Background
The timing of the origins of fetal alcohol syndrome have 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 -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 -7 cell line. The morphological changes and labeling for pluripotency, proliferation and apoptosis demonstrated that ethanol affects how these early cells 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.
The present study evaluated the interactions among pre-cooling, cryoprotectant, cooling, and thawing for rhesus monkey sperm using a four-way factorial design. Specifically, pre-cooling and thawing were evaluated for two conditions: slow vs. fast. Cooling was evaluated at four rates of 5, 29, 200, and 400 °C/min. The types of cryoprotectant involved combinations of egg yolk and glycerol, egg yolk and ethylene glycol, and egg yolk alone without permeable cryoprotectants or buffer alone with glycerol but without egg yolk. Our findings showed strong interactions among cryoprotectants, cooling, and thawing rates, but not pre-cooling rate, on post-thaw motility and forward progression. The optimal combination of cooling and thawing for maximum post-thaw survival depended on the types of cryoprotectant. When glycerol was used as a permeable cryoprotectant in the presence of egg yolk, slow thawing yielded similar success as fast thawing in some males. However, when glycerol was replaced with ethylene glycol for the same treatment, post-thaw motility was significantly lower in samples that were thawed slowly than those that were thawed rapidly. In the absence of permeable cryoprotectant but the presence of egg yolk, fast cooling was always favorable. On the contrary, in the absence of egg yolk but the presence of permeable cryoprotectant (glycerol), post-thaw motility was significantly reduced especially when samples were thawed slowly. Generally, fast thawing was superior to slow thawing regardless of the types of cryoprotectant or cooling rates, and glycerol in the presence of egg yolk yielded the highest post-thaw motility in all treatment groups.
Various antioxidant strategies such as supplementation of antioxidants, limiting oxygen concentration with Oxyrase, and reducing reactive oxygen species (ROS) through mild mitochondrial uncoupling had 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.
Study Objective
To compare cumulus cell structure and timing of oocyte maturation of in vitro and in vivo matured nonhuman primate oocytes.
Design
In vivo (VVM) and in vitro (IVM) maturation of oocytes.
Setting
Animal cell culture laboratory.
Animal(s)
48 female rhesus macaques.
Interventions
15 females were administered FSH and aspirated oocytes were cultured in vitro for 0, 3, 6, 12 or 24 hours (IVM). 33 females were administered FSH and hCG and oocytes were collected 3, 6, 12, or 28–30 hours following hCG (VVM).
Main Outcome Measures
Nuclear maturation and microtubule scores of oocytes and actin and tubulin transzonal processes of cumulus cells. Embryo development was observed for VVM oocytes.
Results
The rate of nuclear maturation was faster for IVM oocytes compared to VVM oocytes. Actin transzonal processes decreased 0 to 12 hours post hCG for VVM oocytes. Tubulin transzonal processes of IVM and VVM oocytes decreased from 0 to 24 hours and 0 to 3 hours respectively. Embryo development improved as VVM time increased.
Conclusions
Nuclear maturation and remodeling of cumulus oocyte complex structural components associated with IVM do not parallel that of oocyte maturation in vivo, indicating that in vitro culture conditions continue to be sub-optimal.
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