本文主要研究内容
作者郑巧园（2019）在《11β-羟化酶（Cyp11b2）在罗非鱼配子发生中的功能研究》一文中研究指出：大多数脊椎动物都具有雌、雄两种性别,雌性的性腺为卵巢,而雄性为精巢。性腺具有配子发生和合成类固醇激素的功能,配子发生是一个由生殖细胞向成熟配子即卵子和精子分化的一系列复杂过程,类固醇激素对硬骨鱼类乃至整个脊椎动物的配子发生、成熟及育性都具有重要作用。11β-羟化酶是一种类固醇合成酶,由隶属于细胞色素P450超家族B亚家族的cyp11b2(Cytochrome P450,family 11,subfamily B,polypeptide 2)编码,cyp11b2在斑马鱼中命名为cyp11c1。在人和哺乳动物,该基因催化糖皮质激素醛固酮的合成,其突变会导致多种疾病症状的产生。在硬骨鱼类中,cyp11b2参与鱼类特有的雄激素11-酮基睾酮(11-ketotestosterone,11-KT)和内源糖皮质激素皮质醇(Cortisol)的合成,分别在精巢和头肾中表达。与哺乳类不同,鱼类主要的雄激素不是睾酮(Testosterone,T),而是11-KT。雄激素能促进雄性生殖器官发育,对精子发生、第二性征的发育及生殖行为等都有重要作用。然而,迄今为止在鱼类,甚至整个脊椎动物,大量有关雄激素作用的报道都集中在激素处理实验及对雄激素受体(Androgen recepter,AR)的敲除实验,还从未有人通过敲除其合成酶来直接研究雄激素对雄性生殖器官的发育、精子发生及成熟和育性等过程的影响,更没有分析过11-KT是否对雌性的卵子发生及成熟有作用。皮质醇是鱼类主要的糖皮质激素,有关鱼类糖皮质激素的研究主要集中在应激反应和能量代谢,以及少量在应激状态下皮质醇水平升高对卵子发生的不良影响,皮质醇水平过低或缺失对鱼类卵子发生的影响鲜有报道。实验室前期克隆了cyp11b2,分析了其表达,制备了其多克隆抗体,发现特异地表达在头肾、肾脏及精巢的莱氏细胞(Leydig cell)细胞,在精巢发育早期检测不到其表达,而在孵化后30天(days after hatching,dah)开始表达,此后表达量一直持续上升直至180 dah。为进一步阐明11-KT及皮质醇在硬骨鱼类配子发生的作用,本论文以尼罗罗非鱼(Oreochromis niloticus)为实验动物,通过CRISPR/Cas9技术成功突变了cyp11b2基因并得到纯合突变鱼,分析了cyp11b2基因突变对性腺表型及配子发生的影响。主要研究结果如下:1、纯合突变鱼的获得。通过CRISPR/Cas成功突变cyp11b2基因,靶位点位于第1个外显子上,同时也是细胞色素P450超家族结构域上,选择位于靶位点上的限制性内切酶MluCI用于筛选F0代阳性鱼。待F0代雄鱼性成熟后,与野生雌鱼交配获得F1代杂合突变鱼。选择删除7 bp造成移码突变的杂合突变雌和雄鱼交配获得F2代纯合突变鱼。2、突变雄鱼的性腺表型和精子发生。通过对F1代杂合突变和F2代纯合突变雄鱼性腺表型、精子发生过程和精子特征分析,发现二者表现出基本相同的表型,F2代纯合突变雄鱼表型更为明显。组织学检测发现,90 dah时对照雄鱼的精巢生殖细胞的减数分裂过程已经启动,可见精原细胞,初级精母及次级精母细胞。纯合突变雄鱼的精巢仅观察到精原细胞,无精母细胞的存在,血清中T水平显著高于对照雄鱼。在180 dah,组织学检测发现,与野生雄鱼一样,纯合突变雄鱼精巢中能检测到各级生精细胞的存在,精子发生部分或完全恢复。然而纯合突变雄鱼血清中T水平远远高于对照雄鱼。此外,无论是90 dah还是180 dah,纯合突变雄鱼血清中都检测不到11-KT的存在。使用T、11-KT和Cortisol回救处理纯合突变雄鱼,在90 dah,组织学检测发现,11-KT处理组可见精原细胞,初级精母及次级精母细胞,与野生型雄鱼表型一致;T处理组仅可见精原细胞,少量初级精母及极少量次级精母细胞;而Cortisol处理组与纯合突变雄鱼表型一致,仅存在精原细胞,无精母细胞。说明在鱼类11-KT是比T更高效的雄激素,对精子发生的启始过程有重要作用;雄激素(而非皮质醇)不足是精子发生延迟的主要原因。在360 dah,组织学检测和各级生精细胞的定量分析发现,纯合突变导致精小叶呈不规则排列,精子和类固醇生成细胞数量均显著减少。统计分析发现,纯合突变鱼的性腺成熟系数(Gonadosomatic index,GSI)和精液量都显著降低。免疫组织化学(Immunohistochenistry,IHC)结果显示纯合突变鱼精巢中Leydig细胞标记基因(Cyp17a2)的阳性信号显著低于对照组。应用计算机辅助精子分析系统检测发现,纯合突变鱼的精子瞬时运动轨迹、精子浓度、精子活力、畸形率、鞭毛拍打频率、平均曲线速率(VCL)、平均直线速率(VSL)、精子质量及育性均正常,与对照雄鱼相比无差异。表明11-KT影响精巢结构及精液量,但不影响精子质量及育性。3、突变雌鱼的性腺表型和卵子发生。分别对F1代杂合突变和F2代纯合突变雌鱼性腺表型和卵子发生过程等进行分析。组织学检测360 dah杂合突变雌鱼性腺,发现卵子发生及成熟均正常,卵巢中能检测到各时相卵母细胞的存在,与野生型雌鱼相比,各时相卵母细胞的数目和GSI均无差异,能够自然排卵和正常受精,但是卵径变小。然而,无论与野生型或突变雄鱼人工授精,其胚胎均在受精后2天(days post fertilization,dpf)停止发育。单独使用11-KT处理不能回救,而单独使用Cortisol处理能够成功回救杂合突变雌鱼,获得纯合突变鱼。对杂合雌鱼的V时相卵子转录组测序分析发现,皮质醇单倍剂量不足影响卵子中大量基因的表达,特别是zar1、zar1l、tdrd5等大量母源基因的表达出现显著变化。说明杂合突变不影响卵子发生,但是影响卵子质量和胚胎发育,母源皮质醇不足是影响卵子质量的重要因素。对360 dah纯合突变雌鱼组织学检测,发现纯合突变雌鱼的卵子发生异常,卵巢中只存在I、II及少量III时相早期的卵母细胞,未发现III时相后期及IV时相卵母细胞的存在,而对照雌鱼卵巢中存在I、II、III和IV时相卵母细胞。统计分析发现,纯合突变雌鱼的GSI显著低于野生型雌鱼。TUNEL细胞凋亡检测发现,纯合突变雌鱼卵巢中阳性信号(红色荧光)明显多于对照雌鱼。