It is well documented that the size of the dominant follicle at deviation is smaller in Bos indicus compared with in Bos taurus breeds. The physiological mechanisms underlying this difference, however, are unknown. The aim of the present study was to evaluate the dynamic of oestradiol production during follicle development close to the expected moment of deviation in Bos taurus and Bos indicus dairy heifers. Intrafollicular concentration of oestradiol (E2) and P450 aromatase gene expression in granulosa cells (GC) were evaluated in Gir (n = 10) and Holstein (n = 10) heifers. Follicular waves were synchronized with an intravaginal progesterone device (1 g, Sincrogest, Ourofino Agropecuária, São Paulo, Brazil) and benzoate oestradiol (2 mg im, Sincrodiol, Ourofino Agropecuária). Ultrasonography evaluations (MyLab30 Vet Gold, Esaote, Genova, Italy, with a 7.5-MHz transducer) were performed every 24 h to detect the emergence of the new follicular waves. The largest follicle of each wave was individually aspirated by ovum pickup before, at the expected diameter, or after deviation in both Gir (4.6 ± 0.2, 6.3 ± 0.2, and 8.5 ± 0.6 mm, respectively) and Holstein heifers (6.0 ± 0.5, 8.6 ± 0.4, and 10.2 ± 0.2 mm, respectively), as previously described (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175). Follicular fluid (FF) samples were centrifuged and the supernatant stored at –20°C until E2 and progesterone (P4) determination by RIA. The pellet of GCs was washed twice with PBS, kept in RNAlater, and frozen at –20°C until RNA extraction and reverse transcription. Relative transcript quantification was performed by real-time PCR. The β-actin gene was used as control. Samples of FF with E2:P4 ratio <1 or presenting contamination by theca cells (detected by the expression of 17α-hydroxylase) were not used for statistical analyses. Concentration of E2 in FF was evaluated between breeds and among follicle size classes by ANOVA and differences among means compared by Student t-test or Tukey’s test, respectively. Within breeds, relative gene expression was accessed by pair-wise fixed reallocation randomization test (software REST®). Results are shown as mean ± SEM. In both breeds, concentration of E2 in FF progressively increased with follicular diameter (P < 0.05). Intrafollicular concentration of E2 (ng mL–1) was greater (P < 0.05) in Holstein than in Gir before (58.5 ± 11.7 v. 8.8 ± 2.0), at expected (226.0 ± 49.9 v. 78.9 ± 21.0), and after follicle deviation (579.1 ± 45.0 v. 185.0 ± 34.9). Interestingly, however, follicles with similar diameters (~6 or 8 mm) showed similar (P > 0.05) E2 concentrations between Holstein and Gir. Moreover, in both breeds, the relative expression of P450 aromatase gene in GC first increased (3.9 ± 2.4 and 67.5 ± 52.8 for Holstein and Gir, respectively; P < 0.05) at the same stage of follicular development (8 mm). The present results suggest that the smaller size of follicles at deviation in Bos indicus is not related to an earlier increase in intrafollicular E2 production.
CNPq, CAPES, and Fapemig (CVZ APQ 02863/09).
Ketamine attenuates cytochrome p450 aromatase gene expression and estradiol-17β levels in zebrafish early life stages
