217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY

Abstract

Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small <6 mm; medium 6 to 8 mm; large >8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P < 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P < 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P < 0.05). Significant (P < 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality.

Funding–National Science Center, grant no. N N302 604438.