Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

斑节对虾精子发生的超微结构

斑节对虾精子发生划分为精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子五个阶段。精子发生中，从精原细胞到精子，染色质经历了从以异染色质为主变为高度凝聚态，再经解聚为弥散絮状的变化过程。同时，核从具有完整核膜变为核膜不完整。成熟的的精子含有核仁。顶体由高尔基囊泡逐渐演化而成，并向外伸长成为棘突。这是斑节对虾精子发生的主要特征。     

KIFC1 participates in acrosomal biogenesis, with discussion of its importance for the perforatorium in the Chinese mitten crab Eriocheir sinensis

Spermatogenesis is a complicated process during which spermatogonia undergo proliferation and divisions leading, after a series of dramatic changes, to the production of mature spermatozoa. Many molecular motors are involved in this process. KIFC1, a C-terminal kinesin motor, participates in acrosome biogenesis and nuclear shaping. We report here the expression profile of KIFC1 during spermatogenesis in the Chinese mitten crab, Eriocheir sinensis. KIFC1 mainly localizes around the nucleus but is also present within the nucleus of the spermatogonium and spermatocyte. At the early spermatid stage, KIFC1 begins to be distributed on the nuclear membrane at the region where the proacrosomal vesicle is located. By the late spermatid stage, KIFC1 is found on the acrosome. Immunocytochemical and ultrastructural analyses have shown that KIFC1 localizes on the perforatorium, which is composed of an apical cap and an acrosomal tubule. We demonstrate that, during spermatogenesis in E. sinensis, KIFC1 probably plays important roles in the biogenesis of the acrosome and in its maintenance. KIFC1 may also be essential for the eversion of the acrosome during fertilization. This work was supported in part by the following projects: the National Natural Science Foundation of China (nos. 30671606 and 40776079) and the National Basic Research Program of China (973 Program; grant no. 2007CB948104).     

泥螺精子发生的超微结构研究

利用透岸民镜观察了泥螺精子发生的过程。结果表明;泥螺精子发生经历了一系列重要的形态和结构变化,主要有核逐渐延长,染色质浓缩,顶体形成,线粒体逐步发达与融合,胞质消除及鞭毛的形成等。泥螺精细胞分化可分为3个时期,在精细细胞分化过程中,细胞核形态及染色质的变化与其他软体动物有较大的差异,核内椭圆形到肾形,再变化为长圆柱形;染色质由絮状颗粒变为细纤维丝状,再变为长纤维丝状,最后向高电子密度均质状态转变,初步探讨了泥螺精子发生过程中核及细胞器的超微结构变化在分类上的意义。     

A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with γ-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.     

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACT

During spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.     

NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila.

The conserved family of NXF proteins has been implicated in the export of messenger RNAs from the nucleus. In metazoans, NXFs heterodimerize with p15. The yeast genome encodes a single NXF protein (Mex67p), but there are multiple nxf genes in metazoans. Whether metazoan NXFs are functionally redundant, or their multiplication reflects an adaptation to a greater substrate complexity or to tissue-specific requirements has not been established. The Drosophila genome encodes one p15 homolog and four putative NXF proteins (NXF1 to NXF4). Here we show that depletion of the endogenous pools of NXF1 or p15 from Drosophila cells inhibits growth and results in a rapid and robust accumulation of polyadenylated RNAs within the nucleus. Fluorescence in situ hybridizations show that export of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs is inhibited. Depleting endogenous NXF2 or NXF3 has no apparent phenotype. Moreover, NXF4 is not expressed at detectable levels in cultured Drosophila cells. We conclude that Dm NXF1/p15 heterodimers only (but not NXF2-NXF4) mediate the export of the majority of mRNAs in Drosophila cells and that the other members of the NXF family play more specialized or different roles.     

