以大鼠黃體初代培養細胞探討類固醇生成基因之調控

Abstract

雌性哺乳動物性成熟之後，卵巢會呈現週期性變化，稱為動情週期，其中包含濾泡的發育、成熟、排卵以及黃體形成。黃體為重要的生殖內分泌組織，含有大小兩種黃體細胞，可分泌大量的助孕酮 (progesterone)，為維護整個懷孕過程所必須。本論文嘗試建立黃體初代培養細胞，用以探討黃體中類固醇生成基因的調控。我們自懷孕三天的大鼠取得黃體組織，並進行細胞分離，發現分離之細胞具有三種型態。大小黃體細胞在細胞型態上有明顯的區別。類固醇生成基因Cyp11a1與Hsd3b在大黃體細胞具有大量表現。然而，在小黃體細胞中只有少量表現。小黃體細胞中的3βHSD酵素活性在體外培養時有逐漸減低的情形，但forskolin的刺激可以促進3βHSD酵素活性提升。 Liver receptor homolog-1 (LRH-1)屬於孤兒核受體NR5A家族，大量存在於黃體組織中。以免疫細胞染色法對初代培養細胞進行分析發現，LRH-1同時存在大小兩種黃體細胞的細胞核中。LRH-1會和轉譯後修飾蛋白SUMO-1產生鍵結，當LRH-1受到SUMO-1修飾，其在細胞核中的位置會產生改變。為探討LRH-1在黃體細胞中調控類固醇生成基因的能力，我們以山羊黃體細胞 (GLC-D細胞株)進行人類CYP11A1啟動子與LRH-1表現質體的共同轉染實驗。結果顯示CYP11A1啟動子在山羊黃體細胞中具有轉錄功能，且LRH-1對於CYP11A1啟動子有增進其轉錄活性的作用。啟動子上兩個功能性的SF-1結合位，分別位於-40與-1610，在GLC-D細胞中都具有影響啟動子轉錄活性的能力。

The process of estrous cycle includes follicle growth, development, ovulation and the formation of corpus luteum. The corpus luteum is a transient steroidogenic tissue and contains two different types of steroidogenic cell. In corpus luteum, large amounts of progesterone are produced for establishment and maintenance of pregnancy. In this study, we attempt to establish a luteal cell culture system to characterize the regulation of steroidogenesis in corpus luteum. Rat corpora lutea were dissected out on day 3 of pregnancy for cell dispersion. There were three types of cell found in the primary cultured luteal cells. Large and small luteal cells could be distinguished from cell morphology. Abundant expression of steroidogenic genes Cyp11a1 and Hsd3b were detected in the large luteal cells. However, a low level expression of Cyp11a1 and Hsd3b was observed in the small luteal cells. The enzyme activity of 3βHSD was decreased in small luteal cells after in vitro culture and that could be stimulated by forskolin. Liver receptor homolog-1 (LRH-1) belongs to the NR5A subfamily, which is highly expressed in the corpora lutea. Our immunostaining data showed that LRH-1 were present in the nucleus of large and small luteal cells. LRH-1 could be conjugated with the post-translational modification protein SUMO-1. The nuclear localization of LRH-1 was changed when LRH-1 was modified by SUMO-1 conjugated. To examine the ability of LRH-1 in the regulation of steroidogenic genes expression in luteal cells, we cotransfected human CYP11A1 promoter construct and LRH-1 expression vector into goat corpus luteal cell (GLC-D cell line). Data showed that the CYP11A1 promoter was functional in GLC-D cells and the promoter activity was stimulated by LRH-1. Two potential SF-1 binding site, located at -40 and -1610, were functional in the GLC-D cells