2. 吉林农业大学生命科学学院, 长春 130118
2. College of Life Sciences, Jilin Agricultural University, Changchun 130118, China
睾丸支持细胞(Sertoli cells,SCs)附着于曲细精管基膜上,是唯一一种与生精细胞直接接触的体细胞,为精原干细胞(spermatogonial stem cells,SSCs)提供生长、发育和自我更新所需的各种细胞因子,并为精子发生提供物理支撑和稳定微环境[1]。小鼠睾丸的SCs均匀地分布在曲细精管中,曲细精管底部的SSCs与SCs以玫瑰花型相连,相邻SCs之间、SCs与SSCs及系列生精细胞之间通过紧密连接、缝隙连接等相互作用的结构关系, 共同维持SSCs的分化、发育、自我更新及精子发生过程[2]。精子发生是个高效、持续、有序的生理过程,SCs与SSCs的结构关系是该过程有效发生的必要条件[3-4]。近年来,借助移植及细胞生物学技术,通过深入研究不同睾丸微环境中SSCs分化、发育和自我更新,探索SSCs复杂有序的发育分化过程以及SCs作为特定微环境的主要构成元素对SSCs分化的影响[5-6]。
1 SCs为SSCs分化发育提供所需的环境SCs呈高度不规则锥体形,侧面和腔面有很多不规则凹陷,镶嵌着各级生精细胞,基部紧贴基底膜,顶部一直伸达腔面。细胞核大都位于基底部,核孔多,核质较均匀[7]。胞质内有较丰富的高尔基体、线粒体、粗面内质网和滑面内质网。巨大的表面积为SCs维持较多生精细胞生存及发挥其相应生理功能提供了结构基础。相邻SCs胞膜通过紧密连接,形成血睾屏障(blood-testis barrier,BTB),将生精上皮分成基底室和近腔室两部分[8-9]。基底室位于SCs紧密连接与基底膜之间,内有精原细胞,而近腔室则位于连接上方的管腔侧,内有细线期之后的初级精母细胞、次级精母细胞及精子细胞等系列生精细胞。基底室内SCs与SSCs相互识别,相互作用,调节SSCs的更新、分化、发育并为其提供营养[10-11]。
SCs是精子发育的基础[12-14],其数量和功能上的任何改变都会影响SSCs的自我更新、发育和分化。由SCs参与形成的BTB,使曲细精管内形成了适于精子发生的局部免疫豁免区域,使SSCs自我更新、发育和分化免受免疫系统干扰。BTB由SCs产生和维持,与微血管距离很远,除紧密连接外,还存在外质特化(ectoplasmic specialization,ES)和管球复合体(tubulobulbar complex)两类结构(图 1)[15]。
外质特化是通过肌动蛋白丝在支持细胞和精子细胞间或支持细胞间形成的黏附结构,分为近腔外质特化和基底外质特化,近腔外质特化的复杂细胞骨架结构形成于血睾屏障和生精细胞顶端黏附位点下区域[16-17],是生殖生理学研究的热点,基底外质特化由夹在SCs质膜和内质网池之间的肌动蛋白丝组成,与连接位点密切相关,在维持细胞间连接方面起重要作用[18-19],生殖细胞和SCs间直接连接方式为桥粒/半桥粒连接(一种常见的细胞连接结构,图 1),作为一种重要的锚定连接装置,外质特化使SSCs实现了对滋养性SCs的机械黏附,并协助发育精子以正确的方向穿越生殖上皮,使其逐渐发育完全并向管腔释放[20]。外质特化的动力学作用是通过紧密链接实现的,是目前唯一已知的闭锁链接模型,在精子成熟和释放过程中起关键作用,研究显示,一种名为SPIRE-1的肌动蛋白对紧密链接具有调控作用,成年雄性Sprague-Dawley大鼠Spire-1基因敲除后,其生精上皮肌动蛋白微丝被破坏,外质特化无法对精子发生过程发挥调控作用,使精子形成受阻[21]。管球复合体是生精上皮顶端和底端复杂的细胞连接结构,其中,管腔端许多精细胞头质膜突出进入相邻SCs质膜,使SCs质膜呈管状内陷,并与SCs肌动蛋白丝相连,形成顶端管球复合体,是促使精子细胞脱除细胞质逐渐成形的功能性结构[22-23],其末端和许多溶酶体囊泡相关联,有助于精子细胞质脱除。同时,该结构将精子细胞锚定在上皮细胞上,并直接参与了精子头部的形成和成形精子的释放[24-25]。SCs中还存在波形蛋白,由SCs核膜向细胞质呈辐射状排布,使SCs与相邻生殖细胞间及SC与SC间形成桥粒连接(细胞间的一种纽扣样连接方式)。将白消安注射到大鼠睾丸22 d后,发现波形蛋白逐渐萎缩,生殖细胞与SCs分离并从曲细精管流失,曲细精管中只剩SCs[26]。