Real-time PCR结果分析发现,纯合突变鱼卵巢中cyp19a1a、foxl2、esr1、esr2b、lhr、fshr、amh、vtg2、vtg3等基因的mRNA表达均显著下调,bmp15、figlα、gdf9、42sp50、zar1、zar1l等基因的mRNA表达均显著上升。与对照雌鱼相比,纯合突变雌鱼血清中雌激素(17β-estradiol,E2)、Cortisol及卵黄蛋白原(Vitellogenin,Vtg)的水平均显著下降,肝脏中Vtg的表达也显著下降。使用外源E2回救处理纯合突变雌鱼,卵巢中出现了IV时相卵母细胞,表明E2可以部分回救性腺表型。综上所述,本研究利用CRISPR/Cas9基因编辑技术获得cyp11b2突变鱼,发现突变导致雄鱼精子发生延迟,精液量减少,但由于前体雄激素T的补偿作用,精子活力及雄鱼育性未受影响,说明在鱼类11-KT是比T更高效的雄激素。此外,杂合突变雌鱼皮质醇不足影响卵子质量,纯合突变雌鱼皮质醇缺失导致卵黄积累障碍。本研究丰富了雄激素11-KT对硬骨鱼类精子发生的认识,创新性发现皮质醇不足或缺失对卵子发生的影响,并初步分析了涉及的分子机制,说明皮质醇在卵子发生中有重要作用。
Abstract
da duo shu ji chui dong wu dou ju you ci 、xiong liang chong xing bie ,ci xing de xing xian wei luan chao ,er xiong xing wei jing chao 。xing xian ju you pei zi fa sheng he ge cheng lei gu chun ji su de gong neng ,pei zi fa sheng shi yi ge you sheng shi xi bao xiang cheng shou pei zi ji luan zi he jing zi fen hua de yi ji lie fu za guo cheng ,lei gu chun ji su dui ying gu yu lei nai zhi zheng ge ji chui dong wu de pei zi fa sheng 、cheng shou ji yo xing dou ju you chong yao zuo yong 。11β-qiang hua mei shi yi chong lei gu chun ge cheng mei ,you li shu yu xi bao se su P450chao jia zu Bya jia zu de cyp11b2(Cytochrome P450,family 11,subfamily B,polypeptide 2)bian ma ,cyp11b2zai ban ma yu zhong ming ming wei cyp11c1。zai ren he bu ru dong wu ,gai ji yin cui hua tang pi zhi ji su quan gu tong de ge cheng ,ji tu bian hui dao zhi duo chong ji bing zheng zhuang de chan sheng 。zai ying gu yu lei zhong ,cyp11b2can yu yu lei te you de xiong ji su 11-tong ji gao tong (11-ketotestosterone,11-KT)he nei yuan tang pi zhi ji su pi zhi chun (Cortisol)de ge cheng ,fen bie zai jing chao he tou shen zhong biao da 。yu bu ru lei bu tong ,yu lei zhu yao de xiong ji su bu shi gao tong (Testosterone,T),er shi 11-KT。xiong ji su neng cu jin xiong xing sheng shi qi guan fa yo ,dui jing zi fa sheng 、di er xing zheng de fa yo ji sheng shi hang wei deng dou you chong yao zuo yong 。ran er ,qi jin wei zhi zai yu lei ,shen zhi zheng ge ji chui dong wu ,da liang you guan xiong ji su zuo yong de bao dao dou ji zhong zai ji su chu li shi yan ji dui xiong ji su shou ti (Androgen recepter,AR)de qiao chu shi yan ,hai cong wei you ren tong guo qiao chu ji ge cheng mei lai zhi jie yan jiu xiong ji su dui xiong xing sheng shi qi guan de fa yo 、jing zi fa sheng ji cheng shou he yo xing deng guo cheng de ying xiang ,geng mei you fen xi guo 11-KTshi fou dui ci xing de luan zi fa sheng ji cheng shou you zuo yong 。pi zhi chun shi yu lei zhu yao de tang pi zhi ji su ,you guan yu lei tang pi zhi ji su de yan jiu zhu yao ji zhong zai ying ji fan ying he neng liang dai xie ,yi ji shao liang zai ying ji zhuang tai xia pi zhi chun shui ping sheng gao dui luan zi fa sheng de bu liang ying xiang ,pi zhi chun shui ping guo di huo que shi dui yu lei luan zi fa sheng de ying xiang xian you bao dao 。