2. 吉林农业大学动物科学技术学院, 长春 130118
2. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China
精子发生是在支持细胞(sertoli cell, SC)和曲细精管基底膜等构成的环境下,精原干细胞(spermatogonial stem cells,SSCs)自我更新、分化发育、减数分裂与精子变态的过程[1]。正常而高效的精子发生对于人类生殖及哺乳动物繁殖都具有重要意义。白消安(1, 4-丁二醇二甲磺酸酯)是一种烷化剂,最早出现在治疗白血病的案例中[2],后经研究发现,在利用白消安治疗癌症过程中,男性患者往往会出现不育现象[3]。目前,越来越多的研究表明,白消安等癌症治疗药物以及环境毒素都可导致睾丸和生殖道感染与炎症反应,从而影响到精子发生,是雄性不育的重要诱因[4-5]。这些诱因导致了以管周淋巴细胞浸润为特征的慢性睾丸炎症,进而损伤了血睾屏障(blood-testis barrier, BTB),危及到精子发生[6-7]。有研究表明,给青春期雄性大鼠注射双酚A,可降低处理鼠睾丸中的occludin、connexin 43和N-cadherin蛋白含量,致使BTB的紧密连接(tight junction, TJ)、缝隙连接(gap junctions, GJ)、桥粒连接(anchoring junction, AJ)等重要结构受损,增加了BTB通透性,从而导致炎症效应,损害了睾丸的正常生精功能[8-9]。用白介素-6(IL-6)处理大鼠体外培养支持细胞,会导致其occludin表达下降,引起细胞膜电阻下降,导致细胞膜通透性增加;向大鼠睾丸注射IL-6,注射后50 d左右,注射鼠逐渐表现出局灶性睾丸炎症反应,紧密连接相关蛋白occludin逐渐减少,BTB通透性逐渐增加[10]。因此推测,白消安是通过引起睾丸炎症毒害睾丸生精机能的。
近年来,为实现繁殖控制,提升有价值种公畜繁殖效率,SSCs移植受体制备越来越受关注[11-13]。SSCs移植受体制备的本质就是高效摧毁睾丸内源性生殖细胞[14]。化疗药物白消安对精子发生有较强破坏作用,是被用作制备SSCs移植受体的理想药剂。但是,最初的腹腔注射法在成功制备移植受体的同时,还伴随了较强的毒副作用,甚至可以致命[15]。所以,Qin等[16]经过深入研究,建立了睾丸内白消安注射法,获得了理想的受体制备效果,并有效避免了白消安的毒副作用,更加安全可行。但是,白消安处理对睾丸细胞的毒害机制以及白消安是否会诱导睾丸产生炎症都还不清楚。
所以,本文旨在深入探讨白消安对睾丸细胞和BTB的毒害作用,为探讨化疗药物和环境毒素作用下雄性生精机能的科学防护与恢复,建立高效安全的受体制备技术提供科学指导。还将促进对哺乳动物精子发生机制的探索和人类生殖医学的研究。
1 白消安对血睾屏障的损伤研究 1.1 血睾屏障及其作用睾丸是一种免疫特权器官,通过形成BTB保护自身免受由内源底物和外源污染物引起的免疫反应伤害[17]。早期研究表明,BTB主要由曲精细管基底膜与相邻的支持细胞共同组成,该特殊结构能对进入睾丸的营养物质、重要调控分子进行筛选和调控,对精子发生起到一定的营养和调控作用[18];同时,还可防止有害外源污染物进入曲细精管,干扰精子发生或损害已形成的精子[19-20];更重要的是,该结构还能防止自身抗体对精子免疫导致的炎症性反应,为精子发生提供免疫豁免环境[21-25]。
