Changes of Kiss-1-GPR54 signaling pathway of growing rats
To determine whether Kiss-1-GPR54 signaling pathway affected by age (P < 0.01), we measured Kiss-1, GPR54, PLC mRNA levels in the hypothalamic. We found similar variation tendency in Kiss-1, GPR54, and PLC mRNA in the hypothalamic during development, it was fluctuated with the growth and development in male rats. All of the three genes first increased and then decreased from PND 21 to PND 56, and reached a peak at PND 43. Additionally, we analyzed Kiss-1 protein expression in the rat hypothalamic ARC via immunofluorescence, we found that the protein expression of Kiss-1 also reached a peak at PND 43, which was consistent with mRNA. But the change of GPR54 protein did not change during development was contrast with Kiss-1 protein. The number of GPR54 protein was more than Kiss-1 protein throughout the developmental process (Fig. 3).
Effect of high-fat-diet on the expression of Kiss-1-GPR54 signaling pathway of growing rats
As shown in Fig. 2B, we found that high-fat diet intervention upregulated the mRNA expression of Kiss-1 in the hypothalamic. Compared with C group, it was shown a significantly increased at PND 56 after high-fat diet intervention (Fig. 2B, P < 0.05). Furthermore, a high-fat diet increased the protein expression of Kiss-1 in the ARC than in the C group at both PND 43 and PND 56 (Fig. 4B, P < 0.05).
PLC is downstream of the Kiss-1 system, as a key factor in stimulating voltage-gated calcium channels. As shown in Fig. 2E and 2F, the expression of PLC mRNA at PND 43 was significantly higher than other period, high-fat diet decreased the expression of PLC mRNA in hypothalamic, especially at PND 43.
Effect of exercise on the expression of Kiss-1-GPR54 signaling pathway of growing rats
To investigate whether the effect of exercise intervention on high-fat diet male rats of different ages are consistent, we examined expression of Kiss-1-GPR54 signaling pathway at the age of PND 43 and PND 56 in both CE and HE groups. We found that the expression of Kiss-1 mRNA in the hypothalamic at PND 43 was significantly increased after continuous 60–70% •VO2max treadmill training in high-fat diet rats (Fig. 2B, P < 0.05). The mRNA levels in GPR54 in the hypothalamic were significantly increased in both CE and HE groups after exercise intervention (Fig. 2D, P < 0.05). Exercise intervention could upregulate Kiss-1-GPR54 signaling pathway in high-fat diet rats at PND 43. But for maturity rats, exercise intervention showed a different result. As shown in Fig. 2B, we found that exercise intervention downregulated the expression of Kiss-1 mRNA and protein in high-fat diet rats at PND 56, which was the maturity period (Fig. 2B, 2H, 2I, P < 0.05,). The expression of GPR54 protein in the ARC of the hypothalamic in the HE group was also remarkable lower than that in the HC group at PND 56 (P < 0.05, Fig. 4D, 4E, 4I).
Effect of high-fat diet and exercise on body mass and reproductive system of male rats
Body weight and body length increased gradually with growth in all groups. High-fat diet intervention significantly upregulated body mass and body length at PDN 43 (P < 0.05, Fig. 5A, 5B), but not at PND 56 (P > 0.05). Exercise intervention significantly downregulated body mass and body length at PND 56 in standard diet rats (P < 0.05), and significantly reduced body mass at PND 56 in high-fat diet rats (P < 0.05). We also found that the body mass and body length showed a tendency to increase after exercise intervention in standard diet rats at PDN 43, which was consistent with then change of Kiss-1.
In C group, we found the testicular mass was significantly increased with the rise of body mass and body length in all groups. The results of HE staining also showed that with the development of the testes, the number of sperm cells and spermatocytes increased in C group of different age stage (Fig. 6A, 6B, 6C), meanwhile, the serum testosterone levels increased at all stages of development from childhood to maturity as the morphology of testis matured, which were significantly increased at PND 56 (Fig. 5D, P < 0.05).
After high-fat diet or by 60–70% •VO2max treadmill training intervention, the weight of the testes did not show any change (Fig. 5C), but HE staining in high-fat diet rats showed that the testicular lipid droplets increased, while, the number of spermatozoa and leydig cells in the testicular decreased. From the staining results, we found that the epithelium of spermatogenic cells were arranged loosely, and in the seminiferous tubules, vacuoles formed between spermatocytes and spermatoblasts due to the deposition of lipid droplets (Fig. 6F, 6G). The serum testosterone levels decreased in high-fat diet rats (P < 0.05, Fig. 5D), but it was mainly occurred at PND 43. Exercise intervention could increase serum testosterone levels in high-fat diet rats (Fig. 5D, P < 0.05), but it also reduced serum testosterone levels in standard diet rats, especially at PND 56. Meanwhile the results of HE staining showed that the vacuoles formed between spermatocytes and spermatoblasts decreased with exercise, and the number of leydig cells in the HE group was also increased compared with those in the HC group (Fig. 6H, 6I).