shi yan shi qian ji ke long le cyp11b2,fen xi le ji biao da ,zhi bei le ji duo ke long kang ti ,fa xian te yi de biao da zai tou shen 、shen zang ji jing chao de lai shi xi bao (Leydig cell)xi bao ,zai jing chao fa yo zao ji jian ce bu dao ji biao da ,er zai fu hua hou 30tian (days after hatching,dah)kai shi biao da ,ci hou biao da liang yi zhi chi xu shang sheng zhi zhi 180 dah。wei jin yi bu chan ming 11-KTji pi zhi chun zai ying gu yu lei pei zi fa sheng de zuo yong ,ben lun wen yi ni luo luo fei yu (Oreochromis niloticus)wei shi yan dong wu ,tong guo CRISPR/Cas9ji shu cheng gong tu bian le cyp11b2ji yin bing de dao chun ge tu bian yu ,fen xi le cyp11b2ji yin tu bian dui xing xian biao xing ji pei zi fa sheng de ying xiang 。zhu yao yan jiu jie guo ru xia :1、chun ge tu bian yu de huo de 。tong guo CRISPR/Cascheng gong tu bian cyp11b2ji yin ,ba wei dian wei yu di 1ge wai xian zi shang ,tong shi ye shi xi bao se su P450chao jia zu jie gou yu shang ,shua ze wei yu ba wei dian shang de xian zhi xing nei qie mei MluCIyong yu shai shua F0dai yang xing yu 。dai F0dai xiong yu xing cheng shou hou ,yu ye sheng ci yu jiao pei huo de F1dai za ge tu bian yu 。shua ze shan chu 7 bpzao cheng yi ma tu bian de za ge tu bian ci he xiong yu jiao pei huo de F2dai chun ge tu bian yu 。2、tu bian xiong yu de xing xian biao xing he jing zi fa sheng 。tong guo dui F1dai za ge tu bian he F2dai chun ge tu bian xiong yu xing xian biao xing 、jing zi fa sheng guo cheng he jing zi te zheng fen xi ,fa xian er zhe biao xian chu ji ben xiang tong de biao xing ,F2dai chun ge tu bian xiong yu biao xing geng wei ming xian 。zu zhi xue jian ce fa xian ,90 dahshi dui zhao xiong yu de jing chao sheng shi xi bao de jian shu fen lie guo cheng yi jing qi dong ,ke jian jing yuan xi bao ,chu ji jing mu ji ci ji jing mu xi bao 。chun ge tu bian xiong yu de jing chao jin guan cha dao jing yuan xi bao ,mo jing mu xi bao de cun zai ,xie qing zhong Tshui ping xian zhe gao yu dui zhao xiong yu 。zai 180 dah,zu zhi xue jian ce fa xian ,yu ye sheng xiong yu yi yang ,chun ge tu bian xiong yu jing chao zhong neng jian ce dao ge ji sheng jing xi bao de cun zai ,jing zi fa sheng bu fen huo wan quan hui fu 。ran er chun ge tu bian xiong yu xie qing zhong Tshui ping yuan yuan gao yu dui zhao xiong yu 。ci wai ,mo lun shi 90 dahhai shi 180 dah,chun ge tu bian xiong yu xie qing zhong dou jian ce bu dao 11-KTde cun zai 。