Estradiol Concentration and Gene Expression of Mrna for CYP19A1, PAPP-A and LHR in Dominant and Subordinate Follicles at Follicle Deviation in Nelore Cows

The main objective of this experiment was to investigate expression of mRNA of genes associated with follicle deviation, CYP19A1 (aromatase), LH receptor (LHR) and PAPPA (Pappalysin 1), using an in vivo model to obtain granulosa cells. Nelore (NEL; n = 10), non-lactating, 3-6 years old females, had follicle wave emergence synchronized by transvaginal aspiration of follicles ≥5 mm in a cross-over design. At the same time, they received an intravaginal progesterone device (CIDR®, Zoetis, Brazil) and 24h later, two doses with 0.5 mg of cloprostenol with 12h interval, with luteolysis monitored by ultrasound. Ovaries were evaluated every 12h to characterize follicular dynamics. A model based on follicular aspiration was used, and cows were distributed among three treatments: group 0h, when the largest follicle reached 6.5 mm, the two largest follicles were aspirated (DF0h and SF0h); group 12h, the two largest follicles (DF12h and SF12h) were aspirated 12h after the largest follicle reached 6.5 mm; and deviation group, the largest follicle (DF0h) was aspirated when it reached 6.5 mm, and the second largest follicle (SF→DF) was aspirated 12h later. Granulosa cells were obtained by washing the follicle cavity by successive aspiration and ejection with 1 mL of sterile saline solution, using an aspiration system with double lumen. The suspension was centrifuged and the supernatant was stored. Follicular fluid estradiol-17β (E2) concentration was measured by ELISA. The cell pellet was suspended in lysis buffer from RNeasy kit (Qiagen, SP, Brazil) and mRNA expression was analyzed by RT-PCR for LHR, CYP19A1 and PAPPA. Statistical analysis for follicular dynamics data and gene expression was performed using the PROC MIXED of SAS. Samples contaminated with blood or E2 concentration below 1 ng/mL were removed from the analysis. Follicular fluid E2 concentration (ng/mL) from SF→DF (176.9 ± 48.8; n = 4) was higher (P≤0.02) than from SF0h (34.2 ± 35.8; n = 7) and SF12h (28.9 ± 46.0; n = 4), and did not differ (P>0.05) from DF0h (188.9 ± 30.3; n = 7) and DF12h (244.4 ± 46.0; n = 4). There was no difference (P>0.05) among groups in relative mRNA expression for PAPPA (DF0h: 0.14 ± 0.05; SF0h: 0.14 ± 0.07; SF→DF: 0.31 ± 0.08; DF12h: 0.14 ± 0.08, and SF12h: 0.14 ± 0.08) and CYP19A1 (DF0h: 0.38 ± 0.13; SF0h: 0.27 ± 0.15; SF→DF: 0.49 ± 0.20; DF12h: 0.47 ± 0.19, and SF12h: 0.06 ± 0.19). There was a tendency for greater LHR mRNA expression in SF→DF follicles (3.86 ± 1.43) compared to group 0h (DF0h: 0.56 ± 0.86, P=0.07, and SF0h: 0.46 ± 1.08; P=0.06). There were no differences in gene expression between SF→DF and the 12h group (DF12h: 5.71 ± 1.43 and SF12h: 1.04 ± 1.43). DF12h LHR expression was highest in groups 0h (DF0h and SF0h; P=0.01) and SF12h (P=0.05). We concluded that increased expression of the LH receptor was the main early mark of dominance in Nelore cows.