生理剂量睾酮通过雄激素受体改善小鼠行为学能力的研究
新型男性避孕药，安全可逆
意外怀孕是一个重要的公共卫生问题，据统计，在全球范围内，意外怀孕率高达50%。
科学合理避孕具有重要的社会经济价值和健康效益。
然而，目前可用的避孕方法中，主要是针对女性，男性可选择的避孕或节育方法很少，只有避孕套和输精管结扎术。
但避孕套容易避孕失败，输精管结扎逆转术成功率不能保证，很容易导致永久性绝育。
几十年来，人们一直致力于开发出有效的男性避孕药。
科学家们发现，精子的生成十分复杂，在男性体内，视黄酸受体（R A R）调控着睾丸中精原干细胞（SS C）的一系列自我更新和分化程序。
要想开发有效的男性避孕药，必须找到一种方法，在抑制精子生成的同时，保留精原干细胞，并且不损害男性的生殖系统。
2月20日，索尔克生物研究所埃文斯博士团队在《美国国家科学院院刊》发表了研究论文。
该研究团队发现了一种可逆的、非激素的男性节育的新药物。
这种药物是一种H D AC（组蛋白去乙酰化酶）抑制剂，可以阻止雄性小鼠的精子产生和生育能力，但不会影响性欲或未来的繁殖。
在研究中，研究团队发现，要想完成精子发生过程，视黄酸受体必须与一种名为S M R T的蛋白质结合，然后，S M R T招募H D AC，这种蛋白质复合物才会继续同步产生精子的基因表达。
于是，研究人员观察了一种特殊的小鼠模型———S M R T转基因小鼠。
这种小鼠的S M R T蛋白发生了突变，无法再与视黄酸受体结合，所以不能产生成熟的精子，然而，它们能表现出正常的睾丸激素水平和正常的发情行为，表明他们的交配欲望没有受到影响。
研究团队希望通过药物干预可以实现同S M-R T转基因小鼠一样的效果，他们使用了一种美国食品药品监督管理局批准的口服H D AC抑制剂M S-275，并通过长期、低剂量地向正常小鼠投喂M S-275观察其表现。
结果发现，M S-275能够发挥类视黄醇和甲状腺激素受体S M R T复合物的抑制作用，成功地破坏了小鼠的精子发生，但是没有降低血清睾酮水平和SS C的存在。
雄激素受体敲除小鼠睾丸生精功能变化及p63的表达
雄激素受体敲除小鼠睾丸生精功能变化及p63的表达作者：郑纯威陈宇丰奕波黄强来源：《中国现代医生》2013年第34期[摘要] 目的研究雄激素受体（AR）敲除小鼠睾丸生精功能及p63在曲细精管中表达的改变。
方法采用雄性Flox-AR小鼠与雌性ACTB-Cre小鼠交配，并敲除胚胎中AR，筛选AR敲除（AR敲除组）和AR未敲除（对照组）雄性小鼠各10只，对比睾丸曲细精管生精功能及p63的表达。
结果两组小鼠曲细精管内径、管周膜厚度、管壁生殖细胞层数、生殖细胞成熟度评分及总Makler评分之间差异均有统计学意义（P < 0.05），p63的阳性表达主要位于精原细胞及精母细胞，精子细胞及成熟精子中表达呈阴性，且AR敲除组表达阳性率及HSCRE评分均显著低于对照组（P < 0.01）。
结论 AR表达与小鼠睾丸曲细精管p63表达关系密切，AR 敲除小鼠睾丸曲细精管生精功能受损、p63表达下调，p63调控生殖细胞的早期分化。
[关键词] 雄激素受体；基因；p63；生精功能[中图分类号] R698.2 [文献标识码] A [文章编号] 1673-9701（2013）34-0001-03The testicular spermatogenic function and expression of p63 in androgen receptor knockout mouseZHENG Chunwei CHEN Yu FENG Yibo HUANG QiangLaboratory Animal Center of Zhejiang Province Traditional Chinese Medicine Academy，Hangzhou 310007， China[Abstract] Objective To study spermatogenesis and p63 expression in the seminiferous tubules of the testicular androgen receptor （AR） knockout mice. Methods Male Flox-AR mice mated with female ACTB-Cre mice， and knocked the AR in addition to embryos， screening AR knockout male mice （ARKO group） and AR did not knock （control group），each of 10 mice. Contrasted testis seminiferous tube spermatogenic function and p63 expression. Results The differences of seminiferous tube diameter， the peritubular film thickness， wall germ cell layers， the germ cell maturity score and Makler score between two groups were statistically significant （P < 0.05）， p63 mainly expressed in spermatogonia and spermatocytes， expressed in sperm cells and mature sperm were negative， and AR knockout group expression the positive rate and HSCRE of scores were significantly lower than the control group （P < 0.01）. Conclusion AR expression in mouse testis seminiferous tubules are closely related p63 expression. AR knock damage in addition to the mouse testis seminiferous tube spermatogenic function of p63 downregulation of p63 regulation of the early differentiation of the germ cells.[Key words] Androgen receptor； Gene； p63； Spermatogenic function雄激素主要由睾丸间质细胞分泌，诱导男性性别的分化，促进内生殖器发育，进入曲细精管促进生精细胞的分化和精子生成[1]。
睾酮对雄性性行为调控机制研究进展