shi yong T、11-KThe Cortisolhui jiu chu li chun ge tu bian xiong yu ,zai 90 dah,zu zhi xue jian ce fa xian ,11-KTchu li zu ke jian jing yuan xi bao ,chu ji jing mu ji ci ji jing mu xi bao ,yu ye sheng xing xiong yu biao xing yi zhi ;Tchu li zu jin ke jian jing yuan xi bao ,shao liang chu ji jing mu ji ji shao liang ci ji jing mu xi bao ;er Cortisolchu li zu yu chun ge tu bian xiong yu biao xing yi zhi ,jin cun zai jing yuan xi bao ,mo jing mu xi bao 。shui ming zai yu lei 11-KTshi bi Tgeng gao xiao de xiong ji su ,dui jing zi fa sheng de qi shi guo cheng you chong yao zuo yong ;xiong ji su (er fei pi zhi chun )bu zu shi jing zi fa sheng yan chi de zhu yao yuan yin 。zai 360 dah,zu zhi xue jian ce he ge ji sheng jing xi bao de ding liang fen xi fa xian ,chun ge tu bian dao zhi jing xiao xie cheng bu gui ze pai lie ,jing zi he lei gu chun sheng cheng xi bao shu liang jun xian zhe jian shao 。tong ji fen xi fa xian ,chun ge tu bian yu de xing xian cheng shou ji shu (Gonadosomatic index,GSI)he jing ye liang dou xian zhe jiang di 。mian yi zu zhi hua xue (Immunohistochenistry,IHC)jie guo xian shi chun ge tu bian yu jing chao zhong Leydigxi bao biao ji ji yin (Cyp17a2)de yang xing xin hao xian zhe di yu dui zhao zu 。ying yong ji suan ji fu zhu jing zi fen xi ji tong jian ce fa xian ,chun ge tu bian yu de jing zi shun shi yun dong gui ji 、jing zi nong du 、jing zi huo li 、ji xing lv 、bian mao pai da pin lv 、ping jun qu xian su lv (VCL)、ping jun zhi xian su lv (VSL)、jing zi zhi liang ji yo xing jun zheng chang ,yu dui zhao xiong yu xiang bi mo cha yi 。biao ming 11-KTying xiang jing chao jie gou ji jing ye liang ,dan bu ying xiang jing zi zhi liang ji yo xing 。3、tu bian ci yu de xing xian biao xing he luan zi fa sheng 。fen bie dui F1dai za ge tu bian he F2dai chun ge tu bian ci yu xing xian biao xing he luan zi fa sheng guo cheng deng jin hang fen xi 。zu zhi xue jian ce 360 dahza ge tu bian ci yu xing xian ,fa xian luan zi fa sheng ji cheng shou jun zheng chang ,luan chao zhong neng jian ce dao ge shi xiang luan mu xi bao de cun zai ,yu ye sheng xing ci yu xiang bi ,ge shi xiang luan mu xi bao de shu mu he GSIjun mo cha yi ,neng gou zi ran pai luan he zheng chang shou jing ,dan shi luan jing bian xiao 。ran er ,mo lun yu ye sheng xing huo tu bian xiong yu ren gong shou jing ,ji pei tai jun zai shou jing hou 2tian (days post fertilization,dpf)ting zhi fa yo 。chan du shi yong 11-KTchu li bu neng hui jiu ,er chan du shi yong Cortisolchu li neng gou cheng gong hui jiu za ge tu bian ci yu ,huo de chun ge tu bian yu 。dui za ge ci yu de Vshi xiang luan zi zhuai lu zu ce xu fen xi fa xian ,pi zhi chun chan bei ji liang bu zu ying xiang luan zi zhong da liang ji yin de biao da ,te bie shi zar1、zar1l、tdrd5deng da liang mu yuan ji yin de biao da chu xian xian zhe bian hua 。