299 EFFECT OF ESTRADIOL-17β AND FOLLICLE STIMULATING HORMONE ON THE IN VITRO MATURATION OF PORCINE OOCYTES DERIVED FROM SMALL FOLLICLES

Abstract

In porcine cumulus-oocyte complexes (COC) from middle follicles (MF: 3–6 mm in diameter), FSH is known to induce the resumption of meiosis and accompanied by transactivate of the EGF receptor and activation of MAPK3/1 in the cumulus cells. The aim of the current study was to examine the effect of oestradiol-17β (E₂: 0.1 μg mL^–1) or FSH on in vitro maturation (IVM) of porcine oocytes derived from small follicles (SF: 1–2 mm in diameter). The COC were aspirated from MF of porcine ovaries obtained at slaughterhouse and cultured for IVM in mPOM (with 1 mM dibutyryl cAMP, 10 IU mL^–1 of eCG, and 10 IU mL^–1 of hCG for 20 h and then without those for 24 h in an atmosphere of 5% CO₂ in air at 39°C) after washing 3 times. The COC from SF, which were aspirated at the same time with COC from MF, were precultured in the absence or presence of E₂ or E₂ plus FSH for 6 h before IVM culture. After the culture, oocytes were denuded from cumulus cells with 0.1% (vol/vol) hyaluronidase and the meiotic stage was observed. Relative transcript abundance of FSH and EGF receptors of CC was also examined by real-time RT–PCR just after preincubation for 6 h. Statistical analysis of data from 3 to 5 replicates was analysed by ANOVA and Tukey's multiple comparison tests. Maturation rate of oocytes from SF (40.6 ± 3.1%) was significantly lower than that of oocytes from MF controls (78.8 ± 2.8%, P < 0.01). Preincubation in the presence of E₂ alone and E₂ plus 0.005 IU of FSH significantly increases the maturation rate of oocytes from SF (56.8 ± 1.5 and 55.7 ± 3.1%, respectively, P < 0.01), although the rate was still lower than MF controls. However, in the presence of E₂ plus a higher concentration of FSH (0.05 and 0.5 IU), oocyte maturation rate was similar (36.3 ± 2.4 and 33.7 ± 1.9%, respectively) to SF controls and lower than those of E₂ alone and E₂ plus 0.005 IU of FSH groups. Relative transcript abundance of FSH receptor of CC increased (P < 0.01) during preincubation in the presence of E₂, but decreased in the presence of 0.05 IU of FSH. There were no significant differences in the transcript abundance of EGF receptors among treatments during preincubation (P = 0.09). In conclusion, preincubation of COC from SF in the presence of E₂ alone and E₂ plus 0.005 IU of FSH improves the maturation rate of the oocytes, whereas the presence of FSH more than 0.05 IU mL^–1 concealed the positive effect. These effects may be yielded by change in the relative transcript abundance of FSH receptor of COC through the treatments.