178 EXPRESSION OF mRNA ENCODING LUTEINIZING HORMONE RECEPTOR AND MEVALONATE KINASE AROUND FOLLICLE DEVIATION IN NELORE HEIFERS (BOS INDICUS)

Abstract

Luteinizing hormone (LH) plays a key role in controlling physiological processes in the ovary, and the expression of LHR by bovine granulosa cells is crucial to the follicular transition from FSH to LH dependency. There are controversies about the time at which follicles acquire LHR in granulosa cells. In Nelore breed (Bos indicus), the morphological divergence occurs, on average, 2.5 days after ovulation when the diameter of the dominant follicle is ~6.0 mm. In our previous work with semiquantitative PCR, the mRNA expression of LHR isoforms was detected more clearly after deviation (Day 3). The LHR mRNA binding protein, mevalonate kinase (MVK), is responsible for the down-regulation of LHR mRNA, thereby controlling the steady-state of LHR mRNA expression. In rats, there is an inverse correlation between the mRNA expression of LHR and MVK in luteal cells; however, there is no evidence about MVK expression in the bovine antral follicle. To gain insight about the involvement of the LHR/MVK system in the control of follicle deviation, we assessed the mRNA expression of LHR and MVK in granulosa cells from dominant and subordinate follicles close to deviation in Nelore heifers. Animals (n = 10) were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (before deviation; n = 3), 2.5 (around deviation; n = 4), and 3 (post-deviation; n = 3) days after ovulation. Granulosa cells were harvested from the 2 largest follicles and submitted to total RNA extraction and reverse transcribed with oligo-dT. The mRNA abundance of LHR and MVK was measured by real-time RT-PCR using the Sybr Green system with bovine-specific primers and normalized by the expression of endogenous gene, cyclophilin A (PPIA), using the ΔΔct method corrected by Pffafl’s equation. Dominant and subordinate follicles were considered those expressing the greatest and second greatest abundance of aromatase mRNA (CYP19) in granulosa cells within each heifer. Effects of the day and follicle status on the mRNA abundance of LHR and MVK were tested by ANOVA and the mean values compared by paired t-test or Tukey test (P < 0.05 indicated significant difference). The LHR mRNA was detected at the predicted time of follicle deviation in Nelore heifers (Day 2.5) and was higher in dominant follicle on Day 3 (32.8 ± 12.6) compared with Day 2.5 (3.2 ± 0.9). The second largest follicle (subordinate follicles) had lower mRNA abundance of LHR when compared with future dominant follicles (largest follicles) on days 2.5 (0.8 ± 0.4 v. 3.2 ± 0.9) and 3 (1.9 ± 0.8 v. 32.8 ± 12.6). In contrast to the mRNA expression of LHR, MVK mRNA was more expressed in the subordinate follicles than in the largest follicles at Days 2.5 (3.1 ± 0.9 v. 0.9 ± 0.3) and 3 (2.6 ± 0.6 v. 0.9 ± 0.1) after ovulation, suggesting that it may be necessary to decrease the MVK expression in future dominant follicles to increase their LHR expression and follow up to ovulation.

Supported by FAPESP.