224 BMP15 during in vitro maturation of prepubertal goat oocytes increases parthenogenetically activated embryo development and EGFR expression in cumulus cells

Oocyte competence for embryo development depends on bidirectional communication with cumulus cells (CCs) (Gilchrist et al. 2008). Oocyte-secreted growth factors (OSFs) such as bone morphogenetic protein 15 (BMP15) induce proliferation and differentiation of CCs and improve embryo development (Sudiman et al. 2014). OSFs are suggested to promote the epidermal growth factor network functionality, which is associated with oocyte competence (Richani et al. 2018). Our aim is to study the effect of adding BMP15 to IVM media on embryo competence of prepubertal goat oocytes and on the epidermal growth factor receptor (EGFR) expression of its CCs. Cumulus-oocyte complexes (COCs) were collected by ovary slicing and matured in TCM-199 with FSH, LH, oestradiol, EGF, and cysteamine during 24 h at 38.5°C with 5% CO₂. IVM media of the experimental group were supplemented with 100 ng/mL of BMP15. Matured oocytes were either in vitro fertilised or parthenogenically activated (PA). IVF consisted of a co-culture of partially denuded oocytes with 4 × 10⁶ sperm/mL frozen-thawed semen in BO-IVF medium (Bioscience) for 19 h. For PA, oocytes were denuded, put on PBS with 5 uM ionomycin 4 min and then on TCM-199 with 2 mM 6-dimethylamino-purine 4 h. Afterwards, presumptive zygotes and activated oocytes were embryo cultured for eight days at 38.5°C with 5% CO₂ and 5% O₂ in BO-IVC medium (Bioscience). Blastocysts were stained with 100 µg/mL propidium iodide (PI) and 1% Triton X-100 for 25 s and transferred overnight to pure ethanol with 25 µg/mL Hoechst 33258. PI-stained cells were counted as trophectoderm (TE) cells and the rest as inner mass cells (ICM). CCs of matured COCs were collected and EGFR protein levels were quantified by Western blotting using a mouse monoclonal anti-EGFR antibody (ThermoFisher). Data were statistically analysed by two-way ANOVA followed by Tukey’s correction. Four replicates were used per group. After PA of 150 COCs of BMP15 group and 180 COCs of control group, BMP15 led to a higher blastocyst yield (15.5% ± 1.8 and 6.3% ± 1.9, respectively; P < 0.05) and ICM number than control (48.2 ± 5.0 and 32.1 ± 3.5; P < 0.05). However, it did not affect total blastocyst cell number (161.5 ± 16.0 and 136.3 ± 19.7) or TE number (113.3 ± 12.2 and 104.2 ± 17.2). After IVF of 210 COCs of BMP15 group and 192 COCs of control group, there was no difference in blastocyst yield (8.3% ± 3.1 and 5.9% ± 2.1, respectively), total blastocyst cell number (161.4 ± 3.8 and 144.4 ± 19.1), TE number (125.1 ± 29.3 and 114.0 ± 17.2), or ICM number (36.3 ± 6.6 and 30.4 ± 5.6). Western blotting in CCs showed that the relative expression over control of EGFR protein was 1.66 ± 0.19 times statistically higher when treated with BMP15. In conclusion, adding BMP15 to IVM medium of oocytes from prepubertal goats increased their EGFR expression in CC and their in vitro development when they were obtained by PA, but it did not affect either in vitro development or the quality of the blastocysts generated by IVF.

This study was funded by the Spanish Ministry of Science and Innovation (PID2020-113266RB-100).