精子发生转录调控机制的研究进展

精子发生过程中的转录调控是由一系列基因表达和调控事件组成的复杂过程,影响精子的形成、质量和功能。转录调控过程介导与精子形成密切相关的基因,包括精子特异性基因、组蛋白基因和其他转录因子的基因表达。这些基因的表达和沉默受到转录因子、表观遗传修饰和非编码RNA等多种机制的调控。此外,转录调控在精子发生的不同阶段起着不同的作用,包括精原干细胞的自我更新和分化、精母细胞的减数分裂和精子细胞的变形成熟。深入理解精子发生中的转录调控机制对于研究精子形成的生物学过程、解析生育障碍的病理机制以及开发生育问题相关的治疗方法具有重要的意义。     

十足目甲壳动物非浓缩精子发生过程中组蛋白动态研究进展

精子发生是一个受到高度调控的发育过程.在精子形成过程中,组蛋白和鱼精蛋白参与的替换过程对于精子核单倍体基因组的重包装具有重要作用.十足目甲壳动物精子具有非浓缩核的特征,目前多项研究已经证明,十足目甲壳动物精子细胞核或顶体结构中存在部分组蛋白及修饰的组蛋白.本综述总结了十足目甲壳动物精子发生过程中组蛋白的动态特征,有助于阐释非浓缩核染色质结构包装调控的机制.     

人Piwi基因突变致男性不育的机制研究

大量遗传学研究表明,Piwi蛋白对于动物生殖系细胞发育具有至关重要的作用,Piwi基因敲除致动物不育。人Piwi(Hiwi)基因特异性地在雄性生殖细胞表达,但目前对其在人精子发生中的作用及其与男性不育的联系还知之甚少。该研究通过筛查临床男性不育样本发现,少弱精症患者Hiwi基因中存在拮抗泛素化修饰的D-box元件突变;通过构建基因敲入小鼠模型证实,该突变导致雄性不育。机制研究表明,小鼠Piwi(Miwi)D-box突变致MIWI蛋白异常稳定存在于后期精子细胞中,导致与其相互作用的组蛋白泛素连接酶RNF8(ring finger protein 8)被扣留于细胞质、不能入核催化组蛋白泛素化修饰,进而抑制组蛋白被鱼精蛋白替换,引发精子形成异常、雄性不育。该研究发现了男性不育的一类新型致病基因突变,并发现了Piwi蛋白具有调控组蛋白泛素化修饰的新功能,揭示了精子形成中调控组蛋白–鱼精蛋白转换的重要机制。     

精子形成中的表观遗传调控

精子形成是指单倍体球形精子细胞分化发育成为精子的过程。这一连续进程包含一系列复杂的生化事件和剧烈的形态变化,涉及顶体和尾部形成、组蛋白-鱼精蛋白转换、细胞核压缩和细胞质丢弃等。近期研究发现,表观遗传调控在精子形成过程中发挥重要作用,对确保精子细胞正常发育和精子生成至关重要。现总结近期的相关研究进展,从DNA甲基化和组蛋白修饰两个方面简介精子形成过程中的表观遗传调控功能和机制。     

精子性染色体相关基因转录本及检测方法研究进展

精子形成过程中,基因表达经历了减数分裂性染色体失活,但有很多基因在减数分裂后期转录又重新被激活。转录产物研究表明,X和Y染色体特异基因在不同物种精子中都发生了不同程度的转录,并且对转录产物的研究方法也取得了巨大进展,产生了微阵列、SAGE及抑制消减杂交等先进的技术手段,随着这些技术的日益成熟和完善,通过深入研究,精子中性别相关基因转录表达调控机理将越来越清晰,并推动性别特异蛋白的研究取得更大进展。     

