199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

Abstract

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO₂ in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL^–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1^a (control), 2.7 ± 0.4^b (Bl-I), 3.2 ± 0.5^b (Cilost) and 2.1 ± 0.3^ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development.

We acknowledge FAPESP and NSERC.