中心体蛋白Cenexin在大鼠精子发生过程中的表达特征

中心体蛋白Cenexin是成熟中心粒的唯一标志分子。为阐明中心粒在大鼠精子发生过程中的成熟以及功能,我们首先通过RT-PCR技术从大鼠睾丸组织中扩增出了Cenexin cDNA片段,原核表达重组蛋白后,用其免疫小鼠制备了高滴度的抗Cenexin的多克隆抗体,然后利用免疫荧光染色、Western Blot和半定量RT-PCR方法,研究了大鼠精子发生过程中Cenexin蛋白和基因的表达特征。结果显示Cenexin mRNA水平在精原细胞和精母细胞中较高,随后表达水平下降,而蛋白质分子在精原细胞到精子细胞中都定位于细胞的一个中心粒上,表示有成熟中心粒的存在,在长形精子细胞中该蛋白位于鞭毛的基体部。附睾的绝大多数成熟精子中Cenexin免疫染色消失。中心体蛋白Cenexin在精子变态期的表达变化可能与精子鞭毛形成的起始有关。     

中国鲎精子发生的研究: Ⅰ.精子的发生过程

利用光镜和电镜技术,结合细胞化学方法研究了中国鲎（Ｔａｃｈｙｐｌｅｕｓ ｔｒｉｄｅｎｔａｔｕｓ）精子发生过程．结果表明：中国鲎精子发生可划分为精原细胞、初级精母细胞、次级精母细胞、精子细胞及精子五个时期。在精子发生中,出现顶体丝结构,这是鲎精子发生的一个重要特征。页体由顶囊和亚顶体间隙组成。顶体丝由顶体囊底部发出,穿过亚顶体间隙,贯穿核中央部分的通道．沿核底部穿出,绕核缠绕,形成约２８匝的百体丝螺圈。这一特点可能与精子在受精时必须穿越厚的卵膜密切相关。成熟精子头部如梨形,前端覆盖有吸盘状后帽,头部长约４μｍ,尾部鞭毛长达３５μｍ、鞭毛为９ ×２结构,在鞭毛基部有一对中心粒。     

山羊精子发生不同阶段的显微与超微结构观察

应用光镜和透射电镜技术研究山羊精子发生不同阶段各级生精细胞显微、超微结构及山羊精子分化成熟过程。结果表明：山羊精子发生经历了精原细胞、初级精母细胞、次级精母细胞、精子细胞及变态精子阶段发育成成熟的精子。精原细胞期核呈椭圆形,染色质凝集成团分布于核质中,线粒体开始出现;精母细胞期有高尔基体分布;精子细胞经过核质浓缩、线粒体迁移等过程发育成成熟精子,成熟的山羊精子头部细长,核质高度浓缩,中段膨大,线粒体丰富。线粒体、中心粒对精子变态发生起重要作用,同时观察到头部与中段脱落的畸形精子。     

长吻鮠精巢发育的分期及精子的发生和形成

长吻鮠精巢的发育分为精原细胞增殖期、精母细胞生长期、精母细胞成熟期、精子细胞出现期,精子完全成熟期和精子退化吸收期。精巢的后1/3不产生也不贮存精子,精子的发生和形成经过精原细胞、精母细胞、精子细胞到精子的一系列过程。精原细胞有两种类型。精子无顶体,有中心粒帽,中片长,核凹窝和线粒体发达,鞭毛具侧鳍。     

长吻wei精巢发育的分期及精子的发生和形成

长吻wei精巢的发育分为精原细胞增殖期、精母细胞生长期、精母细胞成熟期、精子细胞出现期、精子完全成熟期和精子退化吸收期。精巢的后1/3不产生也不贮存精子,精子的发生和形成经过精原细胞、精母细胞、精子细胞到精子的一系列过程。精原细胞有两种类型。精子无顶体,有中心粒帽,中片长,核凹窝和线粒体发达,鞭毛具侧鳍。     

刘氏蝎蛉的精子发生和精子超微结构

【目的】精子超微结构在不同昆虫类群中变异较大,在昆虫种类鉴别和系统发育分析中具有重要意义。但截止目前,长翅目(Mecoptera)昆虫的精子发生和精子超微结构研究还很不充分。【方法】采用光学显微镜和透射电子显微镜技术,观察了刘氏蝎蛉Panorpa liui Hua的精子发生和精子超微结构。【结果】刘氏蝎蛉精原细胞在精囊内共分裂7次,产生128个精子细胞。这些精子细胞同步发育,成熟后结合形成数目略低于128的精子束。精子形成期,精子细胞内高尔基复合体产生的原顶体颗粒物质形成精子顶体;球状细胞核伸长、核内染色质凝集,形成致密的线形细胞核;分散的线粒体聚集、融合产生的副核转化形成2条线粒体衍生物。成熟精子由头部、颈区和长鞭毛组成。精子头部包括双层顶体和具有2条侧沟的细胞核两部分;颈区主要由中心粒和致密的鞘状中心粒侧体组成。鞭毛螺旋状,主要由1条9+2型轴丝、2条大小不等的线粒体衍生物和2条副体组成。【结论】刘氏蝎蛉精子束内精子数目略低于128,可能与精子复杂的形成过程及细胞的吞噬作用有关。线粒体衍生物在不同类群间差异显著,可为长翅目系统发育分析提供有用的特征。     

中国淡水蛏精子发生的超微结构研究

利用透射电镜观察了中国淡水蛏的精子发生和精子结构。描述了精子发生过程中各细胞形态结构的变化;揭示了细胞核、线粒体和项体等的演变过程。中国淡水蛏的精子经过精原细胞、初级精母细胞、次级精母细胞和精子细胞,最终限育为成熟的精子。精子为典型的原生型,由头部、中段和尾部构成。项体复合体位于精子头部前端,呈“⊥”形,精核近圆形;中段包括２个中心粒和４或５个紧密排列的椭圆形线粒体;尾部鞭毛断面为典型的“９＋２”     

