolism , Animals , Apoptosis/drug effects , Blastocyst/metabolism , Cell Line , Cell Proliferation/drug effects , Central Nervous System Depressants/metabolism , Control Groups , Embryonic Stem Cells/pathology , Embryonic Stem Cells/physiology , Estradiol/metabolism , Estrogens/metabolism , Ethanol/metabolism , Macaca mulattaABSTRACT
Various antioxidant strategies such as supplementation of antioxidants, limiting oxygen concentration with Oxyrase, and reducing reactive oxygen species through mild mitochondrial uncoupling had statistically significant beneficial effects on sperm cryopreservation from rhesus monkeys with low cryoresistant ejaculates. Individuals or species that have higher sensitivity to cryodamage may derive the most benefit from these treatments.
Subject(s)
2,4-Dinitrophenol/pharmacology , Antioxidants/pharmacology , Macaca mulatta , Oxygenases/pharmacology , Spermatozoa/drug effects , Animals , Cell Survival/drug effects , Cryopreservation/methods , Ejaculation/physiology , Freezing , Macaca mulatta/metabolism , Macaca mulatta/physiology , Male , Mitochondria/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Semen Analysis , Semen Preservation/methods , Spermatozoa/metabolism , Spermatozoa/physiology , Uncoupling Agents/pharmacologyABSTRACT
OBJECTIVE: To compare cumulus cell structure and timing of oocyte maturation of in vitro-matured (IVM) and in vivo-matured (VVM) nonhuman primate oocytes. DESIGN: In vivo maturation and in vitro maturation of oocytes. SETTING: Animal cell culture laboratory. ANIMAL(S): Forty-eight female rhesus macaques. INTERVENTION(S): Fifteen animals were administered FSH, and aspirated oocytes were cultured in vitro for 0, 3, 6, 12, or 24 hours (IVM). Thirty-three animals were administered FSH and hCG, and oocytes were collected 3, 6, 12, or 28-30 hours after hCG (VVM). MAIN OUTCOME MEASURE(S): Nuclear maturation and microtubule scores of oocytes and actin and tubulin transzonal processes of cumulus cells. Embryo development was observed for VVM oocytes. RESULT(S): The rate of nuclear maturation was faster for IVM oocytes compared with VVM oocytes. Actin transzonal processes decreased 0-12 hours after hCG administration for VVM oocytes. Tubulin transzonal processes of IVM and VVM oocytes decreased from 0 to 24 hours and from 0 to 3 hours, respectively. Embryo development improved as VVM time increased. CONCLUSION(S): Nuclear maturation and remodeling of cumulus-oocyte complex structural components associated with in vitro maturation do not parallel those of oocyte maturation in vivo, indicating that in vitro culture conditions continue to be suboptimal.
Subject(s)
Cell Nucleus/physiology , Cumulus Cells/physiology , Oocytes/physiology , Animals , Cumulus Cells/cytology , Cumulus Cells/drug effects , Female , Follicle Stimulating Hormone/pharmacology , Luteinizing Hormone/blood , Luteinizing Hormone/metabolism , Macaca mulatta , Microtubules/drug effects , Microtubules/physiology , Oligopeptides/pharmacology , Oocytes/cytology , Oocytes/drug effects , PrimatesABSTRACT
OBJECTIVE: To determine the effect on rhesus germinal vesicle-stage oocytes enclosed within cumulus cells (COCs) of cryopreservation either by slow, equilibrium cooling or by rapid, non-equilibrium cooling. DESIGN: Experimental study. SETTING: University primate research center. SUBJECT(S): Twelve female rhesus monkeys. INTERVENTION(S): Monkeys were stimulated with recombinant FSH, and COCs were aspirated from follicles by an ultrasound-guided procedure. MAIN OUTCOME MEASURE(S): Rhesus COCs were examined by confocal microscopy to evaluate integrity of microtubules and intactness of transzonal processes between cumulus cells and the oocytes. RESULT(S): Exposure to 1.5 mol/L propylene glycol + 0.3 mol/L sucrose caused disruption of microtubules in all but 1 of 24 COCs and of transzonal processes in more than half of the COCs. Cryopreservation of 11 COCs by slow freezing disrupted microtubules and transzonal processes in all of them. Exposure alone to 2.7 mol/L ethylene glycol + 2.2 mol/L dimethylsulfoxide + 0.5 mol/L sucrose caused disruption of microtubules and transzonal processes in 7 of 19 COCs. Cryopreservation of COCs by rapid, non-equilibrium cooling caused disruption of microtubules and transzonal processes in 14 of 20 complexes. CONCLUSION(S): Maturation of rhesus COCs at the germinal vesicle stage may be seriously impaired because intracytoplasmic microtubules and transzonal processes are likely to be irreversibly damaged by cryopreservation.
