Effects of Vitamin E on α-Tocopherol Transfer Protein Expression in the Pituitary of Cynoglossus semilaevis

Abstract

α-Tocopherol transfer protein (α-TTP) is a protein that exhibits a marked ligand specificity that selectively recognizes α-tocopherol (vitamin E) and plays a key role in regulating concentrations of vitamin E. The primary function of α-TTP is maintaining adequate vitamin E levels. However, the available data are insufficient to comprehensively understand the mechanisms by which α-TTP regulates vitamin E supplementation. Related studies have been mainly focused on humans, mice, and rats as the primary subjects; however, little is currently known about fish. The pituitary gland plays an important role in the growth and reproduction of fish. It synthesizes growth hormone and gonadotropin after receiving signals from the brain and releases the hormones into the blood through the axons. It is then transported to the target cells (tissues or organs) through the blood to perform the physiological function of hormones. As the pituitary tissue is very small, being nearly half the size of a grain of rice, it is difficult to detect the nutrient contents (such as vitamin E, which has been verified to play an important role in the growth and reproduction of fish) in the tissue. This limits the evaluation of the effect of nutrients on the pituitary gland. Therefore, this experiment was conducted to investigate the effects of vitamin E concentrations on the expression of the α-TTP gene in the pituitary tissue of a half-smooth tongue sole (Cynoglossus semilaevis). DL-alpha-tocopherol acetate (vitamin E, Sigma) was added to the basic diet, which was formulated with fish meal, casein, and wheat meal as protein sources, as well as phospholipids, fish oil, and soybean oil as lipid sources. The finalized crude protein was 56.0%, and the crude lipid was 14.1% at concentrations of 0, 200, 400, 800, and 1 600 mg/kg to feed half-smooth tongue sole (464.0±2.6 g) for 60 d. The fish were maintained in polyethylene tanks (diameter: 150 cm, height: 60 cm) with flowing filtered seawater at a rate of 50 L/min, and during the feeding trial, the water temperature ranged from 24 to 27 ℃, salinity from 30 to 31, and pH from 7.8 to 8.1. Dissolved oxygen was greater than 5.65 mg/L. In addition, the pituitary glands of similar fish were collected, minced into small pieces (about 1 mm3), and digested with 0.25% trypsin-EDTA (Gibco) for approximately 15 min at 25 ℃. Then, the digested glands were filtered through 70 μm nylon mesh (BD Falcon), filtrates were collected and centrifuged at 100 ×g for 10 min at 4 ℃, and cell deposits were resuspended in 1 mL L-15 medium containing 5% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Subsequently, the pre-dissolved vitamin E in ethanol solutions of 0, 18, and 54 μmol/L were added to the L-15 medium for the primary pituitary cell culture of 3 d. The ethanol content in the cell medium (0.1% v/v) was tested to ensure that it was not harmful to the cells, and its purpose was to dissolve the fat-soluble vitamin E in the culture medium. Then, the α-TTP gene was cloned using rapid amplification of cDNA ends PCR (RACE-PCR) technology, and the relative gene expression levels were analyzed via quantitative real-time PCR (qRT-PCR) in the tissues and the primary pituitary cells. The results showed that the total length of the α-TTP gene was 3 964 bp, encoding a total of 293 amino acids. The evolutionary gene tree showed that the α-TTP gene from C. semilaevis had a further genetic relationship with other fish. The α-TTP gene was expressed in all 11 tissues of the half-smooth tongue sole, with the highest expression in the spleen, followed by the kidney, and the lowest expression in the stomach. The feeding experiment indicated that the expression of the α-TTP gene showed an initial increase and then decreased with the increase in dietary vitamin E content. The α-TTP gene expression of 400 mg/kg in this group was significantly higher than that in other groups (P < 0.05). As vitamin E concentrations in the cell culture medium increased, the relative expression of the α-TTP gene increased significantly (P < 0.05), and no declining trend was observed. These findings suggested that vitamin E was involved in the expression of the pituitary α-TTP gene, whose expression level was affected by the vitamin E dose. Since the pituitary α-TTP gene expression could be a potential evaluation indicator to demonstrate the effect of vitamin E on fish growth and reproduction, the association should be further investigated and verified in subsequent related studies. In conclusion, vitamin E supplemented in the diet at the appropriate concentration of 400 mg/kg can significantly increase the expression of the α-TTP gene in half-smooth tongue soles. This study offers novel insights into the underlying mechanisms of α-TTP in mediating the effect of vitamin E on fish growth and reproduction.

Keywords

• vitamin e
• cynoglossus semilaevis
• pituitary
• α-tocopherol transfer protein (α-ttp)