176 GENE EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) ISOFORMS IN GRANULOSA CELLS OF FOLLICLES FROM NELLORE HEIFERS BEFORE, DURING, AND AFTER FOLLICULAR DEVIATION

Abstract

During bovine follicular development, there is a phase known as follicular deviation in which the future dominant follicle grows faster than the other follicles and acquires LH receptors (LHR). In Nellore breed, deviation occurs 2.5 days after ovulation, and at this time, the dominant follicle has in average a diameter of 6.0 mm. Some authors believe that LHRs are present in the future dominant follicle before deviation and are essential for this process. However, others are convinced that LHRs are present only during or after follicular deviation. The aim of the present experiment was to evaluate the expression of 4 LHR isoforms (M1 to M4) in granulosa cells of follicles from Nellore heifers before, during, and after follicular deviation. At a random stage of the estrous cycle (D0), Nellore heifers (n = 21) received a progesterone intravaginal device (1.0 g, Primer^®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin^®, Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg d-cloprostenol, i.m., Prolise^®, ARSA S.R.L., Buenos Aires, Argentina), and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.), and from this point in time, the growth of the dominant follicle growth was observed by ultrasonography (US, Aloka 900, Tokyo, Japan) every 12 h. The animals were allocated in 3 groups: Group 2 (G2, 2 days after ovulation, n = 7), Group 2.5 (G2.5, 2.5 days after ovulation, n = 7), and Group 3 (G3, 3 days after ovulation, n = 7), and were slaughtered 2, 2.5, and 3 days after ovulation, respectively, in order to remove the ovaries. The granulosa cells, obtained from ovarian follicles, were separated for total RNA extraction, and the gene expression of LHR isoforms was measured by semiquantitative RT-PCR. Since LHR expression was not detected in Group 2 (follicles with 4.5 to 6.7 mm), comparisons were performed between groups G2.5 and G3 by ANOVA. The LHR expression was detected only in 2 samples of Group G2 (7.0-mm follicles) and was significantly higher in Group G3 (63.6%; follicles from 8 to 14 mm, P < 0.05). In all samples that expressed LHR, the 4 isoforms were present. It is concluded that LHR expression is present in granulosa cells of follicles from Nellore heifers after follicular deviation.

Support and fellowship from FAPESP (Sao Paulo, Brazil).We are grateful to Tecnopec (Sao Paulo, Brazil) for providing intravaginal devices used in the experiment.