近年来,随着研究的逐渐深入,对BTB的理解也逐渐走向精确。有研究表明,BTB并不包含基底膜,而是一种位于曲细精管中前细线期精母细胞(preleptotene spermatocyte)与粗线期精母细胞(pachytene spermatocyte)之间的结构[26],由支持细胞间及支持细胞和生精细胞间形成的外质特化(ectoplasmic specializations, ES)、TJ、GJ和AJ共同连接构成[27-28]。其中,ES是在支持细胞和特定生殖细胞间(生精上皮周期第Ⅷ阶段后)或相邻支持细胞间,通过肌动蛋白丝相连形成的一种特殊连接结构[29-30],主要是支持细胞与变形期或变形后精子细胞间的连接方式,是SSCs与支持细胞机械黏附的基础,可协助单倍体精子沿正确的发育方向(而不是相反或其他方向)穿越BTB,并使单倍体精细胞逐渐发育完全并向管腔释放[31];在基底部则可借助其粘附作用使各种BTB连接方式紧密结合在一起,对维持BTB稳态起重要作用[32-33]。紧密连接是介于前细线期至粗线期精母细胞间的两个相邻支持细胞的紧密接触点[34],该连接具有严格的选择性通透功能,可根据睾丸生精功能需要,决定通过BTB进入曲细精管近腔小室区域的水、溶质大分子的质与量,其结果是限制了BTB内外侧间蛋白质和脂质的运动,防止了因电荷差引起的细胞极性[35],维护了BTB的特异功能。间隙连接位于相邻支持细胞间及支持细胞和特定生殖细胞(生精上皮周期第Ⅷ阶段之前)间,主要通过间隙连接相关蛋白组成的连接复合体相连,可允许代谢物、第二信使和其他分子量小于1 ku的分子通过,以及离子扩散(ion diffusion)[36-37]。AJ是通过中间丝(intermediate filament,IF)在支持细胞与特定生殖细胞(生精上皮周期第Ⅷ阶段前)间形成的“桥粒样”连接,赋予细胞间粘附力,是重要的信号转导结构[38]。
上述研究使对BTB的认识由泛泛的基底膜与支持细胞组成,深入到了维护微环境的4种连接方式。然而,深入分析发现,ES、GJ和AJ结构,几乎遍布了整个曲细精管,4种BTB连接方式将生精上皮分成基底小室和近腔小室两个重要功能区的划分变得不可操作,也背离了BTB是睾丸生精功能重要保护屏障的定义初衷;同时,几种连接方式都与生精上皮周期第Ⅷ阶段后生精细胞及其相邻支持细胞相关[39],这一细胞阶段较曲细精管基底部的距离已经很远,这样的划分使BTB很难实现其生精功能天然保护屏障的功能目标;另外,曲细精管中BTB无时不刻不存在,但是,目前研究仅以生精上皮周期第Ⅷ阶段的各种连接成分作为BTB的存在标志,不利于深入进行生精过程中BTB作用的研究。基于前人研究发现的4种连接的分布与功能,我们推测,BTB可能就是TJ及与其紧密接触的部分GJ、AJ和ES,指位于前细线期至粗线期精母细胞间相邻支持细胞间特定的各种紧密接触的连接形式。
1.2 白消安对血睾屏障的破坏作用健全的BTB是精子高效正常发生的必要保证,尤其是TJ,它能瞬时打开,使第Ⅷ~Ⅺ阶段生殖细胞迅速穿过BTB进入腔内隔室,还能维持精子发生稳态[26]。现已发现occludin、claudin-1、JAM-A、JAM-B、ZO-1、ZO-2、vinculin等多种紧密连接相关蛋白,这些蛋白对维持哺乳动物BTB稳态发挥了重要调节作用[40-43]。白消安处理小鼠等动物,都可导致BTB蛋白成分的损失,进而影响精子发生。大鼠腹腔注射白消安1周后,ZO-1和波形蛋白含量显著降低,直至第4周试验结束前仍然低于DMSO组,生殖细胞也发生凋亡,示踪试验表明BTB被破坏[44];给DPT过表达小鼠腹腔注射白消安,可降低BTB相关蛋白ZO-1和claudin-11含量,导致生殖细胞凋亡[45]。