shui ming za ge tu bian bu ying xiang luan zi fa sheng ,dan shi ying xiang luan zi zhi liang he pei tai fa yo ,mu yuan pi zhi chun bu zu shi ying xiang luan zi zhi liang de chong yao yin su 。dui 360 dahchun ge tu bian ci yu zu zhi xue jian ce ,fa xian chun ge tu bian ci yu de luan zi fa sheng yi chang ,luan chao zhong zhi cun zai I、IIji shao liang IIIshi xiang zao ji de luan mu xi bao ,wei fa xian IIIshi xiang hou ji ji IVshi xiang luan mu xi bao de cun zai ,er dui zhao ci yu luan chao zhong cun zai I、II、IIIhe IVshi xiang luan mu xi bao 。tong ji fen xi fa xian ,chun ge tu bian ci yu de GSIxian zhe di yu ye sheng xing ci yu 。TUNELxi bao diao wang jian ce fa xian ,chun ge tu bian ci yu luan chao zhong yang xing xin hao (gong se ying guang )ming xian duo yu dui zhao ci yu 。Real-time PCRjie guo fen xi fa xian ,chun ge tu bian yu luan chao zhong cyp19a1a、foxl2、esr1、esr2b、lhr、fshr、amh、vtg2、vtg3deng ji yin de mRNAbiao da jun xian zhe xia diao ,bmp15、figlα、gdf9、42sp50、zar1、zar1ldeng ji yin de mRNAbiao da jun xian zhe shang sheng 。yu dui zhao ci yu xiang bi ,chun ge tu bian ci yu xie qing zhong ci ji su (17β-estradiol,E2)、Cortisolji luan huang dan bai yuan (Vitellogenin,Vtg)de shui ping jun xian zhe xia jiang ,gan zang zhong Vtgde biao da ye xian zhe xia jiang 。shi yong wai yuan E2hui jiu chu li chun ge tu bian ci yu ,luan chao zhong chu xian le IVshi xiang luan mu xi bao ,biao ming E2ke yi bu fen hui jiu xing xian biao xing 。zeng shang suo shu ,ben yan jiu li yong CRISPR/Cas9ji yin bian ji ji shu huo de cyp11b2tu bian yu ,fa xian tu bian dao zhi xiong yu jing zi fa sheng yan chi ,jing ye liang jian shao ,dan you yu qian ti xiong ji su Tde bu chang zuo yong ,jing zi huo li ji xiong yu yo xing wei shou ying xiang ,shui ming zai yu lei 11-KTshi bi Tgeng gao xiao de xiong ji su 。ci wai ,za ge tu bian ci yu pi zhi chun bu zu ying xiang luan zi zhi liang ,chun ge tu bian ci yu pi zhi chun que shi dao zhi luan huang ji lei zhang ai 。ben yan jiu feng fu le xiong ji su 11-KTdui ying gu yu lei jing zi fa sheng de ren shi ,chuang xin xing fa xian pi zhi chun bu zu huo que shi dui luan zi fa sheng de ying xiang ,bing chu bu fen xi le she ji de fen zi ji zhi ,shui ming pi zhi chun zai luan zi fa sheng zhong you chong yao zuo yong 。
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论文作者分别是来自西南大学的郑巧园，发表于刊物西南大学2019-09-24论文，是一篇关于雄激素论文,皮质醇论文,配子发生论文,西南大学2019-09-24论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自西南大学2019-09-24论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：雄激素论文; 皮质醇论文; 配子发生论文; 西南大学2019-09-24论文;