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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 1: 94 - 106

DOI: https://doi.org/10.11978/2023040

Marine Biology

Expression characteristics and function of Anti-Müllerian hormone receptor Ⅱ gene (amhr2) in Paralichthys olivaceus

  • LI Ze , 1, 2, 3 ,
  • WANG Lijuan , 1, 2 ,
  • ZOU Congcong 1, 2, 3 ,
  • SHU Chang 1, 2, 3 ,
  • WU Zhihao 1, 2 ,
  • ZOU Yuxia 1, 2 ,
  • YOU Feng , 1, 2
Expand
  • 1. Chinese Academy of Sciences and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science (Institute of Oceanology, Chinese Academy of Sciences), Qingdao 266071, China
  • 2. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
  • 3. University of Chinese Academy of Sciences, Beijing 100049, China
YOU Feng. email: ;
WANG Lijuan. email:

Copy editor: SUN Cuici

Received date: 2023-03-24

  Revised date: 2023-04-21

  Online published: 2023-05-25

Supported by

National Key R&D Program of China(2022YFD2400402)

National Key R&D Program of China(2018YFD0900202)

Shandong Natural Science Foundation(ZR2022MC026)

Youth Research Fund of Marine Biology and Biotechnology Laboratory, Pilot National Laboratory for Marine Science and Technology (Qingdao)(YQ2018NO01)

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Abstract

Anti-Mullerian hormone receptor Ⅱ (Amhr2) is the specific receptor of Anti-Mullerian hormone (Amh) in the TGF-β pathway. It is critical for gonadal differentiation and development in fish and determination of the sex in some fish species. However, the expression and function of amhr2 in fish has been poorly studied. To clarify the expression characteristics and function of amhr2, a 1536 bp CDS encoding 511 amino acids, was obtained from Paralichthys olivaceus, an important commercially cultured marine fish in China. The flounder Amhr2 protein was clustered with those from other teleosts, with the highly conservative motifs occurring in the C-terminal region. Gene collinearity analysis revealed that the amhr2 collaborates with specificity protein 1 (sp1) upstream, and proline-rich protein 13 (prr13) and poly (rC) binding protein 2 (PCBP2) downstream of the teleost genome. The predicted flounder Amhr2 protein was composed of a signal peptide, two transmembrane domains, and a conservative tyrosine protein kinase domain. Real-time quantitative PCR (qPCR) results showed that amhr2 was mainly expressed in the adult gonads, with significantly higher expression in the testes than in the ovaries (P<0.01). At stages I-V of the testis, the expression of amhr2 remained at a high level, whereas the expression level in the ovaries was significantly higher at stage I than at stages Ⅱ-V (P< 0.05). During the flounder gonadal differentiation period, gynogenesis (control, 20 ± 0.5℃, 100% female) and gynogenesis at high temperature (HT, 28 ± 0.5℃, 100% male) were used to detect the expression of amhr2. The results showed that the expression of amhr2 in the control group was highest at 2 cm total length (TL) and then decreased continuously, while the expression in the HT group first increased and then decreased. The inflection point of amhr2 expression in the HT group was at 6 cm TL, with the highest level. To investigate the effect of Amh on amhr2, we examined the co-localization of Amhr2 and Amh with Hela cells. Examination of subcellular localization showed that both Amhr2 and Amh were mainly located in the cytoplasm. Subsequently, the recombinant protein Amh of the flounder was obtained by prokaryotic expression. After in vitro incubation with the recombinant Amh, the expression of amhr2 in the testes showed no significant difference, whereas the transcription level in the ovaries increased significantly at 12 and 48 h (P<0.05). In addition, a dual luciferase reporter assay in HEK293T cells showed that co-transfection of Amh and Amhr2 could inhibit the promoter activity of the cytochrome P450 aromatase gene (cyp19a) (P<0.01), the key gene for estrogen synthesis. These findings suggest that amhr2 is mainly expressed in the testis, but its expression varies at different stages of the gonadal development. And amhr2 may affect the transcription of cyp19a together with Amh, which plays a role in the onset of the male phenotype formation and gonadal development in the flounder.

Key words： Paralichthys olivaceus; amhr2; gonadal differentiation and development; gene expression; function

Cite this article

LI Ze , WANG Lijuan , ZOU Congcong , SHU Chang , WU Zhihao , ZOU Yuxia , YOU Feng . Expression characteristics and function of Anti-Müllerian hormone receptor Ⅱ gene (amhr2) in Paralichthys olivaceus[J]. Journal of Tropical Oceanography, 2024 , 43(1) : 94 -106 . DOI: 10.11978/2023040