而敲除Occludin 11及claudin基因后,小鼠精子发生停滞于减数分裂期,无法正常进行后续变形与成熟过程[46]。关于睾丸炎症反应的研究表明,向大鼠睾丸注射睾丸匀浆50 d后,成功诱使试验鼠发生自身免疫睾丸炎,曲细精管间出现少量炎性浸润,管腔内出现少量细胞坏死病灶;示踪试验显示,曲细精管内可见到生物素示踪剂(EZ-Link Sulfo-NHS-LC-Biotin),表明BTB功能被破坏,Western blotting结果发现,紧密连接相关蛋白occludin含量显著减少,进一步验证了BTB受损[10]。睾丸炎试验证明,炎症发生伴随了BTB的破坏。白消安试验表明,白消安破坏了BTB的细胞连接,并导致生精细胞大量凋亡,但不知白消安处理是否也诱导了睾丸炎症,因此,BTB的破坏是否与炎症反应有关还需要进一步的探索。
2 白消安对睾丸细胞相关功能蛋白的影响支持细胞是构建BTB的重要结构基础,并与睾丸间质细胞等构成了多种蛋白和细胞因子的强大合成与分泌系统,调控和维护了生精必须的免疫豁免环境[47],并根据精子发生的不同阶段需求,分泌亚精胺/精胺N1-乙酰转移酶2(Sat2)、雄激素结合蛋白(ABP)、转铁蛋白(transferrin)、抑制素B(inhibin B)、激活素(activin)等物质。这些物质在精子发生过程中特异性节律分泌,对生精细胞的增殖、分化、发育等发挥了重要的阶段性调控作用[48-49]。例如,Sat2是支持细胞分泌的一种蛋白,对正常精子发生有一定调节作用,小鼠腹腔注射白消安可使Sat2过表达,G1期细胞比例显著增加,降低了S期细胞比例,表明Sat2过表达阻止细胞周期进展;蛋白质组学分析显示,Sat2过表达通过修饰翻译和蛋白质复合物亚基组织改变,损害细胞周期进程、细胞通讯和蛋白质代谢过程[50]。白消安处理不仅可损害支持细胞,还可损伤管周肌样细胞(PMC),影响其对雄激素受体(AR)、肌样细胞旁分泌因子(P-Mod-S)等支持细胞功能调控因子的合成[51-53],并使PMC数量减少,进而影响支持细胞功能和营养供给,损害精子发生[54]。
睾丸间质中还存在巨噬细胞,可分泌IL-1A、IL-6、和IL-10等淋巴因子,担负着BTB以外睾丸的免疫防护作用[55]。对野生型(WT)小鼠、TLR2和TLR4敲除小鼠腹腔注射白消安后,处理鼠均表现出生精细胞凋亡,同时,睾丸间质中出现巨噬细胞浸润现象[4]。大鼠睾丸注射IL-1α(每个睾丸用量为250 ng)后,超量的IL-1α可使细胞骨架F-肌动蛋白(F-actin)含量下降,导致支持细胞塌陷,并且,在近腔小室中可发现菊粉-异硫氰酸荧光素(inulin-fluorescein isothiocyanate)示踪剂,表明超量的IL-1α损害了BTB结构,导致了睾丸炎症,影响精子发生[56];大鼠睾丸注射IL-6(每个睾丸用量为1 μg)后,可观察到睾丸组织曲精细管出现炎症病灶,间质出现炎性细胞浸润,白细胞数量显著增加[57];向体外培养的大鼠支持细胞添加IL-6,可显著降低支持细胞TJ屏障的跨上皮细胞电阻(TER),增大支持细胞屏障通透性,激活了ERK-MAPK(MAPK14)信号通路,据此推测,IL-6可能激活了MAPK14通路,导致了睾丸炎症,损害了TJ[58]。对睾丸巨噬细胞缺乏IL-10的小鼠和病人的炎性反应研究表明,注射IL-10(每个睾丸用量为0.2 μg,正常睾丸中内源性IL-10浓度范围),可阻断巨噬细胞代谢,促进氧化磷酸化,抑制炎症反应和生精损伤[59]。间质中的巨噬细胞通过淋巴因子分泌量的调节,调控了睾丸的生理与病理状态。
