183 Effects of epidermal growth factor and progesterone on in vitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

Abstract

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that in vitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n = 400) were aspirated from antral follicles (1-3 mm) and cultured for 48 h in control medium (TCM-199 plus 5.0 mg mL⁻¹ LH, 0.5 mg mL⁻¹ FSH, and 5% fetal bovine serum) alone or with EGF (10 ng mL⁻¹), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20 h in TCM-199 containing 5.0 mg mL⁻¹ LH, 0.5 mg mL⁻¹ FSH, 0.4% bovine serum albumin, and 10 μM cilostamide. Then, COCs were matured in vitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P < 0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48 h of culture for COCs in all groups (P < 0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P < 0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P < 0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P < 0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P < 0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration in vitro.