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Cocaine- and Amphetamine-Regulated Transcript (CART):
The Gatekeeper of Follicle Selection
Aritro Sen
Research Assistant Professor, University of Rochester School of Medicine & Dentistry
Division of Endocrinology and Metabolism; 601 Elmwood Ave., Rochester, NY 14642
Regulation of estradiol (E) 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. While the regulation of E production by pituitary gonadotropins,
steroid hormones and growth factors are well established, little is known about intra-follicular
regulators, signaling mediators and intracellular mechanism(s) that inhibit E production. During a
bovine ovarian (oocyte) expressed sequence tag (EST) project, 5 ESTs were identified encoding for a
neuropeptide known as cocaine and amphetamine regulated transcript (CART). Pleiotropic actions of
CART have been well described in the brain and nervous system, including anorexigenic,
neuroendocrine and anti-psychostimulatory effects, while naturally occurring CART mutations in
humans is associated with early onset of obesity and anxiety/depression. However, the intracellular
mechanism(s) mediating biological actions of CART are poorly understood. To date, we have
compelling in vivo and in vitro (CART responsive serum free bovine granulosa cell primary culture
system) evidence that establish CART as a novel intra-follicular negative regulator of steroidogenesis.
Expression of CART mRNA and peptide are detectable in the bovine oocyte and granulosa cells (GC)
of follicles and is temporally and spatially regulated during bovine follicular development. Studies
indicate that CART peptide and mRNA are greater in estrogen-inactive atretic follicles. Also intrafollicular administration of CART in vivo into early dominant follicles result in a dramatic reduction in
follicular fluid E concentrations and GC CYP19A1 mRNA levels. Furthermore there is a pronounced
temporal regulation of GC CART expression near the time of dominant follicle selection and negative
regulation of GC CART mRNA in vitro in response to FSH or IGF1 treatment. Furthermore, in vitro
studies demonstrate the involvement of CART in negative regulation of E production. The negative
actions of CART on E production are mediated by inhibition of FSH-induced cAMP accumulation,
Ca2+ influx and aromatase mRNA expression via a G protein (Go/i) dependent pathway. In addition, the
FSH-induced E production is dependent on Erk1/2 and Akt pathways. CART is a potent inhibitor of
FSH-stimulated Erk1/2 and Akt signaling. Transient CART stimulation of bovine GC shortens the
duration of FSH-induced Erk1/2 and Akt signaling while a prolonged (24 h) CART treatment blocks
Erk1/2 and Akt activation in response to FSH. This CART-induced accelerated termination of Erk1/2
and Akt signaling is mediated both by induced expression and impaired ubiquitin-mediated proteasome
degradation of dual specific phosphatase 5 (DUSP5) and protein phosphatase 2A (PP2A). Results also
support existence of a negative feedback loop where CART via a Go/i-MEK dependent pathway
activates Erk1/2 and the latter induces DUSP5 expression. Moreover, siRNA mediated ablation of
DUSP5 and/or PP2A prevents the CART-induced early termination of Erk1/2 and Akt signaling. Given
the importance of E within and outside the reproductive system, results establish CART as a negative
regulator of FSH –induced E production and support a potential functional role for CART in selection
of dominant follicles during follicular waves in mono-ovulatory species such as the bovine and perhaps
the human. In contrast, CART is not expressed in the ovaries of rats and CART mutant mice are fertile.
These observations suggest that poly-ovulatory species such as rodents may require a less stringent
selection mechanism to ovulate multiple follicles thus providing an evolutionary explanation for the
absence of CART expression in the ovary of polytocous species and support the hypothesis that CART
functions as a “gatekeeper” of the ovulatory quota in monotocous species.