抗缪勒氏管激素(anti-Müllerian hormone, AMH)隶属于转化生长因子β(transforming growth factor β, TGF-β)超家族(Pan et al, 2021), 广泛存在于脊椎动物中, 能够在胚胎发育阶段引起雄性缪勒氏管退化, 并在发育期调节精巢Leydig细胞功能、抑制卵泡早衰(Hart et al, 2021)。硬骨鱼类中不存在缪勒氏管, 但保留了amh基因并高表达于精巢中。已有研究表明, amh能够诱导鱼类精巢分化, 如敲除amh的斑马鱼(Danio reri)会引起雄性性逆转为雌性(Zhang et al, 2020), 在斜带石斑鱼(Epinephelus coioides)中过表达amh会导致性腺向精巢分化(Han et al, 2019)。抗缪勒氏管激素Ⅱ型受体(Amhr2)是Amh的特异性受体, 在一些鱼类性别决定和性腺分化过程中担任关键角色(Pfennig et al, 2015)。如红鳍东方鲀(Takifugu rubripes)的性别就由amhr2的SNP位点决定, 雄性个体为杂合子(His/Asp384), 而雌性个体为纯合子(His/His384) (Kamiya et al, 2012)。在基因型为XY的尼罗罗非鱼(Oreochromis niloticus)中, amhr2突变会导致雄性向雌性的完全性逆转(Li et al, 2015)。
Amhr2作为TGF-β通路受体, 鱼类中其作用的研究主要是围绕配体Amh开展的。研究表明, amh能够通过调控生殖细胞的增殖影响鱼类的性腺分化过程, 敲除amhr2的青鳉(Oryzias latipes)突变体hotei(Morinaga et al, 2007)和敲除amh的斑马鱼(Zhang et al, 2020)性腺生殖细胞均出现了异常地增殖。amhr2与amh mRNA通常在精巢支持细胞和卵巢颗粒细胞中共定位, 如青鳉(Kluver et al, 2007)、红鳍东方鲀(Kamiya et al, 2012)等。同时, 二者表达也存在一定的相关性, 如斜带石斑鱼各时期雌雄性腺中, amh和amhrII基因的表达水平变化趋势一致, 过表达amh会使amhrII基因的表达显著上调(韩玉龙, 2019)。Cyp19a能够编码芳香化酶催化雄激素合成雌激素, 是影响鱼类性别的关键基因之一(He et al, 2016), 目前在斜带石斑鱼中已经证实了amh-amhr2-Smads-cyp19a1a信号通路的存在(韩玉龙, 2019), 但在斑马鱼性腺分化中发现amh与cyp19a不存在调控关系(Rodriguez-Mari et al, 2005)。所以, 对于amhr2信号传导的具体分子机制包括对cyp19a的调控关系还需进一步探究。
牙鲆(Paralichthys olivaceus)隶属于鲽形目、牙鲆科、牙鲆属, 又称比目鱼或偏口鱼, 广泛分布于美洲东西海岸、亚洲东南沿海及俄罗斯远东沿海地区(Shan et al, 2022), 是中、日、韩三国重要的海水增养殖鱼类。其雌性个体体型大、生长速度快, 经济价值显著高于雄性, 因此, 开展牙鲆性别相关研究尤为重要(Zhang et al, 2022)。最新的报道认为amh同源拷贝amhy是牙鲆的性别决定基因, Amh/Amhr2受体系统可影响性别表型(Hattori et al, 2022)。Yamaguchi等(2023)利用CRISPR/Cas9技术构建了牙鲆amhr2缺失突变体, 可导致基因型XY个体的性腺分化为卵巢, 性别分化相关基因也呈现雌性表达模式; 但用芳香化酶抑制剂处理amhr2突变体, 则能够使其精巢分化恢复, 表明Amh/Amhr2受体系统是通过抑制雌激素的合成影响牙鲆性别分化的。但是, 对于Amh/Amhr2受体系统抑制雌激素合成的分子机制和调控方式尚不明确, 且amhr2在牙鲆性腺分化和发育过程中的表达模式也不清晰。因此, 本研究克隆了牙鲆amhr2基因, 分析了其在性腺分化和发育中的表达特征, 明确重组Amh对其表达的影响, 同时探究Amh/Amhr2受体系统是否能直接调控cyp19a启动子, 以期为明晰牙鲆等鱼类性别分化的分子调控机制提供基础数据, 也为牙鲆的性控生产提供理论依据。