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（ Ｒ Ｅ ［） Ｅ 、 Ｒ３ 和雄激素受体 （ Ｒ） 控雄 性个体 的性行 为。早期 的研 究表 路增强 Ｄ Ａ 调 Ａ的释放 同时抑 制其重 吸收从而参与调控雄 鼠的性行为。与正常 明， 在大鼠中 Ｅ ２能够恢复阉割大鼠大多数的交配反应 。此外 ， 下注射 芳 雄 鼠相 比， 皮 阉割雄 鼠内侧视前核 （ Ｎ） Ｏ合成 酶免疫反应减小 ， Ｔ处理 ＭＰ Ｎ 而
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性激素激发实验报告
性激素激发实验报告性激素激发实验报告引言：性激素是一类影响生殖发育和性特征的化学物质，它们在动物和人类的生理过程中起着重要的作用。
本实验旨在探究性激素对生物体的影响，特别是对性行为的激发作用。
通过实验，我们希望能够更深入地了解性激素的功能和机制。
实验设计：本实验采用小鼠作为实验对象，将其分为实验组和对照组，每组均有10只小鼠。
实验组小鼠在实验前通过注射性激素来激发其性行为，而对照组则不进行任何处理。
实验过程中，我们观察小鼠的交配行为以及相关的生理指标变化。
实验过程：1. 实验组小鼠的性激素注射：实验组小鼠在实验前一天，分别注射了雌激素和雄激素。
这些性激素能够模拟小鼠体内的性激素水平，从而激发其性行为。
2. 对照组小鼠的处理：对照组小鼠没有接受任何性激素注射，保持其自然状态。
3. 观察和记录：在注射后的第二天，我们观察和记录小鼠的交配行为。
通过观察交配的频率、时间和持续时间，我们可以评估性激素对小鼠性行为的影响。
同时，我们还测量了小鼠的性激素水平和相关的生理指标，如雄性激素水平和精子质量等。
实验结果：实验结果显示，经过性激素注射后的实验组小鼠表现出明显的性行为激发效应。
与对照组相比，实验组小鼠的交配频率明显增加，交配时间延长，交配持续时间也更长。
这表明性激素的注射能够显著提高小鼠的性行为活跃度。
此外，我们还观察到实验组小鼠的雄性激素水平显著增加，而对照组小鼠的雄性激素水平没有明显变化。
这进一步证实了性激素注射对小鼠性行为的激发作用。
此外，我们还发现实验组小鼠的精子质量有所提高。
经过性激素注射后，实验组小鼠的精子数量增加，精子活力也得到了提高。
这可能与性激素对睾丸功能的促进有关。
讨论：本实验结果表明，性激素注射能够激发小鼠的性行为，并且对其生理指标也有明显的影响。
这与已有的研究结果相符，进一步验证了性激素在性行为调控中的重要作用。
然而，需要注意的是，本实验仅仅是在小鼠上进行的，对于人类的性行为影响还需要进一步的研究。
睾酮对体外培养小鼠卵巢颗粒细胞分泌抗苗勒管激素的影响_张颖
解放军医学院学报 Acad J Chin PLA Med Sch Dec 2013，34（12）1259睾酮对体外培养小鼠卵巢颗粒细胞分泌抗苗勒管激素的影响张 颖1，郑金丹2，刘丽丽21辽宁医学院，辽宁锦州 121000；2辽宁医学院附属第一医院 妇产科，辽宁锦州 121000摘要：目的 探讨睾酮对体外培养的小鼠卵巢颗粒细胞抗苗勒管激素(anti-Mullerian hormone，AMH)分泌的影响。
方法 小鼠卵巢颗粒细胞原代培养，添加不同浓度睾酮，ELISA法检测颗粒细胞AMH的表达。
结果 ELISA法证实颗粒细胞中有AMH表达，不同浓度(10-8 mmol/L、10-7 mmol/L、10-6 mmol/L)睾酮作用24 h可促进颗粒细胞分泌AMH，且随睾酮浓度的增加、作用时间延长，AMH分泌明显增加(P＜0.01)。
结论 睾酮促进小鼠卵巢颗粒细胞分泌AMH，高浓度比低浓度作用更强。
关键词：睾酮；颗粒细胞；抗苗勒管激素中图分类号：R 711.45 文献标志码：A 文章编号：2095-5227(2013)12-1259-03 DOI：10.3969/j.issn.2095-5227.2013.12.019网络出版时间：2013-09-02 10:47 网络出版地址：/kcms/detail/11.3275.R.20130902.1047.003.htmlEffect of testosterone on secretion of anti-Mullerian hormone in mouse ovarian granular cells cultured in vitroZHANG Ying1, ZHENG Jin-dan2, LIU Li-li21Liaoning Medical College, Jinzhou 121000, Liaoning Province, China; 2Department of Obstetrics and Gynecology, First Affiliated Hospital of Liaoning Medical College, Jinzhou 121000, Liaoning Province, ChinaCorresponding author: LIU Li-li. Email: clearsky315@Abstract: Objective To study the effect of testosterone on secretion of anti-Mullerian hormone (AMH) in mouse ovarian granular cells cultured in vitro. Methods Primary mouse ovarian granular cells were cultured in vitro and treated with testosterone at different concentrations. Expression of AMH in mouse ovarian granular cells was detected by ELISA. Results ELISA showed that the AMH was expressed in mouse ovarian granular cells. Twenty-four hours treatment of primary mouse ovarian granular cells with testosterone at the concentrations of 10-8 mmol/L, 10-7 mmol/L and 10-6 mmol/L up-regulated their secretion of AMH in a concentration- and time-dependent manner (P＜0.01). Conclusion Testosterone up-regulates the secretion of AMH in mouse ovarian granular cells in a concentration-dependent manner.Key words: testosterone; granular cells; anti-Mullerian hormone雄激素过多是多囊卵巢综合征(polycystic ovary syndrome，PCOS)最典型的临床特征，PCOS患者同时伴有持续性无排卵，是女性不孕症的主要原因之一[1]。
