Investigating selected Tribulus terrestris L. and Cinnamomum cassia Presl for their testosterone secretion ability from Leydig cells in mice

• Main Authors: Chih-Wei Chang, 張之維
• Other Authors: Jen-Hsou Lin
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/14337236167865181079

碩士 === 國立臺灣大學 === 畜產學研究所 === 90 === The purpose of this study was firstly to isolate purified mice Leydig cells and use enzyme immunoassay (EIA) for testosterone production. Selected Chinese medicinal herbs extracted by hot water extraction procedure were co-cultured with the isolated cells and their effect on testosterone secretion vis a vis basal and stimulants were investigated. Testis were obtained from mature ICR mouse of 6-8 weeks age, decapsulated and finely minced before dissociating with collagenase. The digested tissue is then sieved and Percoll discontinuous gradients method applied to separate the Leydig cells. The harvested Leydig cells were than incubated in M199 in a 48 well culture plate, different doses of Chinese medicinal herbs extracts, various stimulants and signal transductor inhibitors were added to the Leydig cells culture system. The medium was than collected at different time periods and assayed using EIA technique. The results and findings of our present work can be summarized as follows: 1. Investigating for 10, 20, 30, 40 and 50 % Percoll discontinuous gradients and using LH stimulation and 3β-HSD staining it was revealed that 50% Percoll gradients has better results. The Leydig cells when stimulated (LH, cAMP) had good testosterone production both at dose and time response indicating the successful establishment of mouse Leydig cells in vitro cell culture system. 2. Tribulus terrestris L. hot water extracts (H13) stimulated testosterone secretion of mouse Leydig cells, in 100 µg/ml dose almost double to that of basal. It also showed time course (24 and 48 hour) and dose response (1, 10 and 100µg/ml). The activity of protein kinase A (PKA) induced by H13 were inhibited when N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide (H89) was co-cultured. It leads us to presume that the stimulating effect of H13 was mediated by PKA signal transduction pathway. The activity of protein kinase C (PKC) induced by H13 was also inhibited with D-sphingosine explaining the involvement of PKC signal transduction pathway. With the use of actinomycin D and cycloheximide, results showed the actinomycin D and cycloheximide blocked H13 action. It further explains the H13 mediated stimulation via PKA and PKC activation leading to protein transcription and translation. 3. Cinnamomum cassia Presl extracted by hot water extraction (10 and 100 µg/ml) showed decrease in testosterone production over the basal and LH stimulated secretion of mouse Leydig cells in 4 and 24 hour time course. Cinnamomum cassia Presl did not decrease cAMP, as addition of forskolin and cholera toxin stimulated testosterone secretion. Addition of different steroid substrates to study Cinnamomum cassia Presl in steroidogensis enzyme functions revealed that it did not decrease 17α-hydroxyprogesterone, progesterone, pregnenolone and 22R-OHC stimulated testosterone secretion. The events that made us suggest that Cinnamomum cassia Presl decrease LH stimulated testosterone secretion function without acting on the steroidogensis pathway.