1 材料与方法

1.1 实验材料

成体牙鲆(total length, TL) 全长20~40cm, 购自青岛南山市场, 运至中国科学院海洋研究所鱼类培育室中暂养7d, 用于组织差异和性腺发育各时期基因表达分析。期间每日投喂适口商用饵料2次, 换水1次。暂养结束后停食24h, 随机选取雌雄个体各3尾, 经50mg·L-1 3-氨基苯甲酸乙酯甲基磺酸盐(3-aminobenzoic acid ethyl ester methanesulfonate, MS-222)(Sigma, 美国)麻醉后, 采集肾、脾、肝、肌肉、脑和性腺组织。一部分性腺组织于Davison固定液中固定后经组织切片进行发育时期鉴别, 切片染色方法见孙鹏等(2009); 剩余部分经液氮速冻后保存于-80℃冰箱用于后续RNA提取。
雌核发育牙鲆在威海圣航水产科技有限公司进行人工诱导, 诱导方法及参数参照本实验室已有报道(You et al, 2001)。鱼苗培育至1.2~1.5cm TL时, 进行分组处理以获得雌雄表型个体(Wang et al, 2015)。设置对照组与高温处理组(HT组): 随机挑选200尾鱼苗置于90L培育箱中, 每组2个重复。HT组的水温由20℃开始, 每日升高2℃, 最终维持在28 ± 0.5℃; 对照组水温始终维持在20 ± 0.5℃, 至鱼苗12 cm TL时停止实验。其间, 每天换水2~3次、投喂适口商用饵料4~5次, 光照14L: 10D, 连续充氧, 使溶解氧维持在至少6mg·L-1。在2、4、6、8、10cm TL时进行取样, 麻醉后取性腺组织经液氮速冻保存于-80℃低温冰箱中。12cm TL个体性腺固定后通过组织切片进行性别鉴定和性比统计。
组织孵育所用成体牙鲆购自胶南养殖场, 暂养方法同上。选取大小相似的雌雄个体各4尾(30 ± 2cm TL), 麻醉后无菌条件下取出完整性腺组织, 使用含有1%青霉素(10kU·mL-1)和链霉素(10mg·mL-1)混合抗生素的磷酸盐缓冲液(phosphate buffered saline, PBS)冲洗3~5次, 一部分进行固定切片用以鉴别性腺发育时期, 另一部分切成6份约为8.0mm3的组织块放入24孔板中, 分别用于EGFP对照组和重组Amh实验组蛋白孵育期间基因表达检测。
所有实验材料的处理方法均符合中国科学院海洋研究所实验动物福利伦理和动物实验安全审查制度要求。

1.2 总RNA提取及cDNA合成

根据说明书, 使用Trizol试剂(Toroivd, 英国)提取各样品中的总RNA, 利用2%琼脂糖电泳和NanoDrop 2000 分光光度计检测其纯度和浓度, 第一链cDNA使用Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeason, 中国)合成。

1.3 牙鲆amhr2验证及序列分析

根据转录组(Fan et al, 2014)与National Center for Biotechnology Information(NCBI) (amh: XM_ 0200808 98.1; amhr2: XM_020096590.1)相关信息获得牙鲆amh和amhr2基因序列, 利用Primer3 (https://www.yeastgenome.org/Primer3)设计amh和amhr2 CDS扩增引物(表1)。分别以成体牙鲆精巢和卵巢cDNA为模板, 进行amh及amhr2 CDS扩增与验证。反应体系为25μL, 按2×Rapid Taq Mix(康为世纪, 中国)说明书配制体系。反应程序为95℃ 3min; 95℃ 15s, 58℃ 20s, 72℃ 1min, 共35个循环; 72℃ 5min。PCR产物经2%琼脂糖凝胶电泳检测, 并用胶回收试剂盒(Omega, 美国)进行回收, 送至北京睿博兴科生物技术有限公司进行双向测序。
表1 本研究所用引物

Tab. 1 Primers used in this study

引物 序列 NCBI序列号及参考文献
amhr2-CDS-F ATTCCCAGGACCCTCCCTGTT XM_020096590.1
amhr2-CDS-R TCTGCAAAGAGAACAGTGCAAC
amh-CDS-F ATGCCGGTGGTGAACGTCTT XM_020080898.1
amh-CDS-R GCGGCATCCACACTCCTTTG
amhr2-qPCR-F CGTCGTGTTCCTGCCAAAC XM_020096590.1
amhr2-qPCR-R GGCAGCTCATACACCTCCTT
actin- qPCR-F GGAATCCACGAGACCACCTACA EU090804
Hu et al, 2014
actin- qPCR-R CTGCTTGCTGATCCACATCTGC
ef- qPCR-F AGCCAGAAGCGTTTTGAGGAG XM_020104638.1
Zhong et al, 2008
ef- qPCR-R AGATGGGGACGAAAGCAACAC
amh-pc3.1-EcoRV-F CACACTGGACTAGTGGATCCGCCACCATGCCGGTGGTGAACGTC XM_020080898.1
amh-pc3.1-EcoRV-R AGCTTGGTACCGAGCTCGGATCCGCGGCATCCACACTCCT
amhr2-pc3.1-BamHI-F CACACTGGACTAGTGGATCCGCCACCATGATGCTGCAGTGGTGGC XM_020096590.1
amhr2-pc3.1-BamHI-R AGCTTGGTACCGAGCTCGGATCCTGAGCTGCTTTCAGAGACATAAGACTG
amhr2-pCS2-BamHI-F ACGACGACGATAAGGGATCCGCCACCATGATGCTGCAGTGGTGGC XM_020096590.1
amhr2-pCS2-BamHI-R AATTCGAATCGATGGGATCCTGAGCTGCTTTCAGAGACATAAGACTG
amh-pET-EcoRV-F AAGGCCATGGCTGATATGCCGGTGGTGAACGTCTT XM_020080898.1
amh-pET-EcoRV-R GAATTCGGATCCGATGCGGCATCCACACTCCTTTG
将牙鲆amhr2编码的氨基酸序列与NCBI上的部分哺乳类等高等脊椎动物和鱼类同源序列一起, 利用BioEdit (https://thalljiscience.github.io/)和 Mega 7.0 (https://megasoftware.net/)分别进行多序列比对和Neighbor-Joining系统发育树的构建, 并根据NCBI上的基因组信息进行共线性分析。
通过MEME(https://meme-suite.org)、SWISS-MODEL (https://swissmodel.expasy.org/)、ExPASy (https://web.expasy.org/protparam/)、SMART (http://smart.embl-heidelberg.de/)及TMHMM (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0)在线网站分别预测牙鲆Amhr2的保守功能域、3D结构、理化性质、蛋白结构域及跨膜区。
使用ZDOCK (https://www.dockeasy.cn/DockProtein)预测牙鲆Amh与Amhr2的分子间相互作用并对其进行Z-score评分。

