松果腺激素（MELATONIN）對大花鼠睪丸類固醇激素生成之調控：分子作用機制之離體研究

Abstract

松果腺激素（melatonin）主要為松果腺所分泌，具有抑制生殖之作用；早期實驗證明其可直接抑制睪丸助孕素與睪固酮之間的轉化，使睪固酮之生成減少，而增加助孕素的產量。同時，許多實驗證明，melatonin能引發多種組織產生c G.MP （guanosine 3′, 5′-monophosphate）。雖然也有少數證據顯示c GMP能產生類似melatonin之作用，但迄今仍無證據明確指出二者之相關性，甚至尚無以c GMP為second messenger之報告出現。本研究之目的即嘗試證明melatonin是否經由環核?酸作為second messenger，以進一步探討其分子作用方式。 實驗動物為八週齡雄性大花鼠（Long Evans rats）；以酵素分離法取得睪丸精間細胞，作離體培養（Medium 199, pH7.4, 34℃培養4小時，每分鐘震盪90次）。培養液中加入不同劑量之促黃體生成激素（LH）促進睪固酮生成，並加以melatonin, c AMP, c GMP, 2′, 3′-c GMP, c IMP, c UMP, c C M P以及細胞c G M P生成之促進劑或抑制劑。以放射免疫分析法定量所產生的睪固酮、助孕素及c GMP，而以競爭性蛋白質結合分析法定量c AMP及c GMP。 研究結果簡述如下：）在所有測試的環核?酸中，只有c GMP能模擬melatonin之作用，其他則稍有或幾無作用。而且，二者不僅定性上作用型式相同，定量上其抑制L H及c AMP（L H之second messenger）所引發睪固酮生成及增加助孕素產量之幅度亦極為近似；2）Sodium nitroprusside（細胞c GMP生成促進劑）同樣也顯著降低睪固酮之產量，而增加助孕素之分泌；3）在已知一些c GMP生成之抑制劑（methyl paraben, 3′-GMP , cystamine, dithioerythritol, mycophenolic acid及methylene blue）當中，尚無法找到能拮抗melatonin之作用者，因此有待進一步之研究；4）以放射免疫分析法或競爭性蛋白質結合分析法定量c GMP及c AMP，證明melatonin確實也能促進c GMP之產生，但不影響L H所引發c AMP之生成。 綜合以上結果，充分顯示c G M P很可能是melatonin的second messenger，或者至少是melatonin作用機制中一重要之介體（mediator）。此一發現，當有助於吾人對melatonin之分子作用方式作更進一步的瞭解；同時，藉由melatonin之調控作用，也將為性腺類固醇激素生成機制之探討提供一個嶄新的研究方向。

Melatonin, the major hormone secreted from pineal gland, has long been recognized to be antigonadotrophic. Early studies have proved that it inhibits the biotransformation from progesterone to testosterone, resulting in the reduction of testosterone production and the elevation of progesterone secretion. Melatonin has also been proved to be able to stimulate the formation of guanosine 3', 5'-monophosphate (cyclic GMP) in a variety of tissues. Albeit some evidences have also shown that cyclic GMP partially mimics the action of melatonin, no one has, so far, clearly pointed out the interrelations between the two biomolecules. The study was thus directed to prove whether the modulatory effect of melatonin on testicular steroidogenesis is mediated through the formation of such cyclic nucleotide, or more strictly, whether cyclic GMP is the second messenger of melatonin. Testicular interstitial cells, obtained by collagenase dispersion from 8-wk-old male Long Evans rats, were incubated in medium 199 (pH 7.4 , 34℃) with additions of ovine LH, melatonin, cyclic nucleotides, and certain stimulators or inhibitors of cyclic GMP generating system in the cell. After 4h of incubation, testosterone and progesterone were measured by radioimmunoassays (RIA's) while cyclic AMP was measured by the competitive protein binding assay (CPB assay). Cyclic GMP was quantified by both RIA and CPB assay. The results are summarized as follows: 1). among all the cyclic nucleotides tested, cyclic GMP was the only one that could mimic the action of melatonin, while the others had little or no effect. Besides, the mimicry was not only qualitative, but also quantitative. Therefore, cyclic GMP completely mimics the action of melatonin in rat testicular steroidogenesis by inhibiting testosterone formation and promoting progesterone secretion; 2). sodium nitroprusside, a chemical that directly stimulates guanylate cyclase and thus promotes mass production of cyclic GMP, also blocked the conversion from progesterone to testosterone; 3). among those inhibitors of cyclic GMP generating system in the cell (eg. methyl paraben, cystamine, 3'-GMP, dithioerythritol, mycophenolic acid, methylene blue), no one was found to antagonize the action of melatonin. Further investigations are therefore required to elucidate this phenomenon; 4). no matter whether with or without the stimulation of exogenous LH, melatonin stimulated cyclic GMP production by rat testicular interstitial cells and did not affect cyclic AMP formation. In conclusion, the results from the present study strongly imply that cyclic GMP is the second messenger or one of the mediators of the action of melatonin. Such a finding, therefore, helps better understand the molecular mechanism of melatonin. From the study of the modulatory action of melatonin and the inhibitory effect of cyclic GMP, it will also lead us to a new domain in the research of testicular steroidogenesis. Furthermore, the mode of action of melatonin by producing cyclic GMP as its mediator may not only be limited to the testis, but also a universal pattern in all other endocrine organs.