不同剂量的甲睾酮对小鼠生精功能的影响
不同剂量的甲睾酮对小鼠生精功能的影响张克英;苟兴能;张薇;杨兴江【期刊名称】《中国现代医学杂志》【年(卷),期】2008(18)17【摘要】目的探讨不同剂量的甲睾酮对雄性小白鼠生殖系统的发育及精子发生的影响.方法将40只昆明小白鼠随机分为4组,每组10只,每天分别向各组灌喂甲睾酮16mg/kg,32mg/kg,64mg/kg和等量蒸馏水(对照组),连续灌喂7 d.结果睾丸酮灌喂量为16mg/kg时,胸腺显著增大(P＜0.05),而用药量达到32mg/kg时,胸腺增重受到抑制;睾丸指数随用药量的增加而增大,用甲睾酮64mg/kg时睾丸指数极显著地高于其他各组(P＜0.01);精子活率用甲睾酮16 mg/kg达到62.82%显著地高于对照组(P＜0.05);精子畸形率随用药量的增加而提高,达到以64mg/kg时,精子畸形率最高为4.94%,显著地高于对照组和其他各组(P＜0.05).精子密度用甲睾酮64mg/kg最高为3.0×106,显著地高于对照组(P＜0.01).结论适量甲睾酮能提高精子活率,大剂量使精子活率降低,胸腺发育受到抑制.【总页数】4页(P2468-2470,2474)【作者】张克英;苟兴能;张薇;杨兴江【作者单位】四川绵阳市人民医院,内分泌科,四川,绵阳,621000;西南科技大学生命科学与工程学院,四川,绵阳,621010;四川绵阳市人民医院,内分泌科,四川,绵阳,621000;四川绵阳市人民医院,内分泌科,四川,绵阳,621000【正文语种】中文【中图分类】R-332【相关文献】1.非清髓预处理及人脐带血造血干细胞移植对小鼠生精功能的影响 [J], 张萌;姚观平;何方;李玉香;范振海;余丽梅2.甲睾酮对小鼠生殖系统发育及生精功能的影响 [J], 张红军;张克英;杨晋;杨兴江;苟兴能3.杨梅黄酮对铅染毒雄性小鼠生精功能及激素水平的影响 [J], 陈宗耀; 李乐乐; 王振滔; 许世林; 李瑶; 王艳春; 范红艳4.不同剂量的十一酸睾酮对大鼠生精功能影响 [J], 童建孙;王兴海;崔毓桂;马鼎志;蔡瑞芬;张桂元5.19-去甲睾酮对性腺发育不良小鼠附性腺生长的影响 [J], 漆著;Jaskirat SINGH;DavidJ. HANDELSMAN因版权原因，仅展示原文概要，查看原文内容请购买。
雄激素受体敲除小鼠睾丸生精功能变化及p63的表达
【 关键 词】 雄 激 素 受体 ； 基 因； ｐ ６ ３ ； 生精 功 能
【 中图分 类 号】Ｒ ６ ９ ８ ． ２
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睾酮代谢的种属差异研究
睾酮代谢的种属差异研究何芋岐;曾瑶;凌蕾;陆安静;杜艺玫;杨滔;李伯达;彭捷;吴庆【摘要】Objective To investigate the species difference of testosterone metabolism among human and 7 animal species including mice,rats,guinea pigs,rabbits,dogs,pigs,sheep,and cattle.Methods Testosterone was incubated with liver microsomes of humans and investigatedanimals,together with CYP-mediated metabolic reaction factors including NADP Na,glucose-6-phosphate,and glucose-6-phosphate dehydrogenase.Incubation samples were introduced into HPLC to determinate testosterone and its metabolites.Heatmap was used to visualize the metabolic profile while principle component analysis was used to tell the difference of metabolic profile among species.High resolution mass spectrometry was used to assist the identification of metabolite structures.Results 6β-hydroxylation is not the unique metabolic pathway of testosterone.Eighteen NADP-dependent metabolites were observed in humans or other investigated animals.Although 6β-hydroxylation showed positive correlation with the substrate elimination,this specific metabolic pathway could not compensate all substrate consumption.Guinea pig generates the most metabolites while sheep showed the weakest capacity on testosteronemetabolism.Mice,dogs,and rabbits showed the most similar metabolic profile to humans.Conclusion 6β-hydroxylation metabolites of testosterone was not applicable to evaluate the CYP 3A activity of allspecies.Mice,dogs,and rabbits are recommended in pre-clinical trial if the target is proved to be metabolized by human CYP 3A4.%目的研究睾酮在人及小鼠、大鼠、犬、兔、猪、豚鼠、牛、羊等实验动物肝微粒体代谢中的差异.方法睾酮分别于上述种属肝微粒体进行孵育,加入NADP Na、葡萄糖-6-磷酸、葡萄糖-6-磷酸脱氢酶等试剂启动体外P450酶反应.样品采用高效液相色谱紫外检测器及高分辨质谱进行分析.代谢轮廓用热图进行可视化,并用主成分分析进行差异研究.结果6β-羟基化不是睾酮唯一的代谢途径.本研究在不同种属中共发现了18种睾酮的代谢产物.尽管6β-羟基化在不同种属中与睾酮的底物消除基本呈正相关,但该途径无法补偿其他途径造成的底物消除.豚鼠具有最强的睾酮代谢能力,而羊对睾酮的代谢能力最弱.小鼠、犬、兔肝微粒体中,睾酮的代谢轮廓与人最相似.结论6β羟基化途径的定量不能用作所有物种CYP3A活性评判的精准依据.