1.4 实时荧光定量PCR(qPCR)

基于获得的牙鲆amhr2 CDS序列, 利用Primer3设计qPCR引物。模板梯度稀释后, 经qPCR检测其扩增效率为99.3%。反应体系为20μL, 按Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix(Yeasen, 中国)说明书配制体系。反应程序为95℃ 2min; 95℃ 10s, 60℃ 30s, 共40个循环; 95℃ 15s。选择β-actin作为内参基因(Zheng et al, 2011), 每个样本设置3个平行以确保数值的准确, 由目的基因与内参基因拷贝数的差得到ΔCt, 后根据2-ΔΔCt法计算基因的相对表达量。

1.5 Amh和Amhr2的亚细胞定位

以反转获得的牙鲆性腺cDNA为模板, 使用相应引物(表1)扩增amh和amhr2的CDS序列。反应体系为25μL, 按2×Pfu MasterMix(康为世纪, 中国)说明书配制体系。反应程序为94℃ 2min; 94℃ 30s, 60℃ 30s, 72℃ 2min, 共35个循环; 72℃ 5min, 扩增产物经1%琼脂糖凝胶电泳检测后切胶回收。分别利用EcoRV和BamHI限制性内切酶对pcDNA 3.1/myc-His(-)和pCS2质粒进行单酶切, 使用Seamless Assembly Cloning Kit(Clone Smarter, 美国)同源重组构建amh-pc3.1-EcoRV(amh-pc3.1)、amhr2-pc3.1-BamHI (amhr2-pc3.1)和amhr2-pCS2-BamHI(amhr2-pCS2)重组质粒。Hela细胞用含10%胎牛血清的DMEM培养基置于37℃的5%二氧化碳培养箱中培养, 使用前混匀并平铺在24孔板中, 待细胞汇合度达到90%左右进行转染。将2种空载体和重组质粒分别与Hieff Trans® Liposomal Transfection Reagent (Yeason, 中国)混匀后加入孔板中, 培养24h后, 用4%PFA固定10min。经PBS漂洗后用含有0.1%TritonX-100的PBS(PBST)进行透化。再次漂洗后, 用5%BSA封闭30min。分别加入anti-His (1: 200, 鼠源, CWBIO, 中国)和anti-Flag(1: 2000, 兔源, Affinit Y, 中国)作为一抗, 4°C孵育过夜。PBST漂洗后, 分别加入Cy3荧光标记抗体(1: 100, 抗鼠, Roche, 美国)和异硫氰酸(FITC)荧光标记抗体(1: 100, 抗兔, Roche, 美国)进行孵育。使用DAPI显色液进行显色, 并在激光共聚焦显微镜(MRC 1024, 德国)下进行观察。

1.6 牙鲆Amh蛋白重组、表达及活性检测

采用本实验室已报道的方法(Shu et al, 2022), 原核表达并纯化重组Amh: 以pET30a-EGFP融合质粒为载体(Solarbio, 中国), EcoRV限制性内切酶单酶切后, 通过同源重组构建amh-pET-EGFP重组质粒, 后转化至Transetta(DE3)(Transgen, 中国), 挑取单菌落测序并在含有0.2%卡那霉素的自动诱导复合培养基中扩大培养。菌体破碎后, 使用His标记蛋白纯化试剂盒(康为世纪, 中国)对重组蛋白进行纯化, 梯度透析复性后使用NanoDrop 2000检测蛋白浓度。
重组Amh蛋白经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)与蛋白免疫印迹(western blot, WB)进行验证: 取少量对照组和实验组培养基、菌体沉淀、菌体破碎上清、菌体破碎沉淀(重悬于PBS中)及纯化中各步洗脱液, 按比例加入5×蛋白上样缓冲液(碧云天, 中国), 沸水浴12min, 4℃离心10min, 使用10%PAGE凝胶(雅酶, 中国)进行蛋白电泳。电泳后SDS-PAGE实验样品经蛋白胶染液(睿博兴科, 中国)室温染色15min后拍照。相同样品电泳后经半干转膜至硝酸纤维素膜(Whatman, 德国)上进行WB验证: 封闭30min后anti-His(1: 5000, 鼠源, 正能, 中国)一抗4℃孵育过夜, 使用含0.1%Tween-20的PBS清洗5次, HRP标记抗体(1: 5000, 山羊抗鼠, 正能, 中国)室温孵育2h, 清洗5次后用ECL(索莱宝, 中国)显色。
重组蛋白活性检测采用组织孵育方法: 向24孔板精巢和卵巢组织中各加入600μL含有1%抗生素的 L15培养基, 在25°C培养箱预孵育6h。按本实验室已报道蛋白转染方法(Shu et al, 2022), 使用含有100μg EGFP或Amh-EGFP重组蛋白和Hieff Trans® Liposomal Transfection Reagent(Yeason, 中国)的新鲜培养基在25℃下继续孵育48h。分别在孵育12、24、48h取样, 样品经液氮速冻后保存在-80℃冰箱中, 用于后续RNA提取、基因表达检测。

