Effects of roscovitine on in vitro maturation of equine oocytes

Abstract

Roscovitine is a potent inhibitor of Maturation Promotion Factor (MPF) due to its kinase inhibiting mechanism, competing with ATP for its binding site. This effectively maintains oocytes of several species in the germinal vesicle stage of meiotic arrest without decreasing the developmental potential of embryos that result after removal of roscovitine and subsequent fertilization. The first series of experiments was pursued to determine the effective dose of roscovitine for maintaining equine oocytes in the germinal vesicle stage (GV) and its reversibility after release of inhibition. Ovaries were obtained from an abattoir, and follicles ranging between 5-22 mm in diameter were scraped with a bone curette. Recovered oocytes were classified based on surrounding cumulus cells as compact or expanded, and maturation was initiated during transport to the laboratory in a portable incubator. EMM1 (a synthetic oviductal fluid, plus amino acids) was used without additives as a base medium. Roscovitine was added at 22, 66 or 200 μM for the first experiment. LH, FSH, estradiol 17-β, progesterone, EGF, IGF-I, and fetal bovine serum were added to the base medium as controls, and all oocytes were incubated for 30 h. For oocytes with expanded cumulus, 7, 38 and 55% remained in the GV stage with 22, 66, 200 μM roscovitine treatments; for oocytes with compact cumulus, 45, 73 and 56% remained in the GV stage with 22, 66, 200 μM roscovitine treatments. For Experiment 2 of the first series, oocytes were incubated with 66 μM roscovitine for 24 or 32 h followed by an additional 24 h in control medium. MII rates after inhibition and release were similar for all treatment groups and oocyte types, including the control group (mean=72%). The third part of the first series evaluated maturation promoting factor (MPF) activity, using a histone kinase assay of oocytes matured with or without 66 μM roscovitine. A 32 kd phosphorylated band was present in all categories of oocytes incubated in control medium at 16, 30 and 48 h of maturation. Oocytes with expanded cumuli that were incubated with roscovitine for 24 or 32 h and then for additional 24 h in control medium had a band of similar size and density as the oocytes in control medium for 16, 30 and 48 h; oocytes with compact cumulus cells had lower MPF activity (P<0.05) when incubated with roscovitine compared to controls. The second study concerned developmental potential of equine oocytes after culture with 66 μM roscovitine for 24 h. Within 90 min of slaughter, oocytes were placed in either EMMI with 66 μM roscovitine and BSA or control medium with LH, FSH, estradiol 17- P, progesterone, EGF, IGF-I, and fetal bovine serum. A subset of oocytes in the roscovitine treatment was fixed and stained at the end of 24 h of culture; of these, 26/31 (84%) of the oocytes with compact cumulus cells and 16/28 (57%) of the oocytes with expanded cumulus cells had a germinal vesicle. Three culture groups were evaluated: maturation after 30 h in control medium; 24 h in roscovitine medium, and then 30 h in control medium; and 54 h in control medium. There were no differences between maturation rates to metaphase II among the treatment groups and cumulus types. After culture, oocytes with a first polar body were subjected to intracytoplasmic sperm injection (ICSI) using a piezzo drill, and then cultured in G1/2 media for 96 h, assessed for morphological cleavage, and stained with Hoechst 33258 to visualize nuclei. No differences were detected in morphological cleavage rates after ICSI among treatments within cumulus type: percentages of oocytes with compact cumulus cells cleaving after 30 h in control medium, 24 h in roscovitine plus 30 h in control medium, and 54 in control medium were 82, 80, and 83%, respectively; for oocytes with expanded cumulus cells 76, 71, and 62%, cleaved respectively. Significantly more oocytes with compact cumulus yielded embryos having ≥ 8 nuclei in the groups incubated with roscovitine first and then transferred to control medium and those that were cultured for 30 h in control medium (63 and 50%) than for 54 h in control medium (8%; P < 0.01). For oocytes with expanded cumulus cells, the proportion of embryos with ≥ 8 nuclei was higher in oocytes cultured for 30 h in control medium (44%) than for 54 h in control medium (8%; P < 0.05), and higher for oocytes incubated with roscovitine for 24 h and then for 30 in control medium (35%) than for 54 h in control medium (P = 0.08). The average number of nuclei/embryo was higher in oocytes with compact cumulus cells that were incubated with roscovitine for 24 h and then for 30 h in control medium (13.5 ± 2.1) than in any other group (3.6 ± 0.9 to 9.3 ± 1.4, P < 0.05). These results demonstrate that roscovitine can be used to maintain equine oocytes in the germinal vesicle stage for up to 24 h without decreasing their developmental potential. This treatment with oocytes of compact cumulus optimized development of resulting embryos.