水蕨精子发生过程中中心体蛋白和微管蛋白的免疫荧光定位

采用透射电镜技术和免疫荧光标记技术对水蕨精子发生的超微结构以及中心体蛋白和微管蛋白在精子发生过程中的动态表达进行了观察。研究发现：（1）生毛体分化早期周围有放射状微管分布,这与线粒体向生毛体的聚集有关。（2）免疫荧光观察表明,中心体蛋白仅定位于生毛体、基体和鞭毛带上,自生毛体至基体阶段呈现明亮的荧光标记,在核塑形、鞭毛形成至精子成熟阶段,中心体蛋白荧光标记随着鞭毛的发生而逐渐减弱,至游动精子阶段中心体蛋白荧光标记信号几乎消失。（3）微管蛋白早期荧光标记与中心体蛋白标记形相同,在生毛体、鞭毛带、基体等运动细胞器上呈现明亮荧光标记,但微管蛋白随着鞭毛的发生其荧光标记越来越强。从二者的时空表达特征可以推断,中心体蛋白主要是运动细胞器的组织者,而非这些运动细胞器的结构成分,其功能是参与或负责中心粒、基体和鞭毛的发生。     

大黄鱼的精子发生

应用电子显微镜技术观察了大黄鱼（Pseudosciaena crcea）的精子发生过程。其发生经历了初级精原细胞、次级精原细胞、初级精母细胞、次级精母细胞和精子细胞阶段,精子细胞再经过精子形成过程成为精子。在精原细胞阶段,部分核仁物质排出核外,成为拟染色体。拟染色体的主要成分是核糖体。在精子发生中,拟染色体逐渐扩散到生精细胞的胞质中。成熟分裂的前期Ⅰ,同源染色体经历了联会复合体形成和解体的变化。在精子形成过程中,精子细胞先形成鞭毛,随后细胞核逐渐浓缩。     

星豹蛛精子的超微结构和精子发生（蜘蛛目：狼蛛科）

报道星豹蛛(Pardosa astrigera)的精子发生过程和成熟精子的超微结构。研究表明,星豹蛛的精子发生有下列特征与其他蜘蛛的研究结果相同:1.染色质浓缩;2.精子的囊内形成;3.顶体泡出现,并有一直穿到核管的顶体丝;5.轴丝为9×2+3型;6.精子成熟时盘曲;7.精子在输精管内进行包装。同时,我们在研究中获得如下新的资料:1.在精原细胞、精子细胞和精巢外层出现上皮细胞;2.中心粒在精子发生的前期为平行排列,到后期变为垂直排列;3.微管环在前期见于核膜外,随后消失;后期见于细胞膜下,最后消失;4.没有线粒体和鞭毛通道,但在胞质中有成群的电子致密圆泡和一膜状结构;5.顶体泡锥形,部分内陷到核内;6.精子包装为闭精武。上述研究结果为今后深入探讨蜘蛛目的系统发生提供了参考资料。     

Alternative splicing of exon 3b gives rise to ODF2 and Cenexin

ODF2 was first identified as the major component of the sperm tail outer dense fibers. Additionally, ODF2 is a critical component of the mature centriole of the animal centrosome where it locates to the distal appendages. Moreover, generation of primary cilia strictly depends on ODF2. The mature centriole is characterized further by recruitment of Cenexin. Albeit highly similar in sequence the relationship between ODF2 and Cenexin has not been investigated. We demonstrate here that ODF2 and Cenexin are alternative splice products by identifying a novel exon 3b encoding Cenexin specific amino acids. Even though ODF2 is the main isoform in testicular tissue RT-PCR analyses revealed that isoforms are not restricted to specific tissues.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.     

三角帆蚌精子的发生

报道了光镜和透射电镜下三角帆蚌精子的发生过程及其一系列重要的形态变化。包括核延长,染色质浓缩,线粒体逐渐融合并后移;胞质减少及鞭毛形成,精原细胞是精巢中体积最大的细胞,细胞膜界限不明显,内质网发达,精母细胞开始出现中心粒,精细胞分化可分为3个阶段。成熟精子属原生型,由头部、中段和尾部三部分组成。     

Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times of spermatogenesis.     

Asynchronous expression of the homeodomain protein CUX1 in Sertoli cells and spermatids during spermatogenesis in mice

The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.     

Expression of MAEL in nuage and non-nuage compartments of rat spermatogenic cells and colocalization with DDX4, DDX25 and MIWI

The functions of MAELSTROM protein (MAEL) in spermatogenesis are gradually being identified but the precise distribution of MAEL in spermatogenic cells during spermatogenesis has not yet been mapped. We studied the expression of MAEL in rat testis by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence staining showed that MAEL was localized in intermitochondrial cement, irregularly-shaped perinuclear granules and satellite bodies of pachytene spermatocytes, and in chromatoid bodies of spermatids. The SBs appeared exclusively in pachytene spermatocytes at stages IX–X and were stained strongly for MAEL. In step 12–19 spermatids, many granules stained for MAEL but not DDX4. These granules were confirmed to be non-nuage structures, including mitochondria-associated granules, reticulated body, granulated body by IEM. In the neck region of late spermatids and sperm, MAEL-positive small granules were found. MAEL is colocalized with MIWI in nuage and non-nuage. The results suggest that MAEL seems to function in nuage and non-nuage structures and interacts with MIWI.