Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells

Abstract

Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G(o/i)-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.

Evidence That High Variation in Ovarian Reserves of Healthy Young Adults Has a Negative Impact on the Corpus Luteum and Endometrium During Estrous Cycles in Cattle1

“…After 18 h of culture at 378C, 75% of the media was removed and stored at À208C until the progesterone analysis. To isolate luteal cells after the culture, plates were washed three times with ice-cold DPBS, and the cells were treated with trypsin as previously described [17]. All cells greater than 10 lm were counted using the Coulter Counter.…”

“…Ovaries were placed in ice-cold supplemented DPBS and transported to the laboratory. The diameters of dominant follicles were measured with a caliper, and granulosal cells from each dominant follicle were isolated and placed in minimum essential medium alpha (MEMa) [17], which was supplemented as explained above for DPBS and with the following factors: long R3-IGF-1 (1 ng/ml), nonessential amino acids (1.1 mM, Invitrogen), sodium selenite (4 ng/ ml), androstenedione (10 À7 M), bovine insulin (10 ng/ml), apo-transferrin (5 lg/ml), sodium bicarbonate (10 mM), 0.5% (w/v) BSA (Sigma), and Hepes (20 mM). The viability of granulosal cells was determined by trypan blue exclusion dye at the beginning and end of the culture [16].…”

Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells

“…A completely serum-free long-term granulosa cell culture system that permits the induction/maintenance of FSHresponsive E 2 secretion was established for ruminants by Campbell et al (12,13) and has been used in several laboratories (14)(15)(16)(17).…”

Lower Cocaine- and Amphetamine-Regulated Transcript Levels Pose a Risk for Developing PCOS

“…FSH is well known to stimulate E2 synthesis, which, in turn, is crucial for reproduction. Using bovine granulosa cells, Sen and his colleagues reported an inhibitory role of CART on the FSH-induced E2 production [ 17 ] . Folger et al provided further evidence that CART signaling may act to suppress E2 producing capacity of subordinate follicles, thereby steering them to undergo atresia [ 16 ] .…”