Cocaine-and amphetamine regulated transcript (CART) is an endogenous neuropeptide, which is widespread in animals, plays a key role in regulation of follicular atresia in cattle and sheep. Among animal ovaries, CART mRNA was firstly found in the cattle ovaries. CART was localized in the antral follicles oocytes, granulosa and cumulus cells by immunohistochemistry and in situ hybridization. Further research found that secretion of E2was inhibited in granulosa cells with a certain dose of CART, the effect depends on the stage of cell differentiation, suggesting that CART could play a crucial role in regulating follicle atresia. The objective of this study was to characterize the CART expression model and hormones secretion in vivo and vitro in pig follicle granulosa cells, preliminarily studied whether CART have an effect on granulosa cells proliferation and hormones secretion in multiparous animals such as pigs.
The expression levels of CART mRNA in granulosa cells of different follicles were analyzed using qRT-PCR technology. Immunohistochemistry technology was used to localize CART peptide. Granulosa cells were cultured in medium supplemented with different concentrations of CART and FSH for 168 h using Long-term culture system, and observed using a microscope. The concentration of Estradiol (E2) and progesterone (P) in follicular fluids of different test groups were detected by enzyme linked immunosorbent assay (ELISA).
Results showed that expression level of CART mRNA was highest in medium follicles, and significantly higher than that in large and small follicles (P < 0.05). Immunohistochemical results showed that CART were expressed both in granulosa cells and theca cells of large follicles, while CART were detected only in theca cells of medium and small follicles. After the granulosa cells were cultured for 168 h, and found that concentrations of E2increase with concentrations of follicle-stimulating hormone (FSH) increase when the CART concentration was 0 μM. And the concentration of FSH reached 25 ng/mL, the concentration of E2is greatest. It shows that the production of E2needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E2concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively.
These results suggested that CART plays a role to inhibit granulosa cells proliferation and E2production, which induced by FSH in porcine ovarian follicular granulosa cells in vitro, but the inhibition effect is not significant. So we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals.