138 THE EFFECT OF FOLLICLE SUPERSTIMULATION ON mRNA LEVELS IN BOVINE OOCYTES COLLECTED BY OVUM PICKUP

Abstract

A previous study revealed that follicle superstimulation significantly improved the developmental competence of immature bovine oocytes collected by ovum pickup (OPU; Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The aim of the present study was to investigate the effect of follicle superstimulation on the expression of developmentally important genes in bovine oocytes collected by OPU. Follicular oocytes were collected by OPU without (OPU group) or after follicle superstimulation by FSH (FSH/OPU group) by using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner according to Imai et al. (2008). In the FSH/OPU group, after dominant follicle removal from Holstein dry cows by OPU, a CIDR was inserted on Day 5 (dominant follicle removal = Day 0). Cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by IM injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). Oocyte collection by OPU was performed 48 h after PGF administration (Day 11) by the aspiration of follicles larger than 5 mm in diameter. In the OPU group, 3-to-6-mm follicles were aspirated without any previous hormone treatment. In vitro oocyte maturation (IVM) was performed according to Imai et al. (2006 J. Reprod. Dev. 52(Suppl), 19–29). Gene expression was assessed before (0 h IVM) and after IVM (22 h IVM) by RT quantitative PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1 and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using Qiagen RNeasy Micro kit (Qiagen, Valencia, CA, USA). Gene expression was analysed by a Roche Light Cycler 480 device. The relative expression of each gene was normalized to ACTB. Three replications were performed. Data were analysed by ANOVA. At 0 h IVM, PAP and DYNC 1/1 were found to be down-regulated (P < 0.05) in the FSH/OPU group compared with the OPU group, whereas the rest of the studied genes showed similar expression in the FSH/OPU and OPU groups. At 22 h IVM, PAP and DYNC 1/1 remained down-regulated in FSH/OPU oocytes. However, at this time the expression of GDF9 appeared significantly higher (P < 0.05) in FSH/OPU oocytes than in OPU oocytes. The expression of GDF9 was found to decrease during IVM in both groups; however, this decrease was less drastic in FSH/OPU oocytes. The results suggest that follicle superstimulation caused reduced expression of mRNA levels of PAP and DYNC 1/1 irrespective of maturation status and it also moderated the reduction of mRNA levels of GDF9 during IVM.