Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h

Abstract

The aims of the present study were to assess the effect of various substances on meiotic resumption and subsequent development to the blastocyst stage of bovine oocytes. Immature cumulus-oocyte complexes were cultured for 24 h in (a) Medium 199 (M199) alone, or M199 supplemented with (b) 10% fetal calf serum (FCS), (c) 1 μg cycloheximide ml-1, (d) 2 mmol 6-dimethylaminopurine (6-DMAP) l-1, or (e) 0.1 mmol vanadate l-1. After 24 h, groups (a) and (b) were inseminated with frozen-thawed spermatozoa and subsequently cultured, while groups (c-e) were washed and cultured for a second 24 h in M199 + FCS, after which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status, while others were labelled with [35S]methionine to study protein biosynthesis. Incubation with 6-DMAP, cycloheximide or vanadate completely blocked germinal vesicle breakdown with most oocytes remaining at the germinal vesicle stage after 24 h culture (89%, 100% and 85%, respectively). This inhibitory effect was fully reversible in the case of 6-DMAP and cycloheximide; after a second period of incubation, germinal vesicle breakdown occurred in almost all cases (99% and 100%, respectively), and most reached metaphase II (85% and 83%, respectively). In contrast, inhibition with vanadate was only reversible in 56% of oocytes, with only 6% reaching metaphase II. Cleavage rates at 72 h after insemination and blastocyst yields on day 8 of culture were, respectively: (i) M199, 72% and 34%; (ii) M199 + FCS, 80% and 45%; (iii) M199 + cycloheximide, 81% and 19%; (iv) M199 + 6-DMAP, 77% and 14%. 6-DMAP did not modify methionine incorporation. However, cycloheximide completely blocked protein synthesis when present during the period of labelling. Addition of epidermal growth factor to cycloheximide-inhibited oocytes was without effect. In contrast, epidermal growth factor overcame the effect of 6-DMAP in about 50% of oocytes, resulting in lower developmental rates after IVF. These results give an indication of the feasibility of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in acquisition of competence.