Sperm from neonatal mammalian testes grafted in mice

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.     

在单细胞水平建立原位PCR基因鉴别技术平台——以精子基因型鉴别为例

目的精子发生是一个包括有丝分裂和减数分裂的精细调控过程,这一过程是从精原干细胞开始在雄性睾丸的曲细精管中完成的,由哺乳动物雌雄性比例接近1∶1这一现象可以推知:受精时带X基因的精子和带Y基因的精子比率应该是1∶1,但事实上精子发生的过程中,分化出来的X、Y精子的比例是否也为1∶1并不清楚,因为精子发生过程中并不是所有细胞都能最终形成精子,然而要研究雌雄出生比率就需要先研究精子本身是否严格地按照1∶1形成,因此本实验通过建立快速可靠原位PCR技术平台为进一步研究精子发生的调控过程提供支持。步骤用HSL基因优化荧光原位PCR实验方案,包括蛋白酶K作用时间和浓度,原位PCR前先对精子进行解聚,再用SRY基因对精子进行原位PCR鉴定。结果在荧光显微镜下可清楚地看到精子头部的荧光信号,头部有绿色荧光信号的精子为带有SRY基因的Y染色体精子(Y精子),实验检测到昆明小鼠精子共493个,有信号的共273个,X∶Y=55.37%,经卡方检验,X精子与Y精子的实验结果趋近于1∶1。结论可利用荧光原位PCR技术在单细胞水平快速鉴定精子"性别",理论上可用于任何细胞的任何基因的鉴定。     

东方杯叶吸虫精巢和精子发生的超微结构研究

本文应用透射电镜研究了东方杯叶吸虫精巢和精子的超微结构及精子发生过程,并与体外培养成虫进行比较,提出一种精子结构模式图.精子的形成经历了由精原细胞,初级精母细胞、次级精母细胞、精子细胞和精子的发育等过程.精子细胞形成后,胞质突起形成一分化区,并发育成精子鞘,核向鞘中旋转、浓缩和迁移,两根鞭毛轴丝由鞘中基体发出;鞭毛为9+“1”结构.体外培养成虫尚有几种不正常的成熟精子,可作为体外培养的评价指标.     

Boule基因在精子发生过程中的作用

精子发生是一个复杂的细胞分化过程,生精细胞经过有丝分裂和减数分裂产生单倍型的精细胞,精细胞再经过复杂的形态变化而形成成熟的精子。这一过程需要众多基因表达的精确调控。目前已发现了一些与男性不育相关的候选基因,这些候选基因中的DAZ(Deleted in azoospermia)基因家族在精子发生过程中发挥关键性的调控作用。Boule基因是2001年发现的DAZ家族的新成员,是人类精子发生过程中重要的调控因子。Boule表达的改变或BOULE蛋白的缺乏可引起减数分裂阻滞和精子生成障碍,从而导致无精子症并产生不育。     

Review: Actin-based dynamics during spermatogenesis and its significance

Actin can be found in all kinds of eukaryotic cells, maintaining their shapes and motilities, while its dynamics in sperm cells is understood less than their nonmuscle somatic cell counterparts. Spermatogenesis is a complicated process, resulting in the production of mature sperm from primordial germ cell. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. It was proved that all mammalian sperm contain actin, and that F-actin may play an important role during spermatogenesis, especially in nuclear shaping. Recently a new model for sperm head elongation based on the acrosome-acroplaxome-manchette complex has been proposed. In Drosophila, F-actin assembly is supposed to be very crucial during individualization. In this mini-review, we provide an overview of the structure, function, and regulation characteristics of actin cytoskeleton, and a summary of the current status of research of actin-based structure and movement is also provided, with emphasis on the role of actins in sperm head shaping during spermiogenesis and the cell junction dynamics in the testis. Research of the Sertoli ectoplasmic specialization is in the spotlight, which is a testis-specific actin-based junction very important for the movement of germ cells across the epithelium. Study of the molecular architecture and the regulating mechanism of the Sertoli ectoplasmic specialization has become an intriguing field. All this may lead to a new strategy for male infertility and, at the same time, a novel idea may result in devising much safer contraception with high efficiency. It is hoped that the advances listed in this review would give developmental and morphological researchers a favorable investigating outline and could help to enlarge the view of new strategies and models for actin dynamics during spermatogenesis.     

