白消安对睾丸的毒害作用及机制探讨

白消安(busulfan)对精子发生具有较强毒害作用,可导致雄性不育,是制备精原干细胞(spermatogonial stem cells, SSCs)移植受体的理想药剂。睾丸炎对生精机能也有重要影响,甚至能导致雄性不育。然而,白消安损害血睾屏障(blood-testis barrier, BTB),影响精子发生的机制尚不清楚,尚不知其是否会导致非传染性睾丸炎症,并影响细胞因子分泌,损害生育能力。因此,本文综述了白消安对BTB的破坏作用、对睾丸细胞相关功能蛋白的影响,以及白消安损伤睾丸的缓解方法,以深入揭示白消安对睾丸细胞和BTB功能与结构的毒害作用,为研发高效安全的SSCs移植受体制备技术,以及化疗药物治疗和环境毒素作用下生精机能科学防护与恢复提供科学参考。     

白消安致小鼠睾丸损伤的机理及其调控通路的研究进展

白消安因其具有较强精子毒害作用常被用于制备精原干细胞移植受体,但具体毒性机制尚不清楚,影响了其科学使用及效果提升。研究表明,白消安可引起生精细胞产生氧化应激、炎症反应,并抑制细胞自噬、损伤血睾屏障,毒害睾丸生精机能,但其相互之间的关系及调控通路尚不明确。基于核转录因子κB(nuclear transportation factorκB,NF-κB)通路在炎症反应中的重要调控作用,笔者综述了白消安引起的氧化应激、炎症反应,并损伤血睾屏障及抑制睾丸细胞自噬的研究结果,分析了NF-κB信号通路在白消安毒害睾丸中可能发挥的调控作用,以期深入揭示白消安毒害精子发生的分子机理,为科学使用白消安和避免环境毒素伤害提供参考。     

白消安对小鼠睾丸损伤机理的研究

旨在揭示白消安处理对睾丸的损伤机理,提高精原干细胞（SSCs）移植受体制备效率及安全性,缓解或避免因白消安毒性导致的雄性不育。对白消安处理小鼠进行睾丸生物素示踪试验,以验证血睾屏障（BTB）完整性;采用液质联用靶向测定其附睾尾精液中白消安的含量,以验证白消安是否可以直接穿过睾丸BTB进入曲细精管;并对小鼠血清进行炎性因子ELISA测定,以验证其浓度是否发生变化。结果发现,小鼠睾丸注射白消安后,曲细精管内最早24 h可检测到生物素示踪红色荧光,表明BTB已被破坏;注射白消安30 min后,附睾精液中检测到最高水平白消安,随后,其水平逐渐降低;小鼠睾丸注射白消安后,血清中TNF-α、IL-1β和IL-6浓度逐渐增加,36 h后均显著高于对照组（P<0.05）,21 d后达到峰值时,TNF-α浓度为（1 855.51±10.32） pg·mL^-1,IL-1β浓度为（293.59±3.34） pg·mL^-1,IL-6浓度为（340.30±12.55） pg·mL^-1,随后逐渐下降。综上表明,白消安可自由穿越BTB,且于24 h生物示踪素可突破BTB,血清炎性因子TNF-α、IL-1β和IL-6浓度逐渐升高并达显著水平,在21 d达到最高后逐渐下降。     

家畜精原干细胞移植研究进展

精原干细胞（spermatogonial stem cells，SSCs）是精子发生的前体细胞。精原干细胞的移植首先由Brinstre等（1994）报道，他们先将可育小鼠的混合生精细胞移入不育小鼠的曲精细管，结果在受体睾丸内产生了具有受精能力的精子。随后叉将大鼠生精细胞移入免疫缺陷小鼠的曲精细管，结果在小鼠睾丸内产生了大鼠的精于，从而实现了跨物种产生精子的重大突破。精原干细胞移植技术的建立为生殖细胞功能研究、优良物种保存、转基因动物的培育及家畜的遗传育种等开创了新的途径。     

