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Parthenogenetic Activation of Marmoset: MATERIALS AND METHODS(6)

MATERIALS AND METHODS(6)

The concentration of sperm was approximately 10-15 X 106 sperm/ml, and the insemination time ranged from 1220 h. After insemination, oocytes were removed from the insemination media, washed, and placed in drops of MEM with 10% heat-inactivated female marmoset serum and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Cumulus cells were easily removed from oocytes by repeated pipetting with a flame-polished, pulled Pasteur pipette. Fertilization was confirmed by visualization of a second polar body and two or more pronuclei. Embryos were cultured in drops (approximately 50 ^l) of MEM, supplemented with 6 mg/100 ml penicillin, 5 mg/100 ml streptomycin sulphate, and 10% heat-inactivated female marmoset serum. The drops were overlaid with paraffin oil (Merck). Embryos were observed daily and transferred to recipient females at the 4-cell stage, as described above.

Monitoring of Recipient Female Marmosets after Embryo Transfer

Approximately 0.5 ml of blood was drawn in a 1-ml syringe from the femoral vein of recipient female marmosets 2-3 times per week. The blood sample was centrifuged in the syringe casing at —2500 rpm for 10 min. Plasma was aspirated using a glass Pasteur pipette and stored in plastic tubes at -20°C. flovent inhaler

Three hormones—progesterone, mmmosetGG,andim-munoreactive (ir) inhibin—were measured.These ere all diagnostic indicators of pregnancy in the peripheral plasma of recipient female marmosets. Progesterone levels were monitored by weekly ELISA developed for marmoset monkeys. The day of ovulation (Day 0) was determined as the day before progesterone levels rose above 10 ng/ml. CG was measured using a mouse Leydig cell bioassay. Ir-inhibin was measured as previously described, with some modifications. Briefly, levels of ir-inhibin were measured by RIA, using antiserum to the N-terminal sequence of the a-subunit of human inhibin raised in sheep. In the original protocol, the tracer was monomeric inhibin a-subunit. For this study, the 32-kD dimer of inhibin was used as the tracer.