MIWI/piRNA激活小鼠精子细胞mRNA翻译的新功能机制研究

哺乳动物减数分裂后期的精子发生(spermatogenesis),即精子形成(spermiogenesis),是一个剧烈的细胞形态变化过程。伴随精子细胞中细胞核压缩和染色质重构,基因转录活性将逐渐降低直至完全停止,那些为精子细胞后期阶段发育所需的基因都需要提前转录为信使核糖核酸(mRNA),然后以翻译抑制状态储存在精子细胞中,直到特定发育阶段再被激活翻译,以合成蛋白质发挥作用。这个现象被称为“转录–翻译解偶联”,是精子发生中基因表达调控的一个典型特征。然而,目前对于精子细胞中被抑制的mRNA是如何被翻译激活的还知之甚少。我们当前的这项研究发现,MIWI/piRNA通过与翻译起始因子eIF3f、RNA结合蛋白HuR等因子形成功能性翻译激活复合物,特异性地激活小鼠精子细胞中包含AU序列富含元件(AU-rich element,ARE)mRNA的翻译。此项研究揭示了PIWI/piRNA在精子细胞翻译激活中的新功能,并证明此功能为精子细胞发育和功能性精子生成所必需。     

RNA干扰与染色质沉默——生物体内精密的网络调控机制

基因表达受不同层次的调控.RNA干扰通过产生双链小RNA诱导同源mRNA序列降解,从而在转录后抑制特定基因的表达.最新的研究成果显示：RNA干扰产生的双链小RNA可通过与染色质中的重复序列DNA及组蛋白甲基化酶相互作用,引起组蛋白H3 Lys9的甲基化,进一步与异染色质形成相关蛋白结合,导致染色质沉默.综述了RNA干扰,小RNA,组蛋白修饰,染色质沉默及基因表达调控之间存在着精密的网络调控机制.     

对黑斑蛙(Rana nigromaculata)和中华大蟾蜍(Bufo bufo asiatius)精子染色质的碱性蛋白和超微结构的比较研究

在精子形成过程中,精子细胞的细胞核高度浓缩成结构致密,体积很小的细胞核(精子核)。为了研究这种极度浓缩之染色质的组分和结构,我们用凝胶电泳法分析了黑斑蛙和蟾蜍的经过提纯之精子的染色质碱性蛋白和用电镜铺片法观察这些精子染色质的亚显微结构。我们发现黑斑蛙精子含有五种组蛋白,即H_1、H_3、H_(2B)、H_(2A)和H_4。H_3、H_(2A)、H_(2B)和H_4是核小体的主要组分。用电镜观察黑斑蛙精子染色质的结果说明,黑斑蛙精子染色质含有核小体结构,反之电泳分析结果说明,蟾蜍精子含有4条碱性蛋白带,其中1条带染色很深、宽度很大,迁移率远较组蛋白为大而与鱼精蛋白相似,而另外3条带染色既浅、宽度又狭,其中有1条从其迁移率来看相当于H_(2B)。用电镜观察时,这种蟾蜍精子染色质没有核小体结构。     

动物细胞的基因转录调控:来自生化研究的启示

在真核细胞中,转录调控可以发生在多个层面,包括结构和功能迥异的RNA聚合酶、对应的广谱起始因子、基因特异性调控因子(DNA结合蛋白)以及各种共调节因子(coregulatory factors)。这些共调节因子可以通过染色质修饰,如组蛋白乙酰化和甲基化,或更直接地促进转录起始复合物的形成。通过一系列体外转录活性实验的研究,转录相关的酶、蛋白因子的性质和功能以及作用机制正逐步被揭示出来。该文将具体阐述近几十年科学家们在转录共调节因子方面取得的进展。     

长链非编码RNA与表观遗传调控

长链非编码RNA(long non-coding RNA,lncRNA)是指转录本长度超过200 nt且缺乏蛋白编码能力的一类RNA。越来越多的研究表明,lncRNA能在表观遗传、转录及转录后水平调节基因的表达,广泛参与机体的生理和病理过程,在各种疾病的发生和发展中起着重要作用。表观遗传学是研究基因发生可遗传变化而核苷酸序列不变的一门学科,表观遗传现象众多,主要有DNA甲基化(DNA methylation)、组蛋白修饰(histone modification)、染色质重塑(chromatin remodeling)等。本综述对lncRNA在表观遗传调控中的作用进行介绍,以期为进一步研究lncRNA的调控性状提供思路。     

Chromatin and chromosomal fine structure in spermatogenesis of some species of amphibians

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.     

