Mendis-Handagama S M
Department of Animal Science, College of Veterinary Medicine, The University of Tennessee, Knoxville 37996, USA.
Tissue Cell. 2000 Feb;32(1):102-6. doi: 10.1054/tice.1999.0092.
The present study was designed to explore the intracellular cholesterol trafficking in Leydig cells of adult rats following Luteinizing hormone (LH) injection. Histochemical techniques were used to demonstrate distribution of free cholesterol in Leydig cells of control and LH-injected rats. Two groups of sexually mature male Sprague Dawley rats (n=4/group) were used. Fifteen min following an injection of 200 microl of either saline (control) or luteinizing hormone (LH, 500 microg in saline) testes of rats were fixed by whole body perfusion using 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer for 20 min. Fixed testes were cut into 3 mm3 and kept immersed in the fixative for further 15 min. Tissue cubes were then incubated at 37 degrees C in a medium containing cholesterol oxidase, 3,3'-diaminobenzidine tetrahydrochloride, horseradish peroxidase and dimethyl sulfoxide to histochemically localize free cholesterol in Leydig cells and processed for electron microscopy. Thin sections of these tissues were stained with aqueous uranyl acetate and lead citrate and examined with a Philips 201C electron microscope. In Leydig cells of control rats, free cholesterol was detected primarily in lipid droplets and plasma membrane. In the majority of Leydig cells, peroxisomes were unstained for free cholesterol, but occasionally few stained ones were present. Staining was not detected in mitochondria and smooth endoplasmic reticulum (SER) in Leydig cells of control rats. In LH-injected rats, lipid droplets, many peroxisomes, inner and outer mitochondrial membranes and some cisternae of SER in Leydig cells showed staining for free cholesterol. Fusion of Leydig cell peroxisomes with lipid droplets and mitochondria was also observed in the LH treated rats. These findings suggested that peroxisomes in adult rat Leydig cells participate in the intracellular cholesterol trafficking and delivery into mitochondria during LH stimulated steroidogenesis. Lipid droplets are used as one source for cholesterol for this process.
本研究旨在探讨成年大鼠注射促黄体生成素（LH）后，睾丸间质细胞内胆固醇的转运情况。采用组织化学技术，以显示对照大鼠和注射LH大鼠睾丸间质细胞中游离胆固醇的分布。选用两组性成熟雄性Sprague Dawley大鼠（每组n = 4）。在注射200微升生理盐水（对照）或促黄体生成素（LH，500微克溶于生理盐水中）15分钟后，用含0.5%戊二醛和4%多聚甲醛的0.1M二甲胂酸钠缓冲液对大鼠进行全身灌注固定20分钟。将固定后的睾丸切成3立方毫米，并在固定液中再浸泡15分钟。然后将组织块置于含有胆固醇氧化酶、3,3'-二氨基联苯胺四盐酸盐、辣根过氧化物酶和二甲基亚砜的培养基中，于37℃孵育，以组织化学方法定位睾丸间质细胞中的游离胆固醇，并进行电子显微镜处理。这些组织的薄片用醋酸铀水溶液和柠檬酸铅染色，并用飞利浦201C电子显微镜检查。在对照大鼠的睾丸间质细胞中，游离胆固醇主要存在于脂滴和质膜中。在大多数睾丸间质细胞中，过氧化物酶体未被游离胆固醇染色，但偶尔会有少数被染色的过氧化物酶体。对照大鼠睾丸间质细胞的线粒体和平滑内质网（SER）未检测到染色。在注射LH的大鼠中，睾丸间质细胞中的脂滴、许多过氧化物酶体、线粒体内外膜和一些SER池显示出游离胆固醇染色。在LH处理的大鼠中，还观察到睾丸间质细胞过氧化物酶体与脂滴和线粒体的融合。这些发现表明，成年大鼠睾丸间质细胞中的过氧化物酶体在LH刺激的类固醇生成过程中参与细胞内胆固醇的转运并将其输送到线粒体中。脂滴是该过程中胆固醇的一个来源。