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

Spermiogenesis and structure of spermatozoa in the oribatid mite,Hafenrefferia gilvipes (C.L. Koch) (Acari,Oribatida)

The process of spermiogenesis and the structure of spermatozoa in the mite, Hafenrefferia gilvipes (Koch) were studied ultrastructurally. Spermiogenesis was divided into six stages. The spermatids at stage 1 have the usual structure. At stage 2 the structure of the mitochondria and their distribution in the spermatid start to change, leading to the formation of specific mitochondrial derivatives which are subsequently incorporated into the nucleus of the spermatozoon. Parallel to the transformation of mitochondria occurs a reorganization of the nuclear material. The fully formed spermatozoon has a tadpole-like shape, with the cell nucleus located in the distended part of the cell, and containing mitochondrial derivatives in its karyoplasm. Acrosome, flagellum and centrioles are absent. The participation of peripherally distributed microtubules, present in spermatids at stages 4 to 6, in the shaping of the spermatozoon has been suggested.     

金鱼精子发生中的拟染色质小体

拟染色质小体是性细胞特有的细胞器。本文报道:在金鱼精子发生过程中,精原细胞含有大量的拟染色质小体,在它上面不仅聚集着许多线粒体,而且有膜片层附着,其本身还会出现环孔和类似线粒体内嵴状结构。随着细胞的分化,拟染色质小体逐渐变少减小,直至消失。本文对拟染色质小体的形成方式与功能进行了讨论。     

Sea urchin testicular cells evaluated by fluorescence microscopy of unfixed tissue

A procedure is presented for rapid, quantitative evaluation of cell and nuclear types present in the male gonad of the sea urchin. Vitally stained whole mounts of tissue fragments or dissociated cells are prepared, which reveal detailed 3-dimensional chromatin patterns and enough cytoplasmic features to provide reliable markers for most of the somatic and germ line cell types. Representative cellular morphologies are described. Nuclear volume changes during spermatogenesis are quantified. Spermatid nuclei contain an apparently interconnected network of heterochromatin. Regions relatively devoid of chromatin decrease in size as nuclear condensation proceeds and spherical nuclear shape is maintained. The major decrease in nuclear volume occurs prior to the late spermatid stage. The volume of the spermatozoan nucleus is achieved by the smallest late spermatid nucleus before the change from spherical to conoid morphology. The relationship of this morphological transition to sperm histone dephosphorylation is discussed.     

Forced expression of RNF36 induces cell apoptosis

RNF36 (ring finger protein 36; alias Trif), a member of the RING zinc finger protein family, is expressed in germ cells at round spermatid stages during spermatogenesis. RING finger proteins have been implicated in a variety of functions including oncogenesis, viral replication, and developmental processes. Since no germ cell line is presently available to study the function of RNF36, in this research, we expressed RNF36 truncated and full-length proteins in COS-7 and HEK-293 cell lines to study the effect of RNF36 in somatic cells. The full-length RNF36 protein in both cell lines showed a speckled pattern in the nucleus. Truncated RNF36-1 protein with its putative nuclear localization signal (NLS) remained within the nucleus but lost the speckled pattern. The promyelocytic leukemia (PML) protein, another RING finger protein, was previously identified as present in the nucleus with a speckled pattern. Double-staining and coimmunoprecipitation analyses suggested that RNF36 colocalizes and interacts with PML. In vitro phosphorylation analysis further suggested that RNF36 nuclear localization is under the control of phosphorylation, which might be mediated by p38. Treatment with the p38 inhibitor SB203580 resulted in the cytoplasmic translocation of RNF36. Overexpression of full-length RNF36 in cells induced about half of the transfected cells to undergo cell death. The results of DNA fragmentation assays, flow cytometry assay, and TUNEL staining suggest that the death of RNF36-transfected cells was caused by apoptosis. Following further characterization of the molecular mechanism of RNF36-induced apoptosis, we found that the expression of Bax, caspase-2, and receptor-interacting protein were elevated upon RNF36 induction in test cells. These results suggest that RNF36 may interact with PML and induce cell apoptosis. We suspect that RNF36 may play a role in germ cell homeostasis during spermatogenesis.     