精原干细胞的分离纯化方法

精原干细胞(spermatogonial stem cells, SSCs)是永久分化成精子细胞的克隆源, 既可以自我更新生成新的干细胞,又可以增殖分化形成各阶段的生精细胞直至成熟的精子. 由于精原干细胞可以进行体外培养、转基因操作、异体或异种移植重塑精子发生,以及可以 冷冻保存,所以在雄性不育治疗、转基因动物生产、遗传资源保护等方面有着广阔的应用前 景,成为新的研究热点.SSCs的分离富积是对其研究与应用的必要前提条件.然而,SSCs在 睾丸中所占比例极低,选择有效的分离纯化方法,获得富积的SSCs就成为推动其研究的关键 步骤.近年来,随着SSCs移植功能试验的建立,有关SSCs的分离纯化研究取得了长足进步,本文就此作一综述.     

精原干细胞标记及活体追踪的研究进展

研究表明,精原干细胞（spermatogonial stem cells,SSCs）具有重建不育个体精子发生的能力。采用细胞标记试剂标记精原干细胞,可以活体追踪移植至睾丸的精原干细胞命运,即在发育过程中的增殖、运动及分化情况。标记和追踪精原干细胞将有助于了解精子发生恢复的机理并选择最优的移植策略。     

白消安对性成熟公鸡睾丸发育的影响

将96只165日龄健康公鸡随机分为3组,分别行腹腔内单次注射法注射白消安0、50、60 mg/kg鸡重,分别在白消安处理后5、10、15、20、25、30 d研究其对鸡睾丸形态和曲细精管组织学方面的影响.结果表明,白消安(50 mg/kg、60 mg/kg)可显著降低鸡的睾丸质量并严重损伤鸡睾丸曲细精管和生精细胞,是制备精原干细胞移植受体的合适剂量.     

以KnockoutTM SR无血清培养基培养小鼠精原干细胞的研究

精原干细胞(SSCs)是睾丸内具有自我复制和分化为精子潜能的干细胞,它的体外培养是精子发生机理研究和制作转基因动物等的新途径[1-2]。近几年的研究表明,SSCs在体外的自我增殖需要胶质细胞源神经营养因子(GDNF)和饲养层细胞等的支持[3-10],并且睾丸支持细胞和血清均可导致培养的     

精原干细胞的体外培养及其潜在治疗应用价值

精原干细胞(SSCs)能够自我更新,产生大量分化的生殖细胞并在出生以后形成精子,将遗传信息传递给下一代。因为所有雌性生殖干细胞在出生前已停止增殖,所以SSCs是成年哺乳动物唯一的生殖干细胞。将有生育能力的雄性供体睾丸细胞移植到不育症雄性睾丸细胞中,不育症的雄性可以重新进行精子发生和恢复生育能力。这项移植技术已经用来研究SSCs的生物学特性。研究发现,精原干细胞可以在小鼠的曲细精管进行复制,然后迁移,精原干细胞所需自我更新的生长因子保存在哺乳动物中。因此相信该培养技术很快会被应用于人类的精原干细胞中。本文讨论了现有的和潜在的运用移植技术和体外培养精原干细胞的方法。由于辅助的生殖技术能使精子细胞进入卵母细胞使之受精,所以在体外培养分化精原干细胞使之成为成熟生殖细胞的技术,将会促使人的临床精原干细胞的应用得到长足发展。     

Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.     

