Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro

Abstract

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocytes and to elucidate some of the abnormalities occurring during this process. The effects of different intervals of insemination on subsequent embryonic development were also studied. Prepubertal goat oocytes collected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 μg/ml), LH (10 μg/ml) and estradiol-17 β (1 μg/ml) for 27 h at 38.5 °C. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 μg/ml heparin. Representative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemination for a study of sperm penetration, sperm head decondensation, meiotic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some of the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post insemination; decondensation of male and female chromatin and formation of male and female pronuclei occurred at 6 to 8 and 10 to 16 h after insemination, respectively. Highest proportions of oocytes were penetrated after exposure to spermatozoa for 8 h. There were no significant differences in ovum penetration after longer insemination intervals. Cleavage was first observed 24 h after insemination. Three types of abnormalities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due to a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposure to spermatozoa. However, the sperm exposure interval did not significantly affect subsequent embryonic development to the blastocyst stage. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage.