| In mammals, testosterone synthesized by testis interstitial tissue play an important role in the physiological procedures such as malization of embryo development, production of sperm, male sexual behavior and male sex gland development. On physiological condition, Leydig cell steroidogenesis is modulated by LH/hCG, some growth factors and cytokines. Glucose is essential for Leydig cell steroidogenesis and glucose transporters (GLUTs) play a key role in the transport of glucose into the cells. However, there is limited information regarding the regulatory factors and regulating mechanisms of glucose uptake by GLUTs in Leydig cells. In this thesis, the effects of LH/hCG, growth factors and cytokines on glucose uptake in Leydig cells and testosterone formation were studied in order to investigate relationship between steroidogenesis and glucose metabolism in Leydig cells.Firstly, tissue-specific expression of GLUT8 gene in rat testis and Leydig cells were investigated by using RT-PCR. The results revealed that GLUT8 is expressed predominantly in the testis, in smaller amounts in heart and kidney, and in negligible amounts in liver and spleen. Furthermore, GLUT8 mRNA was found to be highly expressed in crude interstitial cells, Leydig cells and testicular and epididymal germ cells. These results indicated that GLUT8 predominantly distribuites in Leydig cells and sperm cells. The data of RT-PCR also indicated that, in prepubertal rat (20-day-old) tissues, GLUT8 expression was comparatively much lower than that in the adult rat tissues.Secondly, the rat Leydig cells, with the purity of 98% and the viability of 95%, were isolated using modified Klinefelter's method. Then, the regulatory effects of hCG, IGF-land cytokines (IL-la, TNF-a and IFN-r) on GLUT8 expression and testosterone formation in primary cultured rat Leydig cells were investigated by comparative RT-PCR and Western blotting. The results showed that hCG caused dose- and time-dependent increases of GLUT8IIImRNA levels. hCG and IGF-1 had synergistic effects on GLUTS mRNA and protein expression (P<0. 001). This result is consistent to the result from the experiment in which IGF-l(100ng/mL) and hCG(10ng/mL) can increase testosterone level synergistically (P<0. 001).Thirdly, GLUT1 and GLUT3 were also found to be expressed in Leydig cells. However, neither GLUT1 nor GLUT3 expression was affected by treatments with hCG, IGF-I, or hCG and IGF-I combined. The treatment of mIL-la (10 ng/mL), mTNF-a (10 ng/mL) and mIFN-y (500 U/mL) separately or in combination significantly decreased the hCG-induced GLUTS mRNA levels in adult rat Leydig cells (P<0. 001).Finally, transfected IP-10 gene was overexpressed in MA-10 mouse Leydig tumor cells. The results showed that IP-10 protein secreted by the IP-10 transfectants into the culture medium was 30-40 fold more than the basal levels as estimated by Western blotting. Transfection of MA-10 cells with IP-10 gene significantly decreased cAMP-induced progesterone synthesis (P<0.01). cAMP (0.2 mmol/L) induced StAR Dl mRNA expression but this StAR Dl mRNA expression induced by cAMP (0.2 mmol/L) was decreased 30-40% by transfection with IP-10, indicating that progesterone synthesis inhibition by IP-10 may be through decreasing StAR Dl expression. Transfection of IP-10 also significantly decreased 3H-thymidine incorporation into DNA induced by insulin-like growth factor (IGF-1, 100 ng/mL). These data suggest that IP-10 also inhibits MA-10 tumor cell proliferation and that it may be possible to use IP-10 in gene therapy for prostate cancer due to its antiangiogenic effects and its inhibitory effects on steroidogenesis.
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