日本沼虾精子发生的研究

对日本沼虾精子发生全过程的电镜观察表明：精原细胞核染色质分散,胞质内有线粒休、内质网的分布。初级精母细胞核染色质块状,不均匀地分布于核中,内质同多小泡多。次级精母细胞核染色质大多分布于核膜内侧,内质网聚集成团,精细胞分化形成精子的早期,胞核增大,核侧形成内质同多小泡的聚合体;中期的核内染色质浓缩,同时形成空囊状结构,     

Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Ultrastructural study of spermatogenesis in the free-living marine nematode <Emphasis Type="Italic">Paracyatholaimus pugettensis</Emphasis> Weiser et Hopper, 1967 (Chromadorida: Cyatholaimidae)

Spermatogenesis and the morphology of mature sperm in the free-living chromadorid Paracyatholaimus pugettensis from the Sea of Japan were studied using transmission electron microscopy. In spermatocytes fibrous bodies (FBs) appear; in spermatids, the synthetic apparatus is located in the residual body, whereas the main cell body (MCB) houses the nucleus, mitochondria, and FBs. The nucleus of the spermatid consists of a loose fibrous chromatin that is not surrounded by a nuclear envelope; centrioles lie in the perinuclear cytoplasm. The plasma membrane of the spermatid MCB forms numerous filopodia. Immature spermatozoa from the proximal part of the testis are polygonal cells with a central nucleus. The latter is surrounded by mitochondria and FBs with poorly defined boundaries. The immature spermatozoa bear lamellipodia all along their surface. Mature spermatozoa are polarized cells with an anterior pseudopodium, which is filled with filaments that make up the cytoskeleton; the MCB houses a nucleus that is surrounded by mitochondria and osmiphilic bodies. In many ultrastructural characteristics, the spermatozoa of P. Pugettensis are similar to those of most nematode species studied so far (i.e., they are ameboid, have no acrosome, axoneme, or nuclear envelope). On the other hand, as in other chromadorids, no aberrant membrane organelles were observed during spermatogenesis of P. Pugettensis.Original Russian Text Copyright © 2004 by Biologiya Morya, Zograf, Yushin.     

RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclear pore complex and functions in nuclear mRNA export

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.     

Ultrastructure of sperm development in the free-living marine nematode Metachromadora itoi (Chromadoria, Desmodorida)

The spermatogenesis of the free‐living marine nematode Metachromadora itoi was studied with electron microscopy. Spermatocytes and early spermatids have no cytoplasmic components specific for nematodes, i.e. membranous organelles (MO) and fibrous bodies (FB). The late spermatids are subdivided into the residual body and the main cell body with a centrally located nucleus devoid of a nuclear envelope. A pair of 9 × 2 centrioles is associated with the nuclei of spermatids and spermatozoa. The nucleus of the mature spermatid is surrounded by a thick mass of radially arranged FB delimited externally by a discontinuous layer of mitochondria, which underlie a thin ectoplasm. Sperm development is accompanied by transfer of FB matter through the mitochondrion layer into the ectoplasm. The immature spermatozoa from the testis have the centrally located nucleus surrounded by a transparent halo with remnants of FB. The halo is delimited by a sphere of mitochondria that underlie the thick fibrous ectoplasm, a derivative of the FB. In the mature spermatozoa the ectoplasm is transformed into the prominent unpolarized pseudopod. The central nucleus is surrounded by a transparent halo and a sphere of mitochondria, which underlie the pseudopod. MO were not found throughout spermatogenesis. In general, spermatogenesis in M. itoi differs from that observed in many nematodes but resembles in some details the sperm development in some chromadorid and tylenchomorph nematodes. The phylogenetic importance of this sperm development is discussed.