粗线期精母细胞和圆形精子细胞分泌的肿瘤坏死因子TNF-α也可通过支持细胞受体,调节支持细胞分泌功能、能量代谢及炎症反应。且白消安处理后,各阶段生殖细胞均发生凋亡现象,包括精母细胞以及圆形精子细胞[44]。研究表明,大鼠注射TNF-α后,可降低其TJ相关蛋白occludin和ZO-1含量,激活p38-MAPK通路,损害BTB[60]。支持细胞和生殖细胞分泌的转化生长因子TGF-β3,也对细胞生长、分化和免疫功能有重要调节作用。小鼠注射TGF-β3后,TGF-β3和TNF-α含量都发生变化,可降低睾丸紧密连接相关蛋白occludin和JAM-A含量,诱导BTB的可逆性损伤[61-62]。TNF-α和TGF-β3都通过炎症通路损伤BTB,影响精子发生。
综上所述,白消安处理可能通过影响生精细胞、支持细胞、管周肌样细胞等的调控关系,改变巨噬细胞的功能状态,影响生精机能。另外,在本课题组之前研究的组织切片中观察到了类似于病灶以及炎性细胞浸润的现象[16]。所以,有必要进一步探索白消安处理与细胞因子变化及睾丸炎症的关系,这将有助于深入揭示白消安对睾丸的损害机制。
3 缓解白消安处理后睾丸损伤的保护措施目前,使用白消安治疗癌症已经越来越普遍,且生存率和治疗成功率逐渐升高。但是,白消安治疗通常会导致生育能力降低。所以,白消安治疗后生育能力的保护问题已引起高度重视。研究表明,白消安可导致内质网应激,诱导产生活性氧(ROS),引发生殖细胞凋亡和睾丸损伤。褪黑素(melatonin)作为松果体合成分泌的吲哚类激素,具有抗氧化、抗炎和抗肿瘤等作用,可通过抑制生殖细胞内质网应激,减少ROS产生,抑制小鼠睾丸氧化应激和精原干细胞凋亡,保护睾丸免受损伤[63-64]。白消安处理后,注射人参皂苷也能明显提高SSCs数量,提高生存能力,并缩短白消安处理后雄性小鼠生殖能力恢复时间[65]。此外,维生素也已用于缓解白消安毒性研究,特别是维生素B12(钴胺素)。为缓解白消安处理引起的PMC数量减少,及导致的支持细胞功能和营养供给损伤,损害精子发生,有试验补充维生素B12刺激精原细胞增生,防止了白消安诱导的生殖细胞死亡[54]。
白消安处理通过诱导生殖细胞凋亡和睾丸损伤,导致雄性不育,上述对于白消安处理后的保护研究所使用的药物均具有抗氧化、抗炎功能,这些药物可有效地降低白消安处理后ROS的产生,且还有可能抑制了炎症反应。所以,探索白消安处理是否会导致炎症,可能有助于开阔有效防控白消安处理引起睾丸损害的思路。
4 结语以往关于BTB的检测研究多集中于生精上皮周期的第Ⅷ阶段,但BTB无时不刻都存在,对于其他阶段BTB以及BTB与TJ、GJ、AJ和ES关系的研究,将会促进BTB结构与功能的深入揭示。另外,对于白消安处理引起的睾丸损伤研究表明,白消安可能是通过破坏BTB的细胞连接,并改变睾丸内炎症细胞因子含量,诱导了睾丸炎症,导致生精细胞大量凋亡。所以,探索白消安对BTB和细胞因子的影响,并研究相关信号通路在减轻或预防睾丸损伤和炎症中的作用,将从炎症角度揭示白消安对精子发生的毒害机制,促进高效安全SSCs受体制备技术研发,并将为治疗性及环境性毒物的防护提供理论指导。
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