Fertilization and cleavage of rhesus monkey oocytes in vitro.

Abstract

Fertilization in vitro of rhesus oocytes has been performed in order to provide a model for the study of primate embryonic development. Rhesus monkeys were superstimulated by daily injections of pregnant mare’s serum gonadotropin (PMSG) for 12 days beginning Days 3-5 of menses. An injection of human chorionic gonadotropin (hCG) was given 24 h after the last PMSG injection to induce oocyte maturation. Laparoscopic aspiration of 106 follicles approximately 30 h after hCG injection yielded 87 oocytes (82% recovery). Fifty-three of these oocytes (62%) were enclosed in loose (expanded) cumulus oophorus. Washed, ejaculated rhesus spermatozoa (20 X 106 /ml) were preincubated for 4-6 h in a modified Tyrode’s solution, then diluted (1/10) into 100-M1 culture drops containing oocytes, and caffeine and dibutyryl cAMP (1 mM each). In later experiments, these chemicals were added to the sperm suspensions during the last 1.5 h of preincubation, so that they became diluted 1/10 (final concentrations 0.1 mM each) in the fertilization drops. Sperm and oocytes were coincubated for 18-24 h, then oocytes were either fixed or transferred to fresh culture medium. In 9 experiments a total of 70 rhesus oocytes were inseminated in vitro. Total results for 5 experiments were 13/30 oocytes (43%) showing signs consistent with fertilization (sperm in vitellus and/or more than 1 pronucleus or early stages of cleavage). Four more experiments yielded confirmatory evidence of fertilization. When oocytes were inseminated with live sperm that had been preincubated with caffeine and dibutyryl cAMP, 23/29 oocytes (79%) cleaved at least once; 20 of these developed to the 8-cell stage or further. A further 11 oocytes, randomly selected from the pool of available eggs in the 4 experiments, were inseminated under identical conditions with killed sperm. None of these oocytes showed activation, cleavage or fragmentation during culture for 48-73 h. Activation of oocytes and cleavage resulted only from interaction with live sperm, indicating that fertilization was in progress or had been completed.