189 Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and expression of maturation-related transcripts in bovine oocytes from medium-size antral follicles

Abstract

The aims of this study were to evaluate the effects of epidermal growth factor (EGF) and progesterone (P4) on maturation and expression transcripts for GDF9, CCNB1, H1FOO, cMOS, PARN, and eIF4E after prematuration of cumulus-oocyte complexes (COCs) from antral follicles. Bovine COCs (3-6 mm) were aspirated and pre-matured for 20 h in control medium [TCM-199 containing 5.0 mg mL⁻¹ LH, 0.5 mg mL⁻¹ FSH, 0.4% bovine serum albumin, cilostamide (10 μM) and follicular hemisections] alone or supplemented with EGF (10 ng mL⁻¹), P4 (100 µM), or both EGF (10 ng mL⁻¹) and P4 (100 µM). After that, COCs were matured for 24 h in the same medium, without EGF, P4, cilostamide, and follicular hemisections. Oocyte diameters were evaluated with the software Nis Elements (Nikon Instruments Inc.). To evaluate meiotic progression, the oocytes were fixed in 4% paraformaldehyde and transferred to 0.5% Triton X-100. The chromatin configuration during meiosis was assessed by 10 μg mL⁻¹ bisbenzimide (Hoechst 33342) and analysed under an epi-fluorescent inverted microscope (DMI4000B; Leica). Oocytes were classified according to the nuclear maturation stage as germinal vesicle, metaphase I, anaphase I, telophase I, and metaphase II. To evaluate mRNA expression, oocytes were stored in micr-centrifuge tubes at −80°C until RNA extraction. RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, mRNA for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH (housekeeping gene) was quantified by real-time PCR and analysed by Kruskal-Wallis test. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test (P < 0.05). The results showed that prematuration of COCs in the presence of P4 and both EGF and P4 promoted an increase in oocyte diameter compared with the control or EGF treatment alone. The presence of cilostamide inhibited early meiotic resumption, benefiting oocyte capacitation, but the presence of EGF, P4, or EGF and P4 together in the prematuration medium did not influence meiosis resumption rates. The presence of EGF or P4 in prematuration medium increased the mRNA levels for cMOS in oocytes (P < 0.05). The H1FOO mRNA levels in oocytes cultured with EGF and P4 increased significantly compared with oocytes cultured in EGF alone (P < 0.05). In contrast, mRNA levels for cyclin B1 in oocytes cultured with P4 were higher than those cultured in the presence of EGF alone (P < 0.05). In addition, levels of mRNA for eIF4E showed a significant reduction in oocytes cultured with P4 compared with those pre-matured with EGF or both EGF and P4. The EGF treatment reduced the levels of mRNA for GDF9 compared with control medium. The mRNA levels of PARN did not differ significantly between treatments. In conclusion, EGF, P4, and EGF and P4 combined did not influence oocyte growth and meiotic resumption. However, EGF or P4 increased the mRNA expression of cMOS, whereas EGF reduced the levels of transcripts for GDF9.