2 SCs的调控作用SCs一方面在结构上为生精细胞分化成熟提供支架,另一方面,还分泌胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor,GDNF)、视黄酸(retinoicacid, RA)等各种蛋白质调节生殖细胞自我更新、分化发育的相关信号通路;另外,SCs通过表达FasL(factor associated suicide ligand)、转化生长因子β(transforming growth factor-β,TGF-β)等蛋白,促进局部免疫豁免的形成,为精子发生提供了稳定的微环境;SCs能够吞噬、消化精子形成过程中脱落的残余胞质,维持正常发育生精细胞的比例。
2.1 SCs对SSCs的增殖调控GDNF属于TGF-β家族的一个分泌型蛋白,是维持SSCs自我更新和增殖的重要因子。GDNF敲除试验发现,GDNF-/-小鼠可以存活,但随着生长发育,敲除鼠曲细精管中的SSCs逐渐减少,到5周龄时,SSCs已完全检测不到;GDNF+/-小鼠成年早期可发育,但生长到5周龄时,睾丸曲细精管内精原细胞逐渐减少,并伴随精子数量逐渐下降,到12月龄,SSCs全部分化,曲细精管内失去了具有自我更新能力的SSCs,只剩SCs,致使小鼠精子生成能力逐渐丧失;而对照组GDNF+/+小鼠并未出现以上现象[27]。对小鼠的毒性试验显示,腹腔注射白消安(10 mg·kg-1)第3周开始,注射组GDNF表达量明显升高,曲细精管内只检测到SCs和精原细胞;第4周注射组GDNF表达量逐渐降低,直至低于对照组水平,并且,曲细精管中开始出现精母细胞;第8周GDNF表达量恢复正常,注射组各级生精细胞数量与对照组无显著差异,注射组生精能力和GDNF含量变化呈白消安依赖性[28]。腹腔注射白消安后,GDNF高表达促进了SSCs增殖,间接抑制了SSCs分化,减少了精子生成,然而,白消安提高GDNF表达的机制尚不清楚。
GDNF是通过SSCs表面受体胶质细胞源性神经营养因子受体α1(glial cell line derived neurotrophic factor receptor alpha1,GFRα1)和受体酪氨酸激酶(receptor tyrosine kinase,RET)实现的[29],GFRα1作用于糖基磷脂酰肌醇锚定蛋白,使GDNF与RET结合,根据机体需要,诱导PI3K/AKT通路、ERK1/2(extracellular regulated protein kinases)、SRC家族激酶通路、蛋白激酶C(protein kinase C,PKC)、促分裂原活化的蛋白激酶(mitogen activated protein kinase,MAPK)等相应信号转导途径的信号级联反应[30-33]。其中,对PI3K/AKT信号通路的研究最广泛深入,PI3K通过RAS和P110直接结合而活化,使质膜上产生第二信使PIP3,PIP3与胞内含有PH结构域的信号蛋白AKT和3-磷酸肌醇依赖性蛋白激酶1(phosphoinositide-dependent protein kinase-1,PDK1)结合,促使PDK1的磷酸集团转移到AKT的308位Thr上,AKT的磷酸化导致了AKT的自身活化。同时,PDK2也可使AKT的Ser473磷酸化而激活AKT[34-35]。AKT的活化可上调转录因子Ets差异基因5(Ets variant gene 5,Etv5)、Pou3f1(POU domain,class-3 transcription factor 1)等转录因子的表达,促进SSCs自我更新[36]。Etv5是SCs分泌的一种转录因子,能促进GDNF受体RET合成,确保GDNF信号通路由胞外向胞内传递[37]。对比Etv5-/-小鼠和Etv5+/+小鼠的睾丸发现,Etv5-/-小鼠的SSCs明显缺失,出现唯支持细胞综合征(SCOS)和无精子症[38],说明Etv5是GDNF通路中维持SSCs增殖更新的重要效应因子。Pou3f1是转录因子OCT结合家族成员,对Pou3f1基因表达的RNA干涉表明,Pou3f1表达水平降低的同时,也伴随了SSCs数量的显著减少,说明Pou3f1对SSCs增殖同样具有重要调控作用[39](图 2)。除GDNF外,SCs分泌的成纤维细胞生长因子2(fibroblast growth factor 2, FGF2)也是SSC的增殖调控因子,FGF2通过与膜上成纤维细胞生长因子受体(fibroblast growth factor receptor, FGFR)结合后,调控Etv5的表达水平,促进SSCs的增殖(图 2)。
