Regulation of Granulosa Cell Cocaine and Amphetamine Regulated Transcript (CART) Binding and Effect of CART Signaling Inhibitor on Granulosa Cell Estradiol Production During Dominant Follicle Selection in Cattle1

Abstract

We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.

Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection

“…Beginning 2 days after the second prostaglandin F2α injection, ovaries were scanned 2–3 times daily using ultrasonography to monitor ovulation and initiation and growth of the first wave of follicles. Follicles were collected at specific stages of the follicular wave (including at emergence [EM; first scan where a follicle ≥4 mm is detected; usually the same day as ovulation; n = 3], pre-deviation [PD; 1.5 d after EM; ∼day 3 of the estrous cycle; n = 5], onset of diameter deviation [OD; first scan where growth of the F1 (largest; future dominant) follicle to >8.5 mm was detected and the F2 (second largest; future subordinate) follicle was still growing; ∼day 4 of the cycle; n = 5], and early dominance stage [ED; first scan where one follicle in the cohort was 2 mm larger than others; ∼day 5 of the cycle; n = 5]) as described previously [9]. Granulosa cells were isolated from the F1 and F2 follicles and immediately lysed and stored at −80°C.…”

“…Enhanced capacity of the dominant follicle to produce estradiol is critical to sustain dominant follicle growth and initiate estrous behavior, the preovulatory gonadotropin surge, resumption of meiosis and ovulation [4]. Estradiol production during follicular development in cattle is regulated by diverse hormones and growth factors including FSH [3], IGF1 [3], [5], [6], CART [7], BMP2 [8] as well as estradiol itself [9]. Recently it has been shown in rodents that estradiol production is regulated by members of the wingless-type mouse mammary tumor virus integration site (WNT) signaling family which modulate FSH action [10], [11].…”

Study on the relationship between expression patterns of cocaine-and amphetamine regulated transcript and hormones secretion in porcine ovarian follicles

“…Granulosa cells were cultured in a humidified environment of 5% CO 2 and air at 37 °C for 168 h, medium was replaced with fresh medium every 48 h. Granulosa cells were harvested after termination of culture, washed by DPBS and digested using tryptase, and cell numbers were determined [26]. The situation of cells were observed and collected images after cultured for 48, 96, 144 and 168 h. At the end of culture, 340 μL medium were pooled from 2 adjacent wells per treatment, stored at − 20 °C for measurement of hormone.…”

Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways

“…To our knowledge, there are no investigations directly examining the effect of amphetamine on sex hormone production in female granulosa cells. However, several reports have revealed that cocaine- and amphetamine-regulated transcript (CART), a neuropeptide protein, is capable of negatively regulating female sex hormone release [ 53 , 54 , 55 , 56 , 57 ]. For example, CART was expressed both in granulosa cells and theca cells [ 54 ].…”