Melatonin regulates the periodic growth of secondary hair follicles through the nuclear receptor RORα

Abstract

Cashmere is the fine bottom hair produced by the secondary hair follicles of the skin. This hair is economically important. Previous studies by our research group have shown that exogenous melatonin (MT) can regulate the periodic growth of secondary hair follicles, induce the secondary development of villi, and alter the expression of some genes related to hair follicle development. Few studies on the regulation of villus growth by MT binding receptors have been published. In this study, MT was implanted subcutaneously behind the ear of Inner Mongolia cashmere goats. RT-qPCR, in situ hybridization, Western blot analysis, immunofluorescence and RNAi techniques were used to investigate the receptors and functions of MT in regulating the development of secondary hair follicles in Inner Mongolia cashmere goats. The results showed that MT binds to the nuclear receptor RORα on dermal papilla stimulates hair follicle development and promotes villus growth. The RORα mRNA expression in the skin of Inner Mongolia cashmere goats was periodic and showed a trend of first increasing and then decreasing. The expression began to increase in February, peaked in April, and reached the lowest level in May. RORα significantly affected the mRNA expression of β-catenin gene, a key gene in hair follicle development, in the presence of MT. It will lay a solid molecular foundation for further research on the regulation mechanism between MT receptor and villus growth and development and to achieve artificial regulation of villus growth time and yield to improve the effect of villus production.

Keywords: Inner Mongolia cashmere goat; RORα; hair follicles grow periodically; melatonin; secondary hair follicle.

Figure 1

Images of 1-year-old Inner Mongolian cashmere goats. Photo taken of Inner Mongolia cashmere goats from Inner Mongolia Jinlai Animal Husbandry Technology Co., Ltd. (A) Inner Mongolia cashmere goats, (B) Inner Mongolia cashmere goat.

Figure 2

RT-PCR electrophoretic map. (A) MTNR1a M: DL2000 DNA marker. 1: Skin amplification results. 2, 3: Results of hypothalamus amplification. 4: Negative control. (B) MTNR1b M: DL2000 DNA marker. 1: Negative control. 2: Results of mouse skin amplification. 3: Results of hypothalamus amplification in cashmere goats. 4: Results of skin amplification in cashmere goats. (C) RORα M: DL2000 DNA marker. 1–4: Results of skin amplification in cashmere goats. 5: Negative control. 6: Results of amplification of the hypothalamus from cashmere goats.

Figure 3

Results of in situ hybridization of the MTNR1a cDNA probe in different tissues. (A) An antisense strand probe was hybridized in situ in hypothalamus tissue. (B) The sense strand probe was hybridized in situ in hypothalamus tissue. (D) The antisense strand chain probe was hybridized in situ in skin tissue. (E) The sense strand probe was hybridized in situ in skin tissue. (C,F) Blank control. Magnification is 100×.

Figure 4

The relative expression of RORα mRNA in the skin of cashmere goats in different months. The expression of RORα mRNA changed dynamically throughout the villus development cycle. qPCR results are the control (n = 3). a, b, and c represent significant differences (p < 0.05).

Figure 5

In situ hybridization of the RORα mRNA probe in skin tissue. (A) Results of in situ hybridization of the antisense strand probe in skin tissue of the MT-implanted group in February. (B) Results of in situ hybridization of the sense strand probe in skin tissue of the MT-implanted group in February. (D) Results of in situ hybridization of the antisense strand probe in skin tissue of the MT-implanted group in August. (E) Results of in situ hybridization of the sense strand probe in skin tissue of the MT-implanted group in August. (C,F) Blank control. Magnification is 40×.

Figure 6

The expression and distribution of RORα protein in primary hair follicles in February, September and December were detected by immunofluorescence. (A–C) The expression of RORα protein in primary hair follicles of the experimental group, control group and negative control group in February. (D–F) The expression of RORα protein in primary hair follicles of the experimental group, control group and negative control group in September. (G–I) The expression of RORα protein in primary hair follicles of the experimental group, control group and negative control group in December. The magnification of (A–E,G,H) is 400×, (F) is 200×, (I) is 100×. The white arrow points to a fluorescent signal.

Figure 7

The expression and distribution of RORα protein in secondary hair follicles in February, September and December were detected by immunofluorescence. (A–C) The expression of RORα protein in secondary hair follicles of the experimental group, control group and negative control group in February. (D–F) The expression of RORα protein in secondary hair follicles of the experimental group, control group and negative control group in September. (G–I) The expression of RORα protein in secondary hair follicles of the experimental group, control group and negative control group in December. The magnification of (A,D,G) is 400×, (E,H) is 200×, (B,C,F,I) is 100×. The white arrow points to a fluorescent signal.

Figure 8

The protein expression of RORα and β-actin in the hair follicle development period of Inner Mongolia cashmere goats was detected by Western blotting. (A) 1, 2, and 3 are RORα proteins in the implanted MT groups of February, September, and December, respectively; 4, 5, and 6 are RORα proteins of the control group in February, September, and December, respectively. (B) 1, 2, and 3 are β-actin proteins in the implanted MT groups of February, September, and December, respectively; 4, 5, and 6 are β-actin proteins of the control group in February, September, and December, respectively. (C) Relative protein expression level of RORα in hair follicles in February, September, and December. The data are presented as the means ± SEMs of triplicates from three independent experiments, using fold change compared to the control group (n = 3). The measure of β-actin was used as Intrinsic reference protein for Western blot. a, b represent significant differences (p < 0.05).

Figure 9

The effect of RNA interference on RORα gene expression in Inner Mongolia cashmere goat skin fibroblasts was detected by qPCR. shRNA was loaded into Inner Mongolia cashmere goat skin fibroblasts by RNA interference technology, and the expression of RORα was detected by qPCR before and after transfection in different groups. qPCR results are presented as means ± SEMs of triplicates from three independent experiments, using fold change compared to the control (n = 3). a, b, c represent significant differences (p < 0.05).

Figure 10

Effect of downregulation RORα expression on its related genes. (A) RNA interference inhibited RORα expression, and qPCR showed that MT could increase the expression of β-catenin. (B) RNA interference inhibited RORα expression, and qPCR showed that MT could lower the expression of TCHHL1. (C) RNA interference inhibited RORα expression, and qPCR showed that MT had no significant effect on SFRP1. qPCR results are presented as means ± SEMs of triplicates from three independent experiments, using fold change compared to the control (n = 3). a, b, c represent significant differences (p < 0.05).