探討脂肪酸降低小鼠萊迪氏細胞之類固醇生成作用

Abstract

不孕症（infertility）是現今社會中常見的一個問題。根據世界衛生組織的定義，一對夫妻或情侶在沒有採取任何避孕措施的情形下，經過一年以上規律的性行為（平均每週1~3次）後仍然無法成功懷孕，即可以被診斷為不孕症。有鑑於現今生活型態的改變與高糖、高脂之飲食習慣的普及化，肥胖已成為造成不孕的其中一個重要原因。在兩個性別中，雄性個體的生殖能力取決於其本身精子的品質與數量，而其中，位於睪丸的萊迪氏細胞（Leydig cell）所分泌之睪固酮（testosterone）內泌素，在精子製造過程中扮演著重要的角色。由於肥胖個體也常常伴隨著高血脂的症狀，其血液中的游離脂肪酸濃度高於正常標準，因此本研究擬探討在肥胖高血脂的情況下，血液中過高的游離脂肪酸是否會導致萊迪氏細胞類固醇生成低下，最終影響其生殖能力。 我們使用MA-10小鼠萊迪氏細胞株作為模式細胞，並添加1,200 µM脂肪酸［600 µM油酸（oleic acid）及600 µM棕櫚酸（palmitic acid）］於細胞培養液內進行培養48小時，以模擬長時間細胞處於高血脂環境的狀態。結果發現長時間的脂肪酸處理除了會顯著降低MA-10細胞的孕酮（progesterone）生成能力之外，也會抑制CYP11A1與3β-HSD這兩個類固醇生成酶的基因表現。此外，我們也利用類固醇生成酶的受質22(R) -羥基膽固醇（22(R)-hydroxycholesterol）與孕烯醇酮（pregnenolone）檢測類固醇生成酶活性，發現高濃度脂肪酸在降低MA-10細胞類固醇生成能力的原因主要是抑制了CYP11A1這個類固醇生成酶的活性。同時，我們也發現MA-10細胞在經過脂肪酸處理後，其細胞自噬（autophagy）的運送蛋白p62與內質網壓力（endoplasmic reticulum stress）的標記CHOP皆顯著的上升；而且增加的p62與CHOP表現量，可以透過以雷帕黴素（rapamycin）活化細胞自噬作用而有回復的情形。值得注意的是，雷帕黴素除了能夠回復高濃度脂肪酸造成的細胞自噬受損之外，它同時也回復了基礎狀態下脂肪酸所抑制的類固醇生成作用。此一結果顯示在MA-10細胞內，脂肪酸會藉由尚未明瞭的機制損壞細胞自噬作用，進而影響到細胞內的類固醇生成作用。 總結上述，高濃度脂肪酸確實是造成MA-10萊迪氏細胞之類固醇生成作用低下的原因之一。首先，脂肪酸會直接抑制CYP11A1此類固醇生成酶之活性，而使類固醇生成作用受到阻礙；而且，脂肪酸也可能透過抑制細胞內的自噬作用，進而影響到類固醇生成作用。然而，脂肪酸是透過什麼機制而影響類固醇生成酶的活性與細胞自噬作用，以及細胞自噬作用在其中所扮演的角色，仍需進一步的研究去釐清。

Infertility is a common problem that bothers both males and females nowadays. According to the definition by the World Health Organization, infertility is “a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse.” Although many factors may be involved, obesity has become a prominent cause of infertility recently due to the high fat/sweet dietary habit and life style changes. Among both sex, male’s fertility relies on the quantity and quality of the sperm, and one of the factors involved in sperm production is the action of testosterone, which is produced by Leydig cells in testis. In addition, one of the signs exist in obese individuals are hyperlipidemia, which indicates the higher plasma free-fatty-acid (FFA) level. The aim of this study is to investigate whether the higher FFA level is the cause for the impaired Leydig cell function and how it induces hyposteroidogenesis. To verify the hypothesis, we used MA-10 mouse Leydig cell line as the cell model, and treated with 1,200 µM fatty-acid (600 µM oleic acid and 600 µM palmitic acid) for 48 hours to mimic the hyperlipidemia situation. Results showed that the steroidogenic ability declined after the FFA challenge, and meanwhile, the expression of two steroidogenic enzymes, CYP11A1 and 3β-HSD, decreased at the transcriptional levels. Furthermore, according to the results of the pregnenolone and 22(R)-hydroxycholesterol supplement experiments, the impaired progesterone production was mainly due to the damaged CYP11A1 function. In addition to the decreased enzyme function, we also observed that the autophagic cargo protein p62 and endoplasmic reticulum (ER) stress marker CHOP significantly increased after fatty-acid treatment, and this increment could be alleviated by adding autophagy activator rapamycin. Additionally, rapamycin could also reverse the basal condition of steroidogenesis in FFA-treated MA-10 cells, indicating FFA would impair the autophagic function thus affecting the steroidogenesis of cells through some unknown mechanisms. In conclusion, our results demonstrated that one of the causes for the hyposteroidogenesis is the higher FFA level, and fatty acids can directly damage the function of CYP11A1, a key steroidogenic enzyme. Besides, fatty acids may also affect other cellular functions including autophagy, which mainly participated in the basal condition of steroidogenesis. However, it is necessary to elucidate how fatty acids impair the function of the enzymes and what the roles of autophagy play in steroid production.