睾丸支持细胞功能的研究进展*

摘要: 支持细胞位于睾丸曲细精管内皮,作为与生精细胞接触的惟一体细胞,在生精过程中起着至关重要的调控作 用。支持细胞在精子发生过程中可为精子细胞提供结构支持; 他分泌的各种蛋白质参与精子发生所需物质的转 运; 同时支持细胞表达一些膜蛋白,促进局部免疫豁免形成,为精子发生提供有利的微环境; 再者,支持细胞通过发 挥粘附与吞噬作用,清除精子发生中凋亡的精子细胞。随着研究的深入,近年来对支持细胞又有了新的认识。     

绒山羊曲细精管生殖细胞的长期培养和精子发生过程的观察

哺乳动物精子发生过程是生殖生物学的重要研究课题之一.为了开展对山羊精子发生过程的研究,我们首次建立了绒山羊睾丸生殖细胞原代培养方法.原代生殖细胞和支持细胞(Sertolicells)由绒山羊睾丸曲细精管组织块产生,在体外共培养超过三个月.在共培养期间,观察到绒山羊的精原干细胞分化为精母细胞和精子细胞的形态变化过程.这一方法的建立为山羊精子发生过程的研究和应用打下了基础.实验结果显示,在没有添加任何生长因子的条件下,绒山羊睾丸生殖细胞长期增生分化,不断产生精子细胞.这一结果暗示了组织块和共生的支持细胞为生殖细胞的增生和分化提供了营养因子和调节因子.生成的游离精子细胞最后全部死亡,暗示这一共培养体系不能提供变态过程所需因子.     

动物精子发生及成熟过程中凝集素受体的分布与变化

对各种动物精子发生与成熟过程中生精细胞表面凝集素受体的变化做了综述 .精子发生过程伴随着不同凝集素受体的出现和变化 ,这些受体是由生精细胞自身合成的 ;精子在成熟过程中 ,质膜凝集素受体的合成、修饰及分布会进一步发生变化 ,这些变化与精子获得与卵子进行识别、粘附、结合等受精能力密切相关 .     

Pluripotency of spermatogonial stem cells from adult mouse testis

Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.     

哺乳动物精子发生及其基因表达调控

精子发生的过程是一个受机体严密调节的过程,其中包括激素调节、睾丸旁分泌因子的调节和其它信号分子的调节,睾丸特异性表达的基因在这一调节过程中发挥着重要的作用。研究睾丸特异性表达基因的功能的最终目的是阐述精子发生的机理和发现新一代高效、特异、可逆的男性避孕药物的靶蛋白基因。     

小鼠精原干细胞的分离、分选、移植和培养

精原干细胞具有自我增殖和分化为精子的能力。精原干细胞的培养、移植和体外诱导分化等研究将使我们最终阐明精原干细胞的自我更新机制和精子发生机理。经过一年多的研究,我们不仅建立了用含血清培养基对分离的乳鼠睾丸细胞进行差异贴壁分选来富集生殖细胞的简单方法,而且成功地对分选出的精原干细胞进行了近一个月的无血清培养。流式分析结果表明,差异贴壁分选能将精原干细胞富集11倍以上。移植分析表明,分选出的精原干细胞具有在受体鼠的曲精小管中产生克隆的能力和正常生精作用。免疫细胞化学和RT-PCR检测表明,培养的细胞表达精原干细胞标志基因。该研究为体外长期培养小鼠或其它哺乳动物的精原干细胞提供了一个范例,也为利用基因修饰的精原干细胞通过移植产生转基因动物奠定了基础。     

Sperm chromatin proteomics identifies evolutionarily conserved fertility factors

Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.     

斜纹夜蛾精巢的发育和精子的发生

采用解剖观察、石蜡组织切片及苏木精-曙红染色方法,利用显微镜照相获取图像,研究了斜纹夜蛾幼虫到成虫精巢的发育及精子的发生.研究结果表明:2个精巢在预蛹期开始融合,并在蛹期融合成1个;在成虫期,随着精子的排出,精巢体积逐渐减小;斜纹夜蛾雄性生殖细胞发育为有核精子和无核精子,2种精子的发生都经过精原细胞囊期、精母细胞囊期和精细胞囊期;精子的形成主要区别在于有核精子的细胞核有一个变形过程,而无核精子形成过程中不经过细胞核变形,且后期将细胞核丢弃.综上所述,有核精子的主要发育时期为6龄到蛹期,而无核精子的主要发育在蛹期.本研究结果可为进一步研究和控制精子形成以及利用生殖机理防治斜纹夜蛾打下基础.     

