Abstract
BackgroundCocaine-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 E2 was 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.MethodsThe 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).ResultsResults 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 E2 increase 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 E2 is greatest. It shows that the production of E2 needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E2 concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively.ConclusionsThese results suggested that CART plays a role to inhibit granulosa cells proliferation and E2 production, 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.
Highlights
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
Li et al Biol Res (2018) 51:6 we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals
CART mRNA expression levels in large, medium and small follicles Quantitative real time PCR analysis revealed that the expression level of CART mRNA was significantly higher in medium follicles than that in large and small follicles (P < 0.05) (Fig. 1)
Summary
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. Further research found that secretion of E 2 was 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. Cocaine-and amphetamine regulated transcript (CART), discovered initially by Douglass et al via differential display RT-PCR analysis of brains of rats administered cocaine [10], is expressed mainly in central nervous system or neuronal origin cells [11], which is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction [12,13,14,15]. As specific CART receptors have not been identified, the regulatory mechanisms of CART biological activities are still unknown
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