如果药物证明由CYP 3A4进行代谢,在临床前期研究中,应结合药理学模型的需要,尽量采用小鼠、犬、兔作为动物模型,以保证药物体内暴露与人体实验尽量接近.【期刊名称】《遵义医学院学报》【年(卷),期】2017(040)005【总页数】6页(P463-468)【关键词】睾酮;种属差异;CYP 3A;代谢【作者】何芋岐;曾瑶;凌蕾;陆安静;杜艺玫;杨滔;李伯达;彭捷;吴庆【作者单位】遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099;遵义医学院第三附属医院内分泌科,贵州遵义563000;遵义医学院药学国家级实验教学示范中心(药学实验室),贵州遵义563099【正文语种】中文【中图分类】R966A right animal model is extremely important for drug discovery and development in particularly in pre-clinical trial stage.Thus,investigation of species difference has to be noted before in vivo experiments. Metabolic stability is an important factor leading failure of drug development [1-2].Weaker metabolic stability of drug candidate in human than in experimental animals,may lead lower exposure of the therapeutic target in drug molecules.Eventually,the significant drug effect observed in animals disappeared in humans.To prevent from misused animal model,understanding the metabolic profile and capacity of specific drug candidates in animals is necessary.The most effective and popular method to understand the metabolic capacities of animals is to check the activity of metabolic enzymes via specific probe substrates [3].CYP 3A is the most important cytochrome P450 isozyme [4].This isozyme is responsible for metabolisms of around half market drugs.Testosterone is a famous CYP 3A4 substrate,usually was used to evaluate CYP 3A4 activity of individual human livers [5].Although testosterone 6β-hydroxylation is proved to be a selective reaction catalyzed by CYP 3A4 in humans[5],whether it’s metabolism in animals is the same or similar as human is still not clear.It means that comparison of CYP 3A activities between human and animals based on testosterone 6β-hydroxylation rate may not or may not fully reflect the real facts.Regarding species difference on testosterone metabolism,comparison between humans and rodents has been discussed [6-7].However,in case of other normally used experimental animals such as rabbit,guinea pig,and dogs,species difference on testosterone metabolism is still not known.In special cases,some large animals,such as pigs,sheep,and cattle,are also used in pharmacology and pharmacokinetics study.Difference on testosterone metabolism between those animals and humans is also worth involving.In the present study,testosterone metabolism in liver microsomes from human and various animal species were studied.This was the first time that species difference on testosterone metabolism was studied systematically.The metabolic profiles would indicate how similar was each investigated animals to human.The result may help to decide the right animal models in case the investigated drugs were potentially metabolized by CYP 3A.1.1 Instruments and Materials An HPLC system (1100,Agilent Co.Ltd.,USA) was used to quantify testosterone,testosterone 6β-hydroxylate,and other testosterone metabolites.A Q-Exactive ultra-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HiMS,Thermo Fisher Co.Ltd.,USA) was used to assist the identification ofthe structure of 6β-hydroxylated testosterone.A C18 column (ZorbaxC18,Agilent Co.Ltd.,USA) was used for separation.Testosterone,HPLC-grade acetonitrile and methanol,NADP Na+,glucose-6-phosphate,and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich(St.Louis,MO,USA).Human liver microsomes were purchased from Shanghai RILD Co.Ltd.