EVIDENCE FOR A LOCAL INHIBITORY EFFECT OF THE CART PEPTIDE ON LH-INDUCED THECAL ANDROSTENEDIONE PRODUCTION

Pleiotropic actions of Cocaine and Amphetamine Regulated Transcript (CART) are well documented in the brain and nervous system. We have previously established a novel local role for CART in negative regulation of bovine granulosal cell estradiol production. Granulosal cell CART mRNA and follicular fluid concentrations of the CART peptide are greater in atretic estrogen inactive subordinate follicles versus healthy estrogen active dominant follicles collected during the early stages of a follicular wave. Furthermore, CART treatment inhibits basal and FSH-stimulated estradiol production by bovine granulosal cells in vitro. However, whether CART can also regulate production of thecal androgen precursors required for estradiol biosynthesis is not known. Thus, the objective of the present studies was to determine the effects of CART on basal and LHstimulated androstenedione production in vitro by thecal tissue collected from bovine follicles at specific stages of a follicular wave. Estrus was synchronized in 24 crossbred beef heifers and follicular growth observed and recorded by daily ultrasonography. Ovaries were removed from heifers at the following stages of the first follicular wave: predeviation (~ d 3 post estrus; n = 8), early dominance (d 4-5 post estrus; n = 4), mid dominance (d 6-9 post estrus; n = 4), loss of dominance (d 10-12 post estrus; n = 4) and an ovulatory wave (preovulatory; 1 d after PGF2α injection on d 7 post estrus; n = 4). The three largest (F1, F2 and F3) follicles were isolated from predeviation and early dominance groups and only the largest follicle was isolated from mid dominance, loss of dominance and preovulatory groups. After harvesting of granulosal cells, theca interna tissue was removed from follicles, cut into 2 × 2 mm pieces and cultured overnight (≥ 4 wells/treatment) in the presence of media alone or media containing 0.01 or 0.1 μM CART (Experiment 1), or in the presence of media alone, LH (4 ng/ml), CART (0.1 μM) or CART plus LH (Experiment 2). Concentrations of androstenedione in culture media were determined by RIA and normalized relative to thecal tissue protein content per well. Irrespective of stage of follicular wave, no effects of CART treatment on basal androstenedione production were observed (Experiment 1). In contrast, potent, stage specific inhibitory effects of the CART peptide on LH-induced androstenedione production were observed (Experiment 2). Irrespective of follicle size or health status, the stimulatory effects of LH on androstenedione production by thecal tissue collected at the predeviation stage were blocked by CART treatment (P < 0.05). Furthermore, a 48% decrease in LH-induced androstenedione production by thecal tissue collected at the early dominance stage was observed in response to CART treatment (P < 0.05). Effects of LH and (or) CART treatment on thecal tissue collected at the mid dominance, loss of dominance and preovulatory stages were not observed. These results support a stage specific local role for CART in negative regulation of LHinduced thecal androgen production during dominant follicle development in cattle. Combined with previous results, the dual local negative actions of CART on androgen and estradiol production by thecal and granulosal cells suggest a key role for this novel peptide in regulation of follicular estradiol production and in turn selection of dominant follicles during follicular waves [Supported by USDA 2005-35203-16011]. (poster)