Microelectrophoretic analysis of basic protein changes during spermiogenesis in the dogfish Scylliorhinus caniculus (L.)

Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form ---S---S--- crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.     

Characterization of the unusual basic proteins of cricket spermatid nuclei on the basis of their molecular weights and amino acid compositions

Typical somatic cell type histones are lost from the nucleus during late spermiogenesis in the house cricket; they are replaced by unusual basic proteins specific to the spermatid. We wish to characterize these proteins because they appear to determine the unusual chromatin structures of the spermatid. Molecular weights of the unusual basic proteins were estimated by chromatographing them on Bio-Gel A 0.5 M agarose columns eluted with 6 M guanidine hydrochloride. Two proteins named TH1 and TH2 have molecular weights in the range spanned by the somatic histones. The molecular weight of TH1 is 17 500 and that of TH2 is 15 500. Three additional spermatid proteins were also analyzed by molecular weight determination. They are called here protamines A, B and C, and they have molecular weights in the range typical of protamines. That of A is 6200, of B is 5500 and of C is 3800. They span the range from the large protamines typical of mammalian sperm to the small protamines of salmonid fish. The molecular weights of the TH proteins were also examined by electrophoresis on SDS-polyacrylamide gels. Amino acid compositions determined for TH1 and TH2 show that both are basic proteins rich in arginine relative to lysine. Their compositions are histone-like, but they appear to be distinct histone types rather than variant forms of the somatic histones.     

Acetylation of histone H4 in complex structural transitions of spermiogenic chromatin

In spermiogenic nuclei of the cephalopod mollusc Sepia officinalis histones are replaced by a precursor-protamine molecule, which is later converted into protamine. Simultaneously, spermiogenic chromatin undergoes a complex structural change. Somatic-like chromatin belonging to the earliest spermatid is progressively reorganized into: (a) granules of 20 nm diameter, (b) fibres of 30-35 nm, and (c) fibres of 40-50 nm. In the final phases of spermiogenesis these fibres of 40-50 nm join to form larger structures of condensed chromatin, and lastly, the uniformly packed chromatin in the sperm nucleus. Using specific antibodies for mono- and hyperacetylated forms of histone H4, in this work we show that the first structural remodelling of chromatin (from somatic-like organization into 20 nm granules) is given concomitantly with a massive mono-acetylation of H4 (acetylation in lysine 12), whereas the structural remodelling from 30-35 to 40-50 nm fibres is produced simultaneously with hyperacetylation of H4 and the nuclear removal of histones.     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

Poly(ADP-ribosyl)ation during chromatin remodeling steps in rat spermiogenesis

In spermiogenesis, spermatid differentiation is marked by dramatic changes in chromatin density and composition. The extreme condensation of the spermatid nucleus is characterized by an exchange of histones to transition proteins and then to protamines as the major nuclear proteins. Alterations in DNA topology that occur in this process have been shown to require the controlled formation of DNA strand breaks. Poly(ADP-ribosyl)ation is a posttranslational modification of proteins mediated by a family of poly(ADP-ribose) polymerase (PARP) proteins, and two family members, PARP-1 and PARP-2, are activated by DNA strand breaks that are directly detected by the DNA-binding domains of these enzymes. Here, we show for the first time that poly(ADP-ribose) formation, mediated by poly(ADP-ribose) polymerases (PARP-1 and presumably PARP-2), occurs in spermatids of steps 11–14, steps that immediately precede the most pronounced phase of chromatin condensation in spermiogenesis. High levels of ADP-ribose polymer were observed in spermatid steps 12–13 in which the highest rates of chromatin nucleoprotein exchanges take place. We also detected -H2AX, indicating the presence of DNA double-strand breaks during the same steps. Thus, we hypothesize that transient ADP-ribose polymer formation may facilitate DNA strand break management during the chromatin remodeling steps of sperm cell maturation.M.L. Meyer-Ficca and H. Scherthan contributed equally to this work     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.