180 EXPRESSION OF CANDIDATE GENES FOR OOCYTE COMPETENCE IN BOVINE CUMULUS CELLS

Abstract

Cumulus cells (CC) are closed connected to the oocytes through a gap junction network during follicular development and ovulation. This reciprocal functional interconnection is essential for oocyte growth, acquitision of competence, and complete maturation. Therefore, CC are a promising source of markers for predicting oocyte developmental potential without damaging the oocyte. There is a clear relationship between follicular size and oocyte competence, being those obtained from large follicles more developmentally competent in vitro. The aim of this study was to identify in CC genes candidates to be involved in the acquisition of competence, using the follicles size model to recovered cumulus–ooctye complexes (COCs) with various levels of competence. Follicles from slaughterhouse ovaries were dissected, selected, and distributed according to their diameter into 4 groups: (1) 1.0 to 3.0 mm, (2) 3.1 to 6.0 mm, (3) 6.1 to 8.0 mm, (4) ≥8.1 mm. The COCs were released by follicle rupture, morphologically classified, matured, fertilized, and cultured in vitro or denuded for the recovery of CC. Then, CC were frozen until gene expression analysis. The developmental potential of oocytes was evaluated by cleavage and blastocyst rates at 48 and 168 h post-insemination, respectively. Transcripts for FSH receptor, GH receptor, epidermal growth factor (EGF) receptor, pentraxin 3, and insulin-like growth factor II (IGF-II) were quantified by real-time RT-PCR and normalized by cyclophilin expression. Data from cleavage and blastocyst rates were analyzed by chi-square test. The relative abundance of mRNA for the 5 genes in CC were evaluated by ANOVA and Tukey’s test. Non-parametric test (Wilcoxon) was used when data were not normally distributed. The results are presented as mean ± SEM, and P < 0.05 was considered statistically different. Cleavage and blastocyst rates were higher in oocytes originated from groups 3 (86 and 62%) and 4 (87 and 60%) than those obtained from groups 1 (20%) and 2 (34%). The lowest level of expression for FSH receptor was observed in CC from 1.0–3.0 mm follicles (1.1 ± 0.07) and the greatest in CC from ≥8.1-mm follicles (2.1 ± 0.09), whereas the expression in the intermediary groups 2 (1.6 ± 0.1) and 3 (1.6 ± 0.2) did not show any difference from the other groups. Similar pattern of expression was observed for EFG receptor gene, except that CC from group 3 (1.7 ± 0.2) already presented a significant increase in mRNA level compared to group 1 (0.9 ± 0.1). Relative abundance of transcript for GH receptor rose gradually in CC according to follicular size, being the higher expression detected in the largest follicles and the lower in the smallest follicles, with means of 0.8 ± 0.1, 2.3 ± 0.6, 2.7 ± 0.6, and 3.9 ± 0.1 for groups 1, 2, 3, and 4, respectively. The other 2 candidates (pentraxin 3 and IGF-II) did not show any significant differences related to follicle size. The results indicate that increases in expression of receptor for FSH, EGF, and GH was coincident with the increase in follicle size and in oocyte developmental potential; therefore, they can be used as markers for bovine oocyte competence. However, additional studies are needed to confirm the markers identified. Financial support from EMBRAPA/CAPES.