SCs可表达维生素A衍生物视黄酸(RA),RA与SSCs表达的RA受体RARα(retinoic acid receptor α)结合,反馈作用于SCs,并使其表达BMP4(bone morphogenetic proteins 4),BMP4与SSCs膜上的骨形态蛋白受体BMPR-ⅠA(bone morphogenetic protenis receptor ⅠA, 又名ALK3)结合,诱导SSCs表达酪氨酸激酶受体c-kit,c-kit与SCs分泌的干细胞因子(stem cell factor,SCF)结合,促进精原细胞分化[40](图 2)。
BMP4是TGF-β家族成员,小鼠出生后,SCs即开始表达。BMP4先与SSCs表面的丝氨酸/苏氨酸激酶受体(TβRⅡ)结合,再与TβRⅠ结合形成复合物,激活TβRⅠ的蛋白激酶,使SSCs细胞内Smad1/5/8磷酸化,磷酸化的Smad1/5/8与共用型调节蛋白Smad4(CO-Smad4)结合形成Smad复合物,该复合物进入细胞核与DNA结合,上调靶基因Sohlh2的特异性转录表达[41],进而促进下游c-kit蛋白表达[42-43]。c-kit与SCs分泌的SCF结合[44],激活PI3K→AKT→mTOR→P70S6K→CyclinD3→CDK4/6→RB p42/p94信号途径,促进G1期精原细胞向S期过渡,诱导SSCs分化。同时SCF/c-kit系统还通过激活MEK→ERK1/2→CyclinE/A2→CDK2通路,拮抗RB磷酸化,负调控SSCs分化[45]。成视网膜细胞瘤蛋白(retinoblastoma protein, RB)为成视网膜母细胞瘤抑制蛋白,可通过磷酸化和脱磷酸化调节细胞周期[46]。RA通过PI3K和MEK正负两条调控通路精确控制了生精过程,两条通路相互独立,互不交叉。而毒性试验表明,白消安处理抑制了CDK2、CDK4、Cyclin E的信号传导功能,影响到小鼠睾丸SCF/c-kit的两条调节通路,导致精子发生障碍[47]。
2.3 SCs的免疫豁免调控睾丸免疫豁免作用的发挥除与BTB有关外,还与SCs分泌的TGF-β和FasL等调节因子有关。根据小鼠膀胱感染可引起睾丸上行性感染原理,将能引起尿道炎的溶脲脲原体(ureaplasma urealyticum,UU)注入DBA/C57b L小鼠膀胱,进行小鼠睾丸感染分析,在第1、2、3周进行FasL和TGF-β基因的RT-PCR检测分析表明,UU感染组与空白对照组比,这两个基因的转录表达逐渐升高,达显著水平后又逐渐恢复到原有水平;对1~3周试验鼠睾丸组织切片进行HE染色发现,对照组生精小管上皮结构基本完整,生精细胞排列整齐,间质内无淋巴细胞浸润;而感染组生精上皮结构混乱,间质中有明显的淋巴细胞浸润,但曲细精管中未见到淋巴细胞[48]。推测FasL和TGF-β基因表达清除了浸润的淋巴细胞,维持了睾丸的免疫豁免作用。为进一步研究TGF-β和FasL与免疫系统的相互作用,将NOD(非肥胖型糖尿病)小鼠SCs与胰岛共培养后,进行同种异体移植,胰岛能在体内存活60 d,而单独移植的胰岛平均存活仅9 d。小鼠血浆TGF-β水平检测发现,共培养胰岛移植后,TGF-β含量((3.73±0.31) ng·mL-1)显著高于单独移植受体鼠((1.97±0.22)ng·mL-1),而移植的共培养胰岛中注入抗TGF-β单克隆抗体后,胰岛移植物只能存活7~17 d[49],说明TGF-β抑制了受体对胰岛移植物的免疫反应。进一步研究表明,Th1主要分泌IFN-γ、IL-2促进T淋巴细胞增殖,介导细胞免疫,而Th2主要分泌IL-4、IL-10,可抑制细胞免疫,促进免疫豁免的形成。SCs分泌的TGF-β使Th1淋巴细胞转变为Th2淋巴细胞,促进了睾丸组织中免疫豁免的形成[50]。同时,将SD大鼠睾丸SCs与脾细胞共培养,以脾细胞单独培养为对照,发现共培养组脾细胞数量显著减少,用SABC法测定SCs的FasL表达发现,共培养组SCs有FasL表达,而脾细胞单独培养组未检测到FasL表达,推测SCs与脾细胞共培养时,SCs表达的FasL与淋巴细胞表面的Fas结合,并杀伤了共培养的活性淋巴细胞[51]。因此推断,睾丸内的SCs通过表达并分泌FasL,与T、B淋巴细胞膜上的Fas受体结合,再与Fas相关死亡域蛋白(fasasso ciating protein with death domain,FADD)聚合并活化FADD,使FADD与Caspase-8(含半胱氨酸的天冬氨酸水解酶8)结合后,激活Caspase-3引起淋巴细胞凋亡,促进睾丸内免疫豁免的形成[52],其过程可简单表示为FasL→Fas→FADD→Caspase-8→Caspase-3。
