松果腺激素（Melationin）對鼠類睾丸雄性素生成之影響

Abstract

本研究之目的有三：(一)探討melatonin與serotonin對鼠類(rodents)睪丸雄性素生成之影響。(二)比較melatonin及serotonin對四種鼠類睪丸雄性素生成影響之種別性差異。(三)研究melatonin對大鼠睪丸雄性素生成抑制之作用方式與機制。 實驗動物為大鼠(rats; 8週齡)中之Long Evans rats (大花鼠)及Levis rats (大白鼠)；倉鼠(Syrian golden hamsters; 8週齡)及小白鼠(mice; 6週齡，ICR strain)。睪丸自動物取出後以collagenase分離精間細胞(interstitial cells)，在34℃連續通氣之狀況下培養4小時。在培養時於培養液(Medium 199, pH 7.4)中加入不等量羊之促黃體生成激素(ovine LH)及melatonin或其他藥物。培養後以放射免疫法測定培養液中與細胞中雄性素與助孕素之含量。 研究結果簡述於下：(一)種別性差異：高劑量之melatonin (10^(-4) M, 5×10^(-5)M)在大花鼠、大白鼠及倉鼠之精間細胞可抑制ovine LH所引發之雄性素生成，而提高助孕素之分泌量。較低劑量之melatonin及各種劑量之 serotonin (10^(-4) M, 10^(-12)M）對雄性素及助孕素均無明顯之影響。各種劑量之melatonin及serotonin對小白鼠精間細胞雄性素及助孕素之生成皆無影響。(二) Melatonin作用方式與機制：以大花鼠為模式發現(l) melatonin不使細胞內之雄性素含量增加；(2) melatonin可抑制cAMP所引發之雄性素生成，並使助孕素增加；(3) cGMP亦可抑制LH或cAMP所引發之雄性素生成，但對助孕素分泌量提高之程度則較melatonin小；(4) cycloheximide （蛋白質生成抑制劑）可同時抑制LH所引發之雄性素及助孕素之生成，因此與melatonin作用之方式不同；(5) melatonin自培養液中移去，其抑制作用迅?消失，表示其作用並非長效性的。 綜合上述之研究結果，本研究首次建立melatonin可直接作用於離體之睪丸精間細胞，抑制LH所引發之雄性素生成。此抑制效果是作用於由助孕素轉變為雄性素之中間步驟。而melatonin不影響精間細胞產生cAMP或整體蛋白質生成作用。cGMP對雄性素生成之影響部分類似melatonin，但不完全相同。

The purposes of this thesis were: (1) to explore the effects of melatonin and serotonin on in vitro testicular androgen formation of rodents; (2) to compare the species difference in the effects of melatonin and serotonin on androgen synthesis in four rodent species; (3) to study the mode of action of the inhibitory effect of melatonin on testicular androgen formation. Four rodent species were used: Long Evans rats and Levis rats (8 weeks old), Syrian golden hamsters (8 weeks old) and mice (ICR strain, 6 weeks old). Testicular interstitial cells were prepared by dispersion with collagenase and were incubated in Medium 199(pH 7.4) with various doses of ovine LH and melatonin or other chemicals for 4 hours under continuous aeration of 95% O, 5% CO at 34C. After incubation, androgen and progesterone in medium and tissue were estimated by radioimmunoassays. The results may be summarized as follows: 1) species difference in melatonin and serotonin actions: higher doses of melatonin (10 M, 5x10 M). inhibited the ovine LH induced androgen formation by the interstitial cells of Long Evans rats; Levis rats and hamsters, while the progesterone formation was considerably elevated. Lower doses of melatonin (10 M, 10 M) and all doses of serotonin (10 M-10 M) had no effect on formation of both androgen and progesterone. The formation of androgen and progesterone by interstitial cells of mice were not affected by all doses of melatonin and serotonin; 2) mode of action of melatonin: Long Evans rats were used for this experiment, the results revealed that a) melatonin did not increase the androgen content within the cells, b) melatonin inhibited the cyclic AMP stimulated androgen formation and elevated progesterone synthesis in a similar pattern to that stimulated by LH. c) cyclic GMP inhibited LH or cyclic AMP- stimulated androgen formation in a similar fashion as was melatonin, but the extend of progesterone elevation was less. d) cycloheximide inhibited the formation of both androgen and progesterone thus it has a different action from melatonin on androgen formation. e) after removal of melatonin from incubation medium, the inhibitory effect of melatonin on androgen formation vanished; this indicates the effect of melatonin is not long-lasting. In conclusion, the present study established for the first time, the in vitro action of melatonin on LH induced androgen formation by isolated testicular interstitial cells of rodents species. Melatonin exerts a direct inhibitory action on testicular androgen synthesis in rats and hamsters, but with no effect in mice, exibiting a species specificity. The site of action of melatonin is not at membrane LH receptors, but at intracellular steps of conversion from progesterone to androgen without affecting the release of androgen from interstitial cells. Serotonin, unlike melatonin, are without effect on testicular androgen formation in the rodent species studied. The action of cyclic GMP partially simulates melatonin in testicular steroidogenesis, whether cyclic GMP is a secondary mediator of melatonin require further studies.