Modulation of Leydig cell androgen biosynthesis and cytochrome P-450 levels during estrogen treatment and human chorionic gonadotropin-induced desensitization.
Modulation of Leydig cell androgen biosynthesis and cytochrome P-450 levels during estrogen treatment and human chorionic gonadotropin-induced desensitization.
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雌激素治疗和人绒毛膜促性腺激素诱导的脱敏过程中间质细胞雄激素生物合成和细胞色素 P-450 水平的调节。
DOI：
10.1016/s0021-9258(19)68732-6
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
M. Dufau
中科院分区：
文献类型：
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作者：
K. Nozu;S. Matsuura;K. Catt;M. Dufau
材料和方法动物处理和Leydk细胞培养-成年雄性大鼠（200 - 250 g）获自Charles River Laboratories。通过皮下注射hCG（Pregnyl，Organon）引起促性腺激素诱导的间质细胞脱敏，其中10 IU相当于在250 μ l磷酸盐缓冲盐水（pH 7.4）中的1 pg纯化hCG。除非另有说明，否则在hCG处理后2天通过断头处死动物。切除垂体的大鼠（50日龄）和完整的对照大鼠购自Hormone Assay Laboratories（芝加哥，IL），并在手术后3天开始激素治疗。每天皮下注射选定剂量的溶于200 μ l芝麻油中的雌二醇-17/3（0.01 - 20 μ g）或hCG（1 μ g），持续3天。在最后一次注射后24小时通过断头处死动物。在一些实验中，如前所述（16），通过将去包囊的性腺与胶原酶孵育来制备睾丸间质细胞。将分离的间质细胞在MIX存在下与100 ng hCG在34 ℃孵育3小时，以测量cAMP和睾酮的产生。当测量双烯醇酮的产生时，通过将Leydig细胞与氰基酮M）和螺内酯M）孵育来抑制其进一步代谢（8）。通过对As-3 P-羟基类固醇脱氢酶进行组织化学染色后，在Levy超平面计数室中计数至少500个细胞来确定Leydig细胞数量（17）。LH/hCG受体通过与“*”I-hCG的结合分析来测定，所述“*”I-hCG通过如前所述的酶促放射性碘化制备（18）。微粒体的制备-去包囊后，用Teflon玻璃匀浆器将睾丸分散在5体积的含有20 mM Tris-HCl（pH 7.4）的0.25 M蔗糖溶液中。将匀浆在9，000 X gina Sorvall冷冻离心机中离心，随后将上清液在Beckman ultracentrifugeL 5 -75型中以105，000 X g沉降1小时。将沉淀的微粒体用0.25 M蔗糖、含有1 nm EDTA和150 mM KCl的20 mM Tris-HCl缓冲液（pH 7.4）洗涤一次，并重悬于含有30%甘油的250 nm磷酸钾缓冲液（pH 7.4）中。通过Lowry等人的方法测定蛋白质。(19)以牛血清白蛋白为标准品。细胞色素P-450的测量-通过分析一氧化碳吸收光谱进行细胞色素P-450的定量（20）。将微粒体悬浮在含有0.2%Emulgen 911（Kao-Atras，Tokyo，Japan）和20%甘油（2.5mg蛋白质/ml）的250 m2磷酸钾缓冲液（pH 7.4）中，并在具有1cm光程的两个比色皿之间等分。一个比色皿用CO缓慢鼓泡10秒，另一个用Nz鼓泡。在添加几格令连二亚硫酸钠后1分钟，在配备有高强度光源的Aminco DW-2记录分光光度计中记录CO差光谱。对于450 nm和490 nm之间的吸收差异，使用91 cm-1的消光系数估计细胞色素P-450的浓度。在所有情况下，CO-450带随时间转化为CO-420带，代表细胞色素P-450分解产物的形成。因此，对于细胞色素P-450的总浓度，将对于420和490 nm之间的变化使用110 m2/cm 2的消光系数测定的细胞色素P-420的量添加到细胞色素P-450的浓度中。反应速率与孵育时间和蛋白质浓度呈线性关系。
MATERIALS AND METHODSTreatment of Animals and Leydk Cell Preparation-Adult male rats (200 to 250 g) were obtained from Charles River Laboratories. Gonadotropin-induced desensitization of Leydig cells was elicited by subcutaneous injection of hCG (Pregnyl, Organon) of which 10 IU was equivalent to 1 pg of purified hCG in 250 pl of phosphate-buffered saline, pH 7.4. Animals were killed by decapitation 2 days after hCG treatment unless otherwise indicated. Hypophysectomized rats (50 days of age) and intact control rats were purchased from Hormone Assay Laboratories (Chicago, IL), and hormone treatments were initiated 3 days after surgery. Subcutaneous injections were performed daily for 3 days with selected doses of estradiol-17/3 (0.01 to 20 pg) dissolved in 200 pl of sesame oil, or with hCG (1 pg). Animals were killed by decapitation 24 h after the last injection. In some experiments, testicular Leydig cells were prepared by incubation of the decapsulated gonads with collagenase as previously described (16). The isolated interstitial cells were incubated at 34" C for 3 h with 100 ng of hCG in the presence of MIX for measurement of CAMP and testosterone production. When pregnenolone production was to be measured, its further metabolism was inhibited (8) by incubation of Leydig cells with cyanoketone M) and spironolactone M). Leydig cell number was determined by counting at least 500 cells in a Levy ultraplane counting chamber after histochemical staining for As-3P-hydroxysteroid dehydrogenase (17). LH/hCG receptors were determined by binding analysis with'*'I-hCG, prepared by enzymatic radioiodination as previously described (18). Preparation of Microsomes-After decapsulation, the testes were dispersed with a Teflon-glass homogenizer in 5 volumes of 0.25 M sucrose solution containing 20 mM Tris-HC1 (pH 7.4). The homogenates were centrifuged at 9,000 X gina Sorvall refrigerated centrifuge, and the supernatants were subsequently sedimented for 1 h at 105,000 X g in a Beckman ultracentrifuge model L5-75. The pelleted microsomes were washed once with 0.25 M sucrose, 20 mM Tris-HC1 buffer (pH 7.4) containing 1 nm EDTA and 150 mM KC1 and resuspended in 250 nm potassium phosphate buffer (pH 7.4) containing 30% glycerol. Protein was determined by the method of Lowry et al.(19) using bovine serum albumin as standard. Measurement of Cytochrome P-450-Quantitation of cytochrome P-450 was performed by analysis of the carbon monoxide absorption spectrum (20). Microsomes were suspended in 250 m~ potassium phosphate buffer (pH 7.4) containing 0.2% Emulgen 911 (Kao-Atras, Tokyo, Japan) and 20% glycerol (2.5 mg of protein/ml), and were equally divided between two cuvettes having an optical path of 1 cm. One cuvette was slowly bubbled with CO for 10 s, and the other with Nz. The CO-difference spectrum was recorded 1 min after addition of a few grains of sodium dithionite, in an Aminco DW-2 recording spectrophotometer equipped with a high intensity light source. The concentration of cytochrome P-450 was estimated using an extinction coefficient of 91 cm" for the absorption difference between 450 nm and 490 nm. In all cases, the CO-450 band was converted with time to a CO-420 band, representing formation of a breakdown product of the cytochrome P-450. For total concentration of cytochrome P-450, therefore, the amount of cytochrome P-420 determined using an extinction coefficient of 110 m"' cm" for the change between 420 and 490 nm was added to the concentration of cytochrome P-450. Reaction rates were linear with time and protein concentration during the incubation …