2.4 细胞的凋亡调控Fas/FasL诱导淋巴细胞凋亡的机制使睾丸内形成了局部免疫豁免,而睾丸内生精细胞也可表达Fas,机体可根据特定生理需求,通过SCs表达FasL, 并利用FasL/Fas信号途径诱导生精细胞凋亡[53],实现SCs对系列生精细胞的凋亡调控。睾丸中除了以Fas/FasL系统为代表的细胞外凋亡途径外,还存在原癌基因蛋白53(proto-oncogeng protein 53,p53)和B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)蛋白等胞内凋亡途径。
四倍体粗线期精母细胞可表达p53,其目标基因是原癌基因蛋白21(p21),而p21是细胞周期素依赖性激酶(cyclin dependent kinase,CDK)的抑制因子,p21与CDK结合,阻止Rb磷酸化,导致p53→p21→CDK→RB→E2F→Bax→CytC→Apaf-1→Caspase-9→Caspase-3等一系列反应,阻滞细胞周期相关基因转录,导致生精细胞凋亡[54-55]。另一方面,生精细胞质中还存在可溶性蛋白Bcl-2相关x蛋白(Bcl-2 associated x protein,Bax),Bcl-2和Bax分别是Bcl-2家族代表性的抑制凋亡和促进凋亡基因,Bcl-2位于线粒体膜,Bax位于细胞质,Bcl-2/Bax比例的改变对该凋亡途径发挥了重要调控作用[56],如果Bcl-2表达下降,Bax表达升高,Bax/Bax同源二聚体增多,Bcl-2活性下降,导致线粒体膜通透性增加,使CytC从线粒体内膜释放,激活半胱氨酸蛋白酶,诱导细胞凋亡,该过程:Bax→CytC→Apaf-1→Caspase-9→Caspase-3[57-58]。生精细胞可根据生理需要通过p53、Bax对生精细胞进行胞内途径凋亡调控(图 2)。
2.5 SCs的吞噬调控SCs具有吞噬生殖细胞残余体的功能,这对于精子生成具有重要意义。生精过程中,大约只有25%的精原细胞能够发育成精子,其余75%的生精细胞则走向凋亡,作为具有吞噬功能的上皮细胞,SCs能够吞噬并降解生精细胞凋亡残体,并形成脂滴,产生能量[59]。同时,凋亡细胞及时清理,也避免了曲细精管内因垃圾堆积影响细胞间信息传递及相关生理功能的正常发挥。细胞凋亡残体未及时清除还将裂解产生有毒物质,毒害其他健康细胞。研究表明,睾丸炎患病个体,其感染的革兰阴性菌裂解产生的LPS会激活TLR4信号通路,诱导细胞因子TNF-α表达,抑制Gas6和Mer表达,阻止向SCs传递吞噬凋亡生精细胞信号,从而抑制SCs的凋亡残体吞噬功能,如果生精细胞凋亡残体没有被及时吞噬,也会诱导曲细精管产生内源性炎症反应,扩大对生精细胞的损害[60]。睾丸内SCs接到吞噬信号后,凋亡细胞膜上的磷脂酰丝氨酸(phosphatidylserine,PS)与清道夫受体B类Ⅰ型(scavenger receptor class B type Ⅰ,SR-BⅠ)第33、191位氨基酸结合,活化胞内的吞噬衔接蛋白(engulfment adapter protein,GULP),使丝裂素活化蛋白激酶p38(p38MAPK)、细胞外信号调节蛋白激酶(extracellular signal regulated kinase,ERK)磷酸化,从而活化AKT(又名RAC),使肌动蛋白骨架重排,吞噬凋亡细胞[61]。
3 小结在精子发生过程中,SCs发挥了重要的调控作用,首先,桥粒、外质特化、管球复合体等特殊物理结构为确保健康高效的精子发生提供了基本的物理支撑和调控基础,这些特殊物理结构的深入研究将为揭示二者间的调控机制提供重要参考。同时,SCs通过增殖分化因子合成与分泌,调控了SSCs的自我更新与分化发育,还通过免疫与凋亡调控以及吞噬作用,确保了精子发生的健康有序。对二者相互作用机制研究的深入,将对治疗临床男性不育及提高动物育种效率等方面具有积极意义。
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