(Shanghai,China).Mice,rats,guinea pigs,rabbits were purchased from the Animal Experimental Center of Zunyi Medical University,and livers of those animals were harvested immediately after euthanasia.Dog (beagle) livers were got from Shanghai University of Traditional Chinese Medicine.Cattle and sheep livers were harvested immediately after deathof animals in food market.1.2 Preparation of animal liver microsomes After liverhomogenation,microsomes were prepared from fresh livers following our previously established method [8].All microsomes products were checked with protein concentration determination kit and diluted to the protein concentration of 10 mg/ml and stored at -80 oC.1.3 Liver microsomes incubation system for CYP-mediated metabolisms Incubation system was set following our previously published method [9].Liver microsomes (0.5 mg/ml) were pre-incubated with testosterone (50 μM),glucose-6-phosphate (1 mM),glucose-6-phosphate dehydrogenase (1 Unit/ml),and MgCl2 (4 mM).Reaction start from adding of NADP Na+ (1 mM).The incubation system was normalized to 100 μl with phosphate buffer saline (PBS,100 mM,pH 7.4).10 min afte r incubation,100 μl of acetonitrile was added to stop the reaction and precipitate proteins.Alltubes were shaken thoroughly and then centrifuged at 10,000×g and 4 oC for 10 min.The supernatant was used for HPLC analysis.A reaction group (n=3) involving all reaction factors,a negative group (n=3) without NADP Na+,and a blank group (n=3) without testosterone were set up.1.4 Determination of testosterone and its metabolites To quantify metabolites of testosterone,supernatant of each incubations from 1.3 were injected to an Agilent HPLC system,eluted by Water (A) - Methanol (B) on a C18 column (Agilent Zorbax C18,4.6×250 mm,5 μm).Elution gradient was shown below:0～15 min,48%～30% of A; 15～22 min,30%～20% of A;22～22.5 min,20%～5% of A,followed by a column wash program.Testosterone and its metabolites were detected at UV 254 nm.To identify which one is testosterone 6β-hydroxylate,the chromatogram of the test group was compared with that of blank and negative controls.The tentatively assigned metabolites was introduced to UPLC-HiMS to obtain the molecular weight and MS/MS spectrums.1.5 Statistics Correlation analysis was done in SPSS 18.0 (IBM Co.Ltd.,USA) program by using the function of linear correlation (pearson).Principle component analysis and visualization at heatmap were done in R program by using mixOmics and gplots package.2.1 CYP-mediated metabolic profile of testosterone in humans and animals Normally,we assumed 6β-hydroxylationIn is the most common metabolic pathway of testosterone.However,in the present study,in human and animal liver microsomes incubation system,we observed 18 