Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h.
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 micrograms 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.
Lonergan
P
P
Station de Physiologie de la Reproduction, INRA, Nouzilly, France.
Khatir
H
H
Carolan
C
C
Mermillod
P
P
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D003864:Depression, Chemical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005307:Fertilization in Vitro; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000187:drug effects; D009866:Oogenesis / Q000187:drug effects*; D014638:Vanadates / Q000494:pharmacology
D003470:Culture Media; D004791:Enzyme Inhibitors; D014638:Vanadates; D004815:Epidermal Growth Factor; C001020:N(6),N(6)-dimethyladenine; D003513:Cycloheximide; D000225:Adenine