1.7 Amh及Amhr2细胞转染

使用1.5中构建好的amh-pc3.1、amhr2-pc3.1重组质粒及本实验室先前构建的cyp19a启动子质粒cyp19a-pGL3(范兆飞, 2017)与Hieff Trans® Liposomal Transfection Reagent(Yeason, 中国)混匀后转染至HEK293T细胞系中, 使用海肾荧光素酶质粒(TK-pRL)作为内参验证细胞转染效率, 使用空载体质粒pcDNA3.1补足并保证每孔的转染DNA总量为1μg。实验分组及转染组分如表2所示。
表2 细胞转染组别及组分

Tab. 2 Cell transfection groups and their components

转染组分(ng)\组别 cyp19a转染对照组 cyp19a+amh共转染组 cyp19a+amhr2共转染组 cyp19a+amh+amhr2共转染组
cyp19a-pGL3 400 400 400 400
amh-pc3.1 0 400 0 200
amhr2-pc3.1 0 0 400 200
TK-pRL 40 40 40 40
pcDNA 3.1 560 160 160 160
在5%二氧化碳培养箱中37℃下培养24h后, 使用Dual-Luciferase Reporter Assay System试剂盒(诺唯赞, 中国)进行荧光酶显色, 检测萤火虫荧光素酶活性和海肾荧光素酶活性。

1.8 数据处理

各项数据均以平均值±标准误(S.E.M.)表示。成体各组织、性腺分化期、性腺发育各时期基因表达的差异及细胞转染荧光强度的变化情况, 通过独立t检验进行显著性分析。性腺孵育实验中, 不同孵育时间的基因表达差异使用成对t检验进行检测。显著阈值均为P< 0.05。

2 结果

2.1 牙鲆amhr2基因克隆及生信分析

通过PCR扩增得到了牙鲆amhr2的CDS序列, 共1536bp, 编码511个氨基酸, 雌雄个体获得的序列没有差异。与NCBI数据库中其他脊椎动物包括其他鱼种Amhr2氨基酸序列进行多序列比对, 显示该蛋白C端具有较高的保守性, 氨基酸平均相似度达到了44.51%, 包含9个α-螺旋结构和9个β-折叠结构, 以及保守的AH×D××××N×L、GL××LH基序(图1)。
图1 牙鲆与其他物种Amhr2氨基酸序列多重比对及二级结构分析

星号, 牙鲆; α, α-螺旋结构; β, β-折叠结构; 黑色区域, 序列完全相同区域; 虚线框, GL××LH保守基序; 实线框, AH×D××××N×L保守基序

Fig. 1 Amino acid sequence alignment and secondary structure prediction of Amhr2 among Paralichthys olivaceus and other species.

P. olivaceus is marked with star. α, α- helices structure; β, β- sheets structure; black area, the sequence completely conserved region; dotted line box, the GL ×× LH conservative motif; full line box, the AH × D ×××× N × L conservative motif

牙鲆Amhr2的预测分子量为57602.34 Da, 理论等电点(PI)为7.48。蛋白总平均疏水性指数GRAVY 小于0(-0.028), 为亲水性蛋白, 不稳定指数大于40(46.16), 提示其蛋白结构不稳定。蛋白结构域分析显示, 其氨基酸序列第1~19位存在信号肽, 5~24、141~159位存在2个跨膜结构, 198~499位存在酪氨酸蛋白激酶结构域(图2a)。通过刚性对接预测Amhr2与配体Amh的复合物模型, Z-score最优模型评分1299.826, 表明二者间大概率存在直接相互作用(图2c)。
图2 牙鲆Amhr2结构域、3D结构及分子对接预测图

a. 结构域预测; b. 3D结构预测; c. Amh与Amhr2分子对接预测

Fig. 2 Functional domains, 3D structure and molecular docking of P. olivaceus Amhr2 protein.

(a) Structure domain prediction; (b) 3D structure prediction; (c) molecule docking prediction of Amh and Amhr2

邻接(neighbor-joining)系统发育树分析发现, 牙鲆Amhr2与其他硬骨鱼类的聚为一枝, 而哺乳类等高等脊椎动物的聚为一枝。共线性分析结果显示, amhr2在各物种中的基因位置相对保守, 和上游特异性蛋白1(sp1)以及下游脯氨酸富集蛋白13(prr13)与聚(rC)结合蛋白2(PCBP2)共定位, 在硬骨鱼中保守性最高(图3)。
图3 脊椎动物Amhr2氨基酸序列系统发生及共线性分析

框, 牙鲆序列; 箭头, 基因转录方向

Fig. 3 Phylogenesis and collinearity analysis of Amhr2 amino acid sequence in vertebrates.