NADP-dependent metabolites.Metabolites were assigned with numbers based ontheir retention time on chromatograms (Fig1).Guinea pigs generates almost all metabolites observed in the present study,showing the highest metabolic capacity among all species investigated in the presentstudy.Sheep generated fewest metabolites.Viewing the whole metabolic profile,obvious species difference is available.2.2 6β-hydroxylated testosterone identification in human liver microsome incubations In total 4 peaks were observed in human liver microsome incubation system.All of the 4 peaks were just obviously observed in reaction group but not negative or blank group (Fig 2A).Based on the retention time,we assigned them as M6,M8,M16,and M18.Among the 4 metabolites,M8 showed the predominant amount.As in most publication,6β-hydroxylation is the major and specific metabolic pathway,M8 was tentatively assigned as testosterone 6β-hydroxylate.Moreover,we introduced M8 into mass spectrometer for MS2 fragment analysis.In MS2 spectrum of M8 (Fig 2B),M+H peak showed consistent value with testosterone 6β-hydroxylate.The fragments 97.07 and 109.07 were successfully aligned to structure of testosterone 6β-hydroxylate.2.3 Correlation analysis between testosterone 6β-hydroxylation and testosterone elimination In humans and 8 animal species,generation of 6β-hydroxylated testosterone and elimination of the substrate (testosterone) were compared with each other.A significant correlation with coefficient (R) of 0.8 (0 ～ 1) was observed (Fig 3).Except for rat,guinea pig,human,and rabbit,testosterone 6β-hydroxylation and testosterone eliminationgenerally showed the same correlation trend.In rats and guinea pigs,the spots shift away the trend line to y-axis.It means that in these two animal species,metabolic pathways other than testosterone 6β-hydroxylation play relative important role for testosterone metabolism.In contrast,in humans and rabbits,the spots shift away the trend line to x-axis,indicating the 6β-hydroxylation play the predominant role in testosterone elimination in these two animals.2.4 Quantitative visualization of testosterone metabolic profile Heatmap visualization divided all metabolites into three groups (Fig 4).The first group includes M8 and M18 which were generated by all investigated species.M2,M3,M12,M17,M7,M4,M1,M10,M15,and M11 were classified into the second group located in middle part of the heatmap.These metabolites just were observed in a few species,and the amount of these metabolites are relatively low.The third class metabolites includesM6,M5,M13,M9,M16,and M14.These metabolites were not observed in all species but in most species or have relative large amount in some species.Regarding individual species,Guinea pig has the most metabolites while sheep generates the fewest metabolites.Heatmap showed clearer information for metabolic profile of testosterone than chromatograms. 