精原干细胞的分选、培养及转基因应用探讨

精原干细胞(spermatogonial stem cells,SSCs)是成年哺乳动物精子发生的基础,具有自我更新和定向分化潜能,能够向后代传递遗传信息。利用SSCs的特点,可以建立转基因动物制作的技术,即分离SSCs、体外培养增殖、基因转染、移植、交配获得转基因动物后代的技术路线,有望应用于家畜经济性状的基因改良。     

Full-term development of offspring using round spermatids produced ectopically from fetal male germ cells

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells, which originate from primordial germ cells in the early embryo. Previously, we reported that the transplantation of fetal male gonadal tissue into the recipient testis was effective obtaining functional sperm. This transplantation technique is a promising new approach for the preservation of testicular function in a mutant animal with embryonic lethality. In the present study, we examined whether spermatogenesis from fetal male germ cells is induced under ectopic conditions in male and female recipients. Nine to 10 weeks after the transplantation of male gonads prepared from embryos at 12.5 or 16.5 days post gestation, male germ cell differentiation occurred under the skin of male and female recipient nude mice. Histological analyses revealed that grafted gonads contained haploid germ cells such as round or elongated spermatids. Furthermore, we succeeded in obtaining normal progeny by injecting the ectopically produced round spermatids into the cytoplasm of oocytes, even when the male germ cells had been generated in female recipients. These results indicate that the transplantation of fetal male gonads under the skin of recipient mice is a useful technique for obtaining functional male gametes.     

成年牛睾丸组织体外无血清培养研究

为研究促进精子发生的体外培养体系,通过无血清睾丸组织培养法,将成年牛睾丸组织块种植于培养板, 观测不同时间睾丸细胞的迁出和形态变化。通过油红O染色鉴定具有分泌功能的支持细胞,通过碱性磷酸酶和免疫组化染色鉴定集落样生长的精原干细胞。结果发现该体系可获得生长良好的多种类型细胞,包括表达AP、Oct-4和GFRα1的精原干细胞及具有分泌功能的睾丸支持细胞,并可获得大量精子。说明,该无血清培养体系可作为一个重要的工具来研究生物学和化学因素对雄性生殖系统的影响,也为研究精子体外发生和移植提供了材料。     

Characteristics of testicular cell development of 5‐day‐old mice in culture in vitro

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO₂ atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.     

The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function

Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility. Bovine testes must be maintained 4-5 °C below body-core temperature for normal spermatogenesis. The effects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identified a cohort of sperm functional proteins differentially expressed between normal vs abnormal sperm, including a testis-specific isoform of Na(+) /K(+) -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na(+) /K(+) -ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function.     

红景天多糖对幼鼠精原干细胞体外增殖的影响

为研究如何在体外获得更好的精原干细胞培养效果,以出生后6～8 d的小鼠为对象,应用两步酶消化法和差速贴壁法分离纯化精原干细胞.分别将浓度为100、200、300和400 ng/mL的红景天多糖加入以Sertoli细胞为饲养层的精原干细胞培养液中,以不添加红景天多糖组为对照,通过RT-PCR法和碱性磷酸酶（AP）染色鉴定细胞,MTT法研究红景天多糖对精原干细胞增殖的影响.RT-PCR和染色结果显示,分离得到的细胞为精原干细胞;MTT结果显示试验组比对照组细胞数量有显著增多（P＜0.05）,增殖率可达152％,且红景天多糖的最适添加量300 ng/mL.说明红景天多糖能显著促进小鼠精原干细胞的体外增殖.     

雌激素对睾丸发育及生精功能的影响

雌激素与睾丸的功能密切相关.人和哺乳动物体内雌激素的合成需芳香化酶(Ar)的参与,而雌激素与特异受体(ERα、ERβ)结合后才发挥生物学效应.在睾丸所有发育阶段,芳香化酶和雌激素受体几乎都有表达.在睾丸发育过程中,雌激素的缺乏对胎儿睾丸的发育有利.但对成年动物来说,大剂量的雌激素可以引起睾丸功能紊乱和生精障碍,而体外条件下小剂量的雌激素又能促进精原细胞的分裂、增生和分化,诱导精子发生和精子成熟,减少细胞凋亡.文章主要从睾丸内雌激素的来源、芳香化酶和雌激素受体的分布、雌激素对胎儿和新生儿睾丸发育以及对精子发生的影响这几个方面进行了综述.