Role of Cocaine- and Amphetamine-Regulated Transcript (CART) in Regulation of Antral Follicle Development in Cattle: Demonstration of Inhibitory Effects of the CART Peptide on Follicular Estradiol Production and Granulosa Cell Aromatase mRNA In Vivo and Potential FSH Regulation of CART Receptor Binding.

We have previously established a novel potential local role for CART in negative regulation of estradiol production by bovine granulosa cells. CART is a potent inhibitor of multiple components of the FSH signal transduction cascade in bovine granulosa cells culminating in markedly reduced estradiol production and aromatase mRNA in response to CART treatment in vitro. In addition, CART expression (follicular fluid CART peptide concentrations and granulosa cell mRNA abundance) is elevated in subordinate versus dominant follicles collected at specific stages of a follicular wave. Collectively above results imply a role for CART in dominant follicle selection. CART's negative actions in vitro are blocked by an inhibitor of the o/i subclass of inhibitory G-proteins, implying existence of the CART receptor on granulosal cells. However, the identity, binding characteristics and regulation of the CART receptor are relatively unknown. Furthermore, the lack of robust effects of CART on basal estradiol production suggests that granulosa cell CART binding may be FSH regulated. Based upon these results, we hypothesized that 125I-CART specifically binds to granulosa cells and that CART binding is FSH regulated. To test these hypotheses, granulosa cells collected from 2-5 mm follicles (freshly isolated or after 7 d of culture) were incubated with 125I-labeled CART (61-102) in the presence (non-specific binding) or absence (specific binding) of a 1000-fold excess of unlabeled CART (55-102) for 45 min at 37C. The cells were washed twice and total and specific binding of 125I-CART quantified. Specific, saturable binding of 125I-CART to granulosa cells was detected with a Kd of 0.47 nM and a Bmax of 325 sites per cell. Furthermore, specific binding of 125I-CART was ~7-fold higher for granulosa cells incubated with FSH for 7 d compared to cells incubated in media alone or freshly isolated granulosa cells. These results suggest that the transient increase in FSH accompanying each follicular wave may upregulate granulosa cell CART binding sites and thus explain the robust inhibitory effects of CART on FSH-stimulated but not basal estradiol production observed in vitro. Based on these results, coupled with the negative effects of CART on estradiol production in vitro, we hypothesized that an intra-follicular injection of CART peptide in vivo would decrease granulosa cell aromatase mRNA and follicular fluid estradiol concentrations in dominant follicles. To test this hypothesis, CART (55-102) or vehicle were injected into first wave dominant follicles during the early dominance stage (Day 4-5 of the bovine estrous cycle) using an ultrasound-guided injection approach. Twenty four hours later the contents of the follicle were aspirated and follicular fluid concentrations of CART and estradiol and abundance of aromatase mRNA in granulosa cells were determined. Results showed that intra-follicular injection of CART decreased aromatase mRNA abundance 96% and follicular fluid estradiol concentrations 54% compared to vehicle injected controls demonstrating similar inhibitory effects of CART on granulosa cell aromatase mRNA expression and estradiol production in vivo as previously observed in vitro. Collectively results of above studies strongly support a potential key role for CART in selection of dominant follicles during follicular waves in single-ovulating species like cattle. Supported by USDA 2005-35203-16011 to GWS. (platform)