2.5 Principle component analysis Metabolites data matrix was inputted into R program for principle component analysis.Score plots (Fig5A) showed that metabolic profile of testosterone in guinea pigs and rats are far away from other species.However,guinea pigs shifted away to right side while rats shifted away to downside.Human generally located in the centerof the score plots.Mice and rabbits located close to humans.All of these phenomenon observed in the score plots indicated that the metabolic profile of testosterone in rabbits and mice are more similar to human than other animals.Guinea pigs and rats showed so different metabolic capacities from human,however,they are also different from eachother.M16,M13,and M8 got larger loading values at PC1 (Fig5B).As guinea pigs shift to right side along PC1,it means that M16,M13,and M8 were generated more in guinea pigs than in other species.With the same method,we identified that M15 and M11 have higher amount in rats than in other species.All of those findings are consistent with the directly observation on the heatmap.For convenience and experimental cost saving,in pharmacokinetics study,rodents and rabbits are usually selected.For sampling,in order to get the blood samples at various time points from a single animal,rats were used more frequently than mice.In some cases,large animals were also used,such as dogs,pigs,as well as sheep.Although rats were used more frequently,at genomic DNA level,rats showed different sequence of CYP 3A from humans [10-11].In rats,the enzyme with similar function with human CYP 3A4,is called as CYP 3A1 [12].In mice,this enzyme is called as CYP 3A11 [13].At the function level,testosterone is always used to probe the activity of CYP 3A in various animal species [14-16].However,we found that,not like usually assumed,rats showed so different metabolic profile on testosterone metabolism from humans.In contrast,testosterone metabolic profile in mice close to human more than rats.Other than rodents,in relatively largeanimals,rabbits and dogs showed more similar metabolic profile of testosterone to human more than sheep,cattle,and pigs.In some animal model for special disease modulation,guinea pigs were used[17].However,in the present study,regarding CYP 3A activity,guinea pigs showed so higher capacity than human and other animal species.It implies that,once a drug candidate is proved to be metabolized by CYP 3A4 in human liver microsomes or human primary hepatocyte cultures,in the pre-clinical trial stage,to make sure the same drug exposure in vivo,mice should be used in the early stage,rats and guinea pigs should be avoided.If larger animals are needed,rabbits and dogs were recommended.Taken together,there is huge species difference in testosterone metabolism mediated by CYP3A among human andanimals.Mice,dogs,rabbits are recommended to be used as animal model to promise the similar drug exposure,if a drug has already been proved to be metabolized by human CYP 3A4.[1] Baranczewski P,Stanczak A,Sundberg K,et al.Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development [J].PharmacolRep,2006,58(4):453-472.