Li Pengfei, Meng Jinzhu, Jing Jiongjie, Hao Qingling, Zhu Zhiwei, Yao Jianbo, Lyu Lihua
College of Life Science, Shanxi Agricultural University, Taigu, 030801, Shanxi, China.
Wujiang College, Tongren University, Tongren, 554300, Guizhou, China.
Biol Res. 2018 Feb 26;51(1):6. doi: 10.1186/s40659-018-0154-y.
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 E 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.
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 (E) 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 E 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 E is greatest. It shows that the production of E needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E concentration 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 E 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.
可卡因-安非他明调节转录肽（CART）是一种内源性神经肽，广泛存在于动物体内，在牛和羊卵泡闭锁的调节中起关键作用。在动物卵巢中，CART mRNA最早在牛卵巢中被发现。通过免疫组织化学和原位杂交技术发现，CART定位于腔前卵泡的卵母细胞、颗粒细胞和卵丘细胞中。进一步研究发现，一定剂量的CART可抑制颗粒细胞中E的分泌，其作用取决于细胞分化阶段，提示CART在调节卵泡闭锁中可能起关键作用。本研究的目的是表征猪卵泡颗粒细胞体内和体外CART的表达模式及激素分泌情况，初步研究CART对猪等多胎动物颗粒细胞增殖和激素分泌是否有影响。
采用qRT-PCR技术分析不同卵泡颗粒细胞中CART mRNA的表达水平。利用免疫组织化学技术定位CART肽。使用长期培养系统将颗粒细胞在添加不同浓度CART和促卵泡激素（FSH）的培养基中培养168小时，并用显微镜观察。通过酶联免疫吸附测定（ELISA）检测不同试验组卵泡液中雌二醇（E）和孕酮（P）的浓度。
结果显示，CART mRNA在中等卵泡中的表达水平最高，显著高于大卵泡和小卵泡（P < 0.05）。免疫组织化学结果表明，CART在大卵泡的颗粒细胞和卵泡膜细胞中均有表达，而在中、小卵泡中仅在卵泡膜细胞中检测到CART。颗粒细胞培养168小时后发现，当CART浓度为0 μM时，E的浓度随促卵泡激素（FSH）浓度的增加而增加；当FSH浓度达到25 ng/mL时，E的浓度最高。这表明猪卵巢卵泡颗粒细胞中E的产生需要FSH的诱导。当培养基中FSH浓度分别为25和50 ng/mL时，随着CART浓度（0.01、0.1、1 μM）的增加，E浓度呈下降趋势。
这些结果表明，CART在体外对猪卵巢卵泡颗粒细胞中由FSH诱导的颗粒细胞增殖和E产生起抑制作用，但抑制作用不显著。因此，我们推测CART可能不是猪卵泡发育过程中的主要局部负调控因子，这与单胎动物不同。