小鼠卵巢颗粒细胞条件培养液中蛋白质和激素水平

测定用于精子顶体反应和早期胚胎离体培养的小鼠卵巢颗粒细胞条件培养液中的蛋白质和激素水平。结果表明：单层培养的颗粒细胞体外培养后生长良好,能稳定分泌蛋白质、雌二醇（Ｅ２）和孕酮（Ｐ）。在培养液中加入促卵泡素（ＦＳＨ）和胰岛素（ＩＮＳ）时,Ｅ２的分泌量无显著变化。     

Germ cell transplantation in infertility mouse

This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules. Donor germ cells were isolated from male FVB/NJ-GFP trensgenic mice. Seminiferous tubule microinjection was applied to achieve intratubular germ cell transfer. The germ cells were injected into exposed testes of the infertility mice. We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker. The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjection. The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60 （68.33%） of the injected recipient mice using allogeneic donor cells. In the colonized testes, matured spermatozoa were seen in the lumen of the seminiferous tubules. In this research, BALB/c-nu infertility mouse model, the recipient animal, was used to avoid immunological rejection of donor cells, and germ cell transplantation was applied to overcome infertility caused by busulfan treatment. These results demonstrate that this technique of germ cell transplantation is of great use. Germ cell transplantation could be potentially valuable to oncological patients.     

短尾鮠精子发生的超微结构研究

用透射电镜观察了短尾鮠的精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子的形态以及细胞核、拟染色体和线粒体等的结构变化规律．结果表明：精原细胞时期细胞器较丰富，到精子细胞后期，细胞器的形态和数量发生了较大变化；随着精母细胞的发育，核质凝聚程度逐渐增强．短尾鮠的精子具有椭圆的头部和复杂的中片，近侧中心粒和远侧中心粒靠近核的中央，鞭毛呈9＋2轴丝结构，并具有由外膜折迭形成的波浪形的结构．表明短尾鮠精子发生中具有某些区别于其它鱼类精子的不同结构．     

人参皂甙Rb1对体外培养的小鼠支持细胞增殖作用的研究

观察人参皂甙Rb1(ginsenoside Rb1,GRb1)对体外培养的小鼠支持细胞增殖能力的影响,探讨其促进精子发生的作用机制.采用多次贴壁法纯化原代培养的支持细胞,取体外培养第2代第3天的支持细胞,利用MTT比色分析法和Brdu增殖细胞标记法,检测不同质量浓度GRb1对体外培养支持细胞增殖的影响.20 mg/LGRb1对支持细胞增殖有明显促进作用.GRb1能维持小鼠支持细胞的存活,并在适当质量浓度范围内可以明显促进支持细胞的增殖,从而为进一步探讨GRb1的药理作用及精子发生的机理提供一些研究基础.     

北方山溪鲵精巢支持细胞的显微与超微结构观察

用光镜和电镜观察了山溪鲵精巢内支持细胞的显微和超微结构．结果表明，在精原细胞增生期，支持细胞间存在镶嵌连接，与精原细胞增生以及生精小囊的体积变大相适应；在精子形成期，支持细胞具有典型的类固醇激素分泌特征；精子成熟后，支持细胞退化．并对生精细胞和支持细胞的关系进行了讨论．     

孔雀鱼精子发生的显微与超微结构

对卵胎生硬骨鱼孔雀鱼精子的发生进行了显微与超微结构研究.成鱼精巢呈肾形,腹面有输精管伸出,内部构造为小管型.孔雀鱼精子发生经过精原细胞,初级精母细胞,次级精母细胞,精子细胞及变态精子阶段;初级精母细胞和精子细胞高尔基体和囊泡丰富;从精母细胞到精子细胞有细胞质不完全分裂现象;成熟精子头部较长,袖套部分长度是头部的2倍,直径与精子头部相同,线粒体多.鞭毛有侧鳍,其轴丝为典型的“9+2”结构.     

小鼠精原细胞体外培养分化的研究

目的 探讨小鼠精原细胞的分离纯化及体外培养条件。方法采用组合酶消化法分离纯化小鼠精原细胞和支持细胞,在添加FSHC和TC的基本培养基中进行体外培养。采用反转录PCR方法检测精子细胞特异性基因过渡蛋白9（Tnp2）的表达以对分化细胞进行鉴定。结果培养5d后有分化的圆形精子细胞出现,培养7d后收集的细胞可见Tnp2基因表达。结论通过体外培养小鼠精原细胞可迅速完成减数分裂过程和减数分裂后的分化过程,并可获得更接近成熟阶段的精子细胞。