[2] Eddershaw P J,Beresford A P,Bayliss M K.ADME/PK as part of a rational approach to drug discovery [J].Drug Discov Today,2000,5(9):409-414. [3] Streetman D S,Bertino J S Jr,Nafziger A N.Phenotyping of drug-metabolizing enzymes in adults:a review of in-vivo cytochrome P450 phenotyping probes [J].Pharmacogenetics,2000,10(3):187-216.[4] Quattrochi L C,Guzelian P S.Cyp3A regulation:from pharmacology to nuclear receptors [J].Drug Metab Dispos,2001,29(5):615-622.[5] Wang R W,Newton D J,Scheri T D,et al.Human cytochrome P450 3A4-catalyzed testosterone 6 beta-hydroxylation and erythromycin petition during catalysis [J].Drug MetabDispos,1997,25(4):502-507.[6] Maenpaa J,Syngelma T,Honkakoski P,et parative studies on coumarin and testosterone metabolism in mouse and humanlivers.Differential inhibitions by the anti-P450Coh antibody and metyrapone [J].Biochem Pharmacol,1991,42(6):1229-1235.[7] Swales N J,Johnson T,Caldwell J.Cryopreservation of rat and mouse hepatocytes.II.Assessment of metabolic capacity using testosterone metabolism [J].Drug Metab Dispos,1996,24(11):1224-1230.[8] He Y Q,Liu Y,Zhang B F,et al.Identification of the UDP-Glucuronosyltransferase Isozyme Involved in Senecionine Glucuronidation in Human Liver Microsomes [J].Drug Metabolism andDisposition,2010,38(4):626-634.[9] 鲁艳柳,邓红,潘虹,等.山姜素对人肝微粒体中CYP1A2的选择性抑制研究[J].遵义医学院学报,2015,38(5):454-459.[10]Hashimoto H,Toide K,Kitamura R,et al.Gene structure of CYP3A4,an adult‐specific form of cytochrome P450 in human livers,and its transcriptional control [J].The FEBS Journal,1993,218(2):585-595.[11]Nagata K,Ogino M,Shimada M,et al.Structure and expression of the rat CYP3A1 gene:isolation of the gene (P450/6betaB) and characterization ofthe recombinant protein [J].Arch Biochem Biophys,1999,362(2):242-253. [12]Kuzbari O,Peterson C M,Franklin M R,et parative analysis of human CYP3A4 and rat CYP3A1 induction and relevant gene expression by bisphenol A and diethylstilbestrol:implications for toxicity testing paradigms [J].Reprod Toxicol,2013,37 (3):24-30.[13]Zimmermann C,Van Waterschoot R A,Harmsen S,et al.PXR-mediated induction of human CYP3A4 and mouse Cyp3a11 by the glucocorticoid budesonide [J].Eur J Pharm Sci,2009,36(4-5):565-571.[14]Piver B,Berthou F,Dreano Y,et al.Inhibition of CYP3A,CYP1A and CYP2E1 activities by resveratrol and other non volatile red wine components [J].Toxicol Lett,2001,125(1-3):83-91.[15]Huan J Y,Miranda C L,Buhler D R,et al.The roles of CYP3A and CYP2B isoforms in hepatic bioactivation and detoxification of the pyrrolizidine alkaloid senecionine in sheep and hamsters [J].Toxicol Appl Pharmacol,1998,151(2):229-235.[16]Li C C,Yu H F,Chang C H,et al.Effects of lemongrass oil and citral on hepatic drug-metabolizing enzymes,oxidative stress,and acetaminophen toxicity in rats [J].Journal of Food and DrugAnalysis,2017.DOI:10.1016/j.jfda.2017.01.008.[17]Chang M Y,Gwon T M,Lee H S,et al.The effect of systemic lipoic acid on hearing preservation after cochlear implantation via the round window approach:A guinea pig model [J].Eur J Pharmacol,2017,799:67-72.。