Box, sequence of P. olivaceus; arrow, the direction of transcription

2.2 牙鲆amhr2组织差异表达

根据性腺组织切片观察, 实验所用牙鲆精巢的发育时期为Ⅲ期, 卵巢为Ⅱ期。qPCR结果显示, amhr2主要表达在雄鱼的精巢中, 在雌鱼肾、脾、肝中有低水平表达, 卵巢中表达极显著低于精巢(P< 0.01)(图4)。
图4 牙鲆amhr2雌雄个体组织差异表达

K, 肾; S, 脾; M, 肌肉; L, 肝; B, 脑; G, 性腺; n=3; **表示雌雄性腺具有极显著差异, P< 0.01

Fig. 4 The expression of amhr2 in different tissues of male and female P. olivaceus.

K, kidney; S, spleen; M, muscle; L, liver; B, brain; G, gonad. n=3. ** indicates the difference between the testes and ovaries, P< 0.01

2.3 amhr2 在I—V期性腺的差异表达

牙鲆I—V期的性腺样品经组织切片确定时期后, 进行qPCR分析。如图5所示, 牙鲆amhr2在卵巢I期时表达较高, 但在Ⅱ—V期几乎不表达, 且表达持续下降(P< 0.01); 在精巢I—V期表达均维持在较高水平。精巢与卵巢amhr2表达有明显差异, 除了I期卵巢中表达极显著高于同期精巢(P< 0.01)外, Ⅱ—V期时卵巢中表达均显著低于同期精巢(P< 0.05)。
图5 牙鲆amhr2在性腺发育各时期的表达

n=3。不同字母表示不同性腺发育时期卵巢中amhr2的表达具有显著差异, P< 0.05。*表示同一性腺发育时期精巢、卵巢中具有显著差异, P< 0.05; **, P< 0.01

Fig. 5 The expression of amhr2 in P. olivaceus during the gonadal development. n=3. Different letters indicate the difference in the ovaries of the fish at different gonadal development stages. * indicates the difference between the testes and ovaries of the fish at the same gonadal development stages, P< 0.05; **, P< 0.01

2.4 amhr2在牙鲆性腺分化期中的差异表达

对照组和HT组12cm TL幼鱼性腺组织切片观察表明, 对照组鱼苗均发育为雌性, 而HT组均为雄性, 即两个组的雄性比例分别为0%和100%。
QPCR检测两组性腺分化期鱼苗性腺的amhr2转录水平。结果显示, 该基因在对照组(雌性组)2cm TL时表达最高, 后表达持续下调, 并在4~10cm TL下调显著(P< 0.05); 在HT组(雄性组)中6 cm TL前表达逐步增加, 在6cm TL时达到最高, 而在8、10 cm TL时下调至微弱表达。两组相比, 对照组在2、8、10cm TL时的相对表达量极显著高于HT组(P< 0.01), 但在4、6cm TL时没有显著差异(图6)。
图6 牙鲆amhr2在性腺分化期的表达

n=3。不同字母表示不同全长雌核发育对照组牙鲆性腺中amhr2表达具有显著差异, P< 0.05。**表示在相同全长时精巢、卵巢中具有极显著差异, P< 0.01

Fig. 6 The expression of amhr2 in P. olivaceus during gonadal differentiation.

n=3. Different letters indicate the differences at different TLs of the gynogenesis control group during the gonadal differentiation, P< 0.05. ** indicates the difference between the testes and ovaries of the fish at the same TLs, P< 0.01

2.5 Amhr2与Amh亚细胞定位

通过细胞转染实验来分析牙鲆Amh及Amhr2在Hela细胞中的表达部位。结果显示, Amh及Amhr2主要定位在Hela细胞质中(图7A, B)。将Amh和Amhr2二者共同转染至Hela细胞中, 观察到二者在细胞中的表达位置一致, 均定位于细胞质中(图7C)。
图7 牙鲆Amh和Amhr2的亚细胞定位

A, Amh; pcDNA3.1, 空载体质粒; amh-pc3.1, amh重组质粒; B, Amhr2; pCS2, 空载体质粒; amhr2-pCS2, amhr2重组质粒; C, Amh和Amhr2的共定位 (a. DAPI; b. anti-Flag; c. anti-His; d. anti-Flag+ anti-His); 红色框, 白色框放大后图像, bar=50 μm

Fig. 7 Subcellular localization of Amh and Amhr2 of P. olivaceus.

(a) Amh, pcDNA3.1, blank vector plasmid; amh-pc3.1, recombinant plasmid of amh; (B) Amhr2, pCS2, blank vector plasmid; amhr2-pCS2, recombinant plasmid of amhr2; (c) the co-localization of Amh and Amhr2 (a. DAPI; b. anti-Flag; c. anti-His; d. anti-Flag+ anti-His). Red box, the enlarged image of the white box, bar=50 μm

2.6 重组Amh蛋白对牙鲆离体性腺amhr2表达的影响

利用体外原核表达获得牙鲆Amh-EGFP重组蛋白, 经SDS-PAGE电泳检测, 融合蛋白约为80 kDa, 为包涵体。WB结果显示, 未破碎菌体和破碎后菌体沉淀中存在目的条带(图8)。利用His-Tag镍柱亲和层析法进行纯化, 获得了纯度较高的重组蛋白。
图8 牙鲆Amh蛋白的原核表达及纯化

a. Amh原核表达SDS-PAGE结果(M, marker; 1, 空白培养基; 2, 实验培养基; 3, 空载菌体沉淀; 4, Amh菌体沉淀; 5, 空白菌体上清; 6, Amh菌体上清); b. 重组Amh纯化SDS-PAGE结果(M, marker; 1-7, 洗脱目的蛋白); c. Amh原核表达WB检测(M, marker; 1. 空白培养基; 2. 菌体沉淀; 3. 菌体破碎后上清; 4. 菌体破碎后沉淀；框, 重组Amh)。

Fig.8 Expression and purification of the recombinant P. olivaceus Amh.

