Transformation of sperm nuclei into metaphase chromosomes in maturing pig oocytes penetrated in vitro.

Abstract

Cumulus-free pig oocytes at the germinal vesicle (GV) stage were incubated in modified Brackett & Oliphant's medium with 5% fetal calf serum and 5mM caffeine with or without cryopreserved, ejaculated spermatozoa. When oocytes were transferred into modified tissue culture medium (TCM-199B at pH 7.4) supplemented with 10 IU/ml eCG, 10 IU/ml hCG and 1 microg/ml oestradiol-17beta after 8 h of incubation with spermatozoa and cultured for 0-48 h, 86-99% of oocytes were penetrated. Most (95-100%) oocytes penetrated 0-16 h after transfer had decondensed sperm chromatin. However, 24 h after transfer 47% and 33% of penetrated oocytes contained recondensed sperm chromatin and sperm metaphase chromosomes, respectively. The proportion of penetrated oocytes containing sperm metaphase chromosomes increased after 36-48 h of transfer (51-65%). The transformation of sperm nuclei to metaphase chromosomes was obtained in 75% and 79% of anaphase I (AI) to telophase I (TI) and metaphase II (MII) oocytes, respectively, but only in 38% of metaphase I (MI) oocytes. Moreover, such transformation was observed only in 1 of 30 oocytes at the stages of GV breakdown to prometaphase I and none of 69 oocytes at the GV stage. The transformation of sperm nuclei into metaphase chromosomes was completely inhibited in oocytes penetrated by eight or more spermatozoa. Well-developed male and female pronuclei were observed in only 3 (4%) of 77 oocytes penetrated 48 h after transfer. The proportion of oocytes reaching MII was greatly inhibited by sperm penetration; only 18% of penetrated oocytes, but 87% of non-inseminated oocytes, reached MII by 48 h after transfer. None of the oocytes penetrated by seven or more spermatozoa reached MII. Most (75%) oocytes were inhibited from the transition from MI to MII even though they were cultured for 48 h. The present results indicate that: (1) the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes, (2) immature pig oocytes penetrated by spermatozoa can undergo meiotic maturation to MI, and (3) the transition of such oocytes from MI to MII is inhibited, suggesting that an activity of mitogen-activated protein kinase may be retarded.