(a) SDS-PAGE results of Amh prokaryotic expression (M, marker; 1, blank medium; 2, experimental medium; 3, control thallus precipitation; 4, Amh thallus precipitation; 5, blank cell supernatant; 6, Amh cell supernatant); (b) SDS-PAGE results of recombinant Amh purification (M, marker; 1-7, protein elution in each step); (c) WB results of Amh prokaryotic expression (M, marker; 1, blank medium; 2, Amh thallus precipitation; 3, supernatant after the bacterial fragmentation; 4, precipitation after the bacterial fragmentation. Box, the recombinant Amh)

重组Amh经复性后孵育牙鲆Ⅱ期卵巢和Ⅲ期精巢。结果显示, 卵巢中amhr2的表达在孵育后持续上升, 且在12、48h表达显著高于对照组(P< 0.05)。精巢中amhr2在孵育后12h表达略微上升, 而24和48h后表达下降, 但都没有显著性(图9)。
图9 重组Amh孵育后雌雄性腺中amhr2的表达变化

a. 卵巢组织切片及孵育12、24、48h后amhr2表达变化; b. 精巢组织切片及孵育12、24、48h后amhr2表达变化。n=4; bar=50μm; *表示不同孵育组间具有差异, P< 0.05。

Fig. 9 The changes of amhr2 expression in the ovary and testis incubated with the recombinant Amh.

(a) Histological section of the ovary and the expression of amhr2 after 12, 24, and 48 h incubation with recombinant Amh in the ovaries; (b) histological observation of the testis and the expression of amhr2 after 12, 24, 48 h incubation with the recombinant Amh in the testes. n=4. bar=50μm. * indicates the difference between different groups, P< 0.05

2.7 Amh与Amhr2对cyp19a活性的调控作用

利用双荧光素酶报告系统检测牙鲆Amh和Amhr2对cyp19a启动子的调控。结果显示, 与仅转染cyp19a启动子质粒的对照组相比, 单独转染amh-pc3.1、amhr2-pc3.1及二者共同转染极显著地抑制了cyp19a启动子的活性(P< 0.0001), 且单独转染amh-pc3.1比单独转染amhr2-pc3.1对cyp19a启动子的抑制作用更显著(P< 0.05)(图10)。
图10 牙鲆Amh与Amhr2对cyp19a的调控

不同字母代表各实验组间存在显著差异, P< 0.05。****表示对照组与各实验组间存在极显著差异, P< 0.0001

Fig. 10 The regulation of Amh and Amhr2 on cyp19a in P. olivaceus.

Different letters represent significant differences among the experimental groups, P< 0.05. **** indicates significant difference between the control group and experimental groups, P< 0.0001

3 讨论

Amh/Amhr2受体系统能够调控脊椎动物性别和性腺发育, 其中Amhr2为该系统的关键组分(Duan et al, 2021)。相关研究已在多种硬骨鱼类中展开, 如青鳉、黑鲷(Acanthopagrus schlegeli)、牙汉鱼(Odontesthes bonariensis)、腔棘鱼(Latimeria menadoensis)等(Pfennig et al, 2015)。本文克隆了雌雄牙鲆amhr2基因的CDS序列, 发现不存在性别相关SNP, 这与红鳍东方鲀、暗纹东方鲀(Takifugu obscures)等鲀科鱼类不同(高莹莹 等, 2019)。牙鲆amhr2共编码511个氨基酸, 含有一个N端信号肽、两个跨膜区和一个近C端保守的酪氨酸蛋白激酶结构域。经预测, Amhr2为亲水性不稳定跨膜蛋白, 模拟分子对接能够与牙鲆Amh互相结合, 这与Hart等(2021)利用X射线衍射技术得到人AMH与AMHR2胞外结构域相结合的结论一致。
牙鲆amhr2主要表达于性腺中, 在雌性其他组织也有微弱表达, 这与金钱鱼(Scatophagus argus)(王妹 等, 2018)和尼罗罗非鱼(赵九娥, 2015)等鱼的表达模式类似。性腺发育各时期中, 牙鲆amhr2与amh表达特征大致相同(Wang et al, 2020): 精巢发育过程中, 表达先升高后下降, 在Ⅱ期达到最高; 卵巢发育过程中仅在I期高表达, 而在Ⅱ—V期微弱表达, 也与金钱鱼类似(王妹 等, 2018)。Hela细胞系是一个商品化的细胞系, 转染效率高, 被用于多种鱼类蛋白的亚细胞定位研究, 如斑马鱼(Mindnich et al, 2005)、鲤鱼(Cyprinus carpio L.)(Shan et al, 2018)、红鳍东方鲀(Fu et al, 2018)、鳜鱼(Siniperca chuatsi)(Li et al, 2020)等。本研究在Hela细胞中通过共定位分析发现, 牙鲆Amh和Amhr2共定位在细胞质中, 与暗纹东方鲀(高莹莹 等, 2019)在线预测和小鼠(Mus musculus)(Sedes et al, 2013; Cimino et al, 2016)中利用特异抗体所得到的结果一致。Amh作为一种由Sertoli细胞表达的激素(Yoshinaga et al, 2004), 随体液与其跨膜受体Amhr2结合激活Smad蛋白磷酸化发挥功能(Josso et al, 2022)。牙鲆amhr2与amh表达时期与部位的同步性暗示了二者在性腺发育过程中共同起作用, 并通过Amh/Amhr2受体系统行使功能。
性腺分化过程中, amhr2在精巢分化过程中的表达模式与amh一致, 而与卵巢分化过程中amh的表达模式相反(Wang et al, 2020; Yamaguchi et al, 2023)。基因表达不一致的情况也发生在经雌二醇诱导的黑鲷卵巢分化中, 即早期低水平表达的amh对应了高水平amhr2, 后逐渐下调表达(Wu et al, 2010)。Yamaguchi等(2023)也在分化期遗传雌性牙鲆中检测到了相同的结果, 这可能与雌性牙鲆amhr2起始转录水平较高有关。本实验室先前研究表明, 牙鲆卵巢和精巢分别于3.8cm TL和6.3cm TL时开始分化(孙鹏 等, 2009)。在本研究中, 对照组amhr2于4cm TL时显著下调, HT组则在6cm TL前表达持续上升, 后显著下降。已有结果提示牙鲆amh可能会启动精巢分化过程(Wang et al, 2020), Hattori等(2022)也发现amhr2的表达在精巢分化前逐渐上调, 这与本研究的结果一致, 表明amhr2能够在卵巢分化启动前表达下调, 并在精巢分化启动前上调, 影响牙鲆性别表型形成。
Amh和cyp19a在虹鳟 (Oncorhynchus mykiss) (Yano et al, 2012)、鲤鱼(Wang et al, 2023)等多种鱼类中被认定为性别差异基因, 呈现两性差异表达模式。本研究发现, amh和amhr2共转染至HEK293T细胞中能够极显著地抑制cyp19a的转录。尽管目前已在多种哺乳动物和斜带石斑鱼等鱼类中证实了amh-Smads-cyp19a1a信号通路的存在(韩玉龙, 2019), 但调控关系因鱼种而异, 如在哈奇氏牙汉鱼(Odontesthes hatcheri)中敲降amhY可以上调cyp19a的表达(Hattori et al, 2012), 而在斑马鱼中, amh与cyp19a在精巢和卵巢中独立表达, 不存在调控关系(Rodriguez-Mari et al, 2005) 。目前, Amh/Amhr2受体系统对cyp19a的调控缺乏直接证据。本研究首次给出了牙鲆Amh能够通过与Amhr2结合直接抑制cyp19a启动子转录的证据, 但这只是细胞中的研究结果, 还需要进一步的体内研究予以证实。
近年来, Amh/Amhr2受体系统在牙鲆中的研究引起了关注, Hattori等(2022)的研究发现了位于amh启动子区的雄性特异缺失片段, 并通过敲除amh成功引起了F0代遗传雄性牙鲆的性逆转, 提示了amh在牙鲆中的性别决定作用。此外, Yamaguchi等(2023)对amhr2的敲除实验也获得了F0代性逆转牙鲆, 同时伴随雌性相关基因cyp19a和foxl2表达的显著上调, 并通过芳香化酶抑制剂成功逆转了amhr2缺失突变体的雌性化, 明确了Amh/Amhr2受体系统通过抑制雌激素的合成从而影响性别表型。在本研究中, amhr2在卵巢分化前表达下调, 在精巢分化前和发育过程中与amh同步高表达, 且与Amh在胞质中表达共定位, 提示amhr2通过与amh结合, 从而在牙鲆精巢的分化和发育过程中发挥作用; 同时配体Amh能够在成体卵巢中的促进amhr2转录, 并在精巢中的抑制其表达; 细胞转染进一步明确了Amh和Amhr2对芳香化酶基因cyp19a的抑制作用, 侧面佐证了二者对性别表型形成的影响是通过抑制雌激素合成实现的。
综上所述, 本文克隆了牙鲆amhr2基因序列, 预测了其二、三级结构和蛋白的化学性质与结构特点, 检测了其在各组织和性腺分化、发育中的表达特征及在细胞中的表达部位, 探究了配体Amh对其在成体性腺中表达的影响。发现amhr2在牙鲆中的表达具有组织和时期特异性, 可能与amh共同调控了精巢分化的启动和后续发育, 并通过直接抑制cyp19a的转录发挥功能, 这些研究结果对于解析牙鲆等鱼类性腺分化和发育中Amh/